10 1038@nbt 4201

letters
Efficient proximity labeling in living cells and organisms

with TurboID
Tess C Branon1–4, Justin A Bosch5, Ariana D Sanchez4, Namrata D Udeshi6 , Tanya Svinkina6, Steven A Carr6,
Jessica L Feldman4, Norbert Perrimon5,7 & Alice Y Ting1–4,8
© 2018 Nature America, Inc., part of Springer Nature. All rights reserved.
Protein interaction networks and protein compartmentalization for proteomic analysis. This precludes the use of BioID for studying
underlie all signaling and regulatory processes in cells. dynamic processes that occur on the timescale of minutes or even a
Enzyme-catalyzed proximity labeling (PL) has emerged few hours. Furthermore, the low catalytic activity makes BioID dif-
as a new approach to study the spatial and interaction ficult or impossible to apply in some contexts, such as in worms, flies,
characteristics of proteins in living cells. However, current PL or the endoplasmic reticulum lumen of cultured mammalian cells.
methods require over 18 h of labeling time or utilize chemicals Recently, new promiscuous biotin ligase variants, BioID2 (ref. 26)
with limited cell permeability or high toxicity. We used yeast and BASU27, have been reported, but the former still requires >16 h
display-based directed evolution to engineer two promiscuous of labeling26,28–30, while BASU enriched a proteome of only two pro-
mutants of biotin ligase, TurboID and miniTurbo, which teins27. Further characterization (see below) shows that the activities
catalyze PL with much greater efficiency than BioID or BioID2, of BioID, BioID2, and BASU are all comparable.
and enable 10-min PL in cells with non-toxic and easily A new PL enzyme that combines the simplicity and non-toxicity of
deliverable biotin. Furthermore, TurboID extends biotin-based BioID with the catalytic efficiency of APEX2 would greatly enhance PL
PL to flies and worms. applications. To achieve this, we undertook the directed evolution of
E. coli biotin ligase (BirA) to generate new promiscuous mutants. To
Enzyme-catalyzed PL is an alternative to immunoprecipitation and begin, we compared BioID (BirA-R118G) to seven other mutations at the
biochemical fractionation for proteomic analysis of macromolecular R118 position. We found that R118S is about twofold more active than
complexes, organelles, and protein interaction networks1. In PL, a R118G under identical conditions (Supplementary Figs. 1 and 2 for all
promiscuous labeling enzyme is targeted by genetic fusion to a specific full blot images and Supplementary Note 1), and hence we selected this
protein or subcellular compartment. Addition of a small-molecule mutant rather than BioID as our starting template for evolution.
substrate, such as biotin, initiates covalent tagging of endogenous pro- As in previous work3,31, we combined yeast surface display of our
teins within a few nanometers of the promiscuous enzyme (Fig. 1a). enzyme library with fluorescence-activated cell sorting (FACS) to per-
Subsequently, the biotinylated proteins are harvested using streptavi- form the evolution. We used error-prone PCR to mutagenize BirA-
din-coated beads, and identified by mass spectrometry (MS). R118S, generating a library of ~107 mutants, each with an average of
Two enzymes are commonly used for PL: APEX2, an engineered about two amino acid mutations relative to template. This library was
soybean ascorbate peroxidase2,3; and BioID, a promiscuous mutant then displayed on the yeast surface as a fusion to the Aga2p mating
of Escherichia coli biotin ligase4,5. The advantages of APEX2 are its protein (Fig. 1b). We added biotin and ATP to the yeast pool to initi-
speed—proximal proteins can be tagged in 1 min or less6,7—and its ate promiscuous biotinylation, followed by streptavidin-fluorophore
versatility, as APEX2 also captures endogenous RNAs8 and generates to stain biotinylation sites on the surface of each yeast cell. FACS was
contrast for electron microscopy9. However, APEX labeling requires the used to enrich cells displaying a high degree of self-biotinylation over
use of H2O2, which is toxic to living samples. By contrast, BioID labe- cells displaying low or moderate self-biotinylation (Fig. 1b). We grad-
ling is simple and non-toxic: only biotin needs to be supplied to initiate ually reduced the biotinylation time window from 18 h to 10 min over
tagging. This feature has resulted in >100 applications of BioID since 29 rounds of selection, in order to progressively increase selection
its introduction 5 years ago, in cultured mammalian cells5,10,11, plant stringency (Supplementary Note 1 and Supplementary Fig. 3).
protoplasts12, parasites13–21, slime mold22,23, mouse24, and yeast25. We encountered some technical hurdles during the evolution. First,
The major disadvantage of BioID, however, is its slow kinetics, the activity of our starting template (R118S) and input library were too
which necessitates labeling with biotin for 18–24 h (and some- low to be detected on the yeast surface. Thus, we used tyramide signal
times much longer24) to produce sufficient biotinylated material amplification (TSA32) on the yeast surface to boost the biotin signal
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. 2Departments of Genetics, Stanford University, Stanford,
California, USA. 3Department of Chemistry, Stanford University, Stanford, California, USA. 4Department of Biology, Stanford University, Stanford, California, USA.
5Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA. 6Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. 7Howard
Hughes Medical Institute, Boston, Massachusetts, USA. 8Chan Zuckerberg Biohub, San Francisco, California, USA. Correspondence should be addressed to A.Y.T.
([email protected]).
Received 23 September 2017; accepted 2 July 2018; published online 20 August 2018; doi:10.1038/nbt.4201
nature biotechnology advance online publication

letters
(Fig. 1c and Supplementary Fig. 3b) until the activity of the pool was the same when we ran the same experiment with 50 µM biotin instead
high enough to no longer require it. Second, to avoid enriching mutants of 500 µM biotin for labeling (Supplementary Fig. 6c–e).
that strongly tagged their own lysine residues but failed to biotinylate To compare ligases by a different modality, we also fixed ligase-
neighboring proteins, we treated yeast with the reducing agent TCEP expressing HEK 293T cells after biotinylation, stained them with neu-
in some rounds of selection, to cleave off the ligase after the biotinyla- travidin-fluorophore, and performed confocal microscopy. TurboID
tion reaction (Supplementary Fig. 3c). Finally, we introduced negative and miniTurbo gave clearly detectable biotinylation in most trans-
selection to deplete mutants that exhibited strong biotinylation activity fected cells after 10 min of biotin labeling (Supplementary Fig. 7).
before exogenous biotin addition, because the ability to utilize the low By contrast, BioID, BioID2, and BASU-catalyzed biotinylation was
levels of biotin naturally present in yeast media would result in loss of undetectable even at 1 h, and only dimly detectable at 6 h in a small
user control of the labeling window (Supplementary Fig. 3f). fraction of transfected cells.
Our engineering efforts yielded two promiscuous ligases: 35 kD Different organelles have distinct pH, redox environments, and
TurboID, with 15 mutations relative to wild-type BirA; and 28 kD endogenous nucleophile concentrations, which may influence PL
miniTurbo, with the N-terminal domain deleted and 13 mutations activity. We therefore compared TurboID, miniTurbo, and BioID in
relative to wild-type BirA (Fig. 1d, Supplementary Table 1 and the nucleus, mitochondrial matrix, ER lumen, and ER membrane of
Supplementary Note 2). Fig. 1e and Supplementary Fig. 4 show HEK 293T cells (Fig. 2c). We found that the absolute activities of each
the activity of these ligases on the yeast surface in a side by side com- ligase, as well as the relative activities between ligases, varied across
parison to BioID, BirA-R118S, and various intermediate clones from compartments (Supplementary Note 3). However, TurboID signal
our evolution (G1-G3∆). was clearly detectable after 10 min in each compartment, and even
To test TurboID and miniTurbo in mammalian cells, we expressed stronger than BioID 18-h labeling in the mitochondrial matrix and
them in the cytosol of HEK 293T cells. Labeling was initiated with the ER lumen. TurboID was superior to miniTurbo in each of these four
addition of 50 or 500 µM (Cf ) exogenous biotin and terminated by organelles. Given our observations, we recommend that users test
cooling cells to 4 °C and washing away excess biotin (Supplementary both TurboID and miniTurbo for PL applications, given the context-
Fig. 5). Streptavidin blot analysis of whole cell lysates showed that dependent variations in their activities.
TurboID and miniTurbo biotinylated endogenous proteins much We next evaluated TurboID and miniTurbo in full-scale proteomic
more rapidly than BioID, giving ~3- to 6-fold difference in sig- experiments. We asked whether 10-min labeling with these ligases
nal at early time points, and ~15- to 23-fold difference in signal at would produce proteomic data sets of similar quality to BioID labe-
later time points (Fig. 1f,g, Fig. 2a,b, Supplementary Fig. 4b, and ling for 18 h, in terms of specificity, coverage, and labeling radius
Supplementary Fig. 6). (Supplementary Note 4 and Supplementary Fig. 8). We selected
We included the newer promiscuous ligases BioID2 (ref. 26) and three mammalian organelles for the analysis: the mitochondrial
BASU27 in the comparison, and after normalization to account for matrix, nucleus, and ER membrane (ERM)-facing cytosol (Fig. 2d–h
differences in ligase expression levels, their activities were similar and Supplementary Figs. 9–11). Because the ERM is continuous with
to that of BioID (Fig. 2a,b). Notably, TurboID gave nearly as much the cytosol, it is valuable for assessing labeling radius; a good PL
biotinylated product in 10 min as BioID/BioID2/BASU gave in 18 h enzyme should strongly enrich ERM-localized proteins over imme-
(Fig. 2a,b). Overall, miniTurbo was 1.5- to twofold less active than diately adjacent cytosolic proteins.
TurboID, but exhibited less labeling before addition of exogenous The HEK293T samples we prepared for proteomic analysis are
biotin; this feature makes miniTurbo potentially superior for precise depicted in Figure 2d and Supplementary Figure 10a. TurboID and
temporal control of the labeling window. The resulting trends were miniTurbo labeling were each performed for 10 min, whereas BioID
Figure 1 Directed evolution of TurboID. (a) Proximity-dependent biotinylation catalyzed by promiscuous biotin ligases. Ligases catalyze the formation
of biotin-5′-AMP anhydride, which diffuses out of the active site to biotinylate proximal endogenous proteins on nucleophilic residues such as lysine.
(b) Yeast-display-based selection scheme. A >10 7 library of ligase variants is displayed on the yeast surface as a fusion to mating protein Aga2p. All
ligases have a C-terminal myc epitope tag. Biotin and ATP are added to the yeast library for between 10 min and 24 h. Ligase-catalyzed promiscuous
biotinylation is detected by staining with streptavidin-phycoerythrin and ligase expression is detected by staining with anti-myc antibody. Two-
dimensional FACS sorting enables enrichment of cells displaying a high ratio of streptavidin to myc staining. (c) tyramide signal amplification (TSA)32
improves biotin detection sensitivity on the yeast surface. In the top row, the three indicated yeast samples (G1 is the winning ligase mutant from the
first generation of evolution) were labeled with exogenous biotin for 18 h then stained for FACS as in b. The y-axis shows biotinylation extent, and the
x-axis quantifies ligase expression level. In the second row, after 18 h of biotin incubation, yeast were stained with streptavidin-HRP, reacted with
biotin-phenol2,32 to create additional biotinylation sites (TSA protocol), then stained with streptavidin-phycoerythrin and anti-myc antibody before
FACS. The third row omits biotin. Percentage of cells in upper right quadrant (Q2/(Q2+Q4)) shown in top right of each graph. This experiment was
performed once, but each yeast sample was analyzed under identical conditions at least twice in separate experiments with similar results. (d) E. coli
biotin ligase structure (PDB: 2EWN) with sites mutated in TurboID (left) and miniTurbo (right) shown in red. The N-terminal domain (aa1–63) is also
removed from miniTurbo. A non-hydrolyzable analog of biotin-5′-AMP, biotinol-5′-AMP, is highlighted in yellow. (e) FACS plots summarizing progress of
directed evolution. G1-G3 are the winning clones from generations 1–3 of directed evolution. G3∆ has its N-terminal domain (aa1–63) deleted. ‘Omit
biotin’ samples were grown in biotin-deficient media for the entire induction period (~18–24 h). This experiment was performed twice with similar
results, except G3∆ ‘omit biotin’, which was performed once. All results shown here were performed side by side in a single experiment. (f) Comparison
of ligase variants in the HEK cytosol showing that TurboID and miniTurbo are much more active than BioID, as well as the starting template BirA-R118S
and various intermediate clones from the evolution. Indicated ligases were expressed as NES (nuclear export signal) fusions in the HEK cytosol. 50 µM
exogenous biotin was added for 3 h, then whole cell lysates were analyzed by streptavidin blotting. Ligase expression detected by anti-V5 blotting.
U, untransfected. S, BirA-R118S. Asterisks indicate ligase self-biotinylation. BioID labeling for 18 h (50 µM biotin) shown for comparison in the
last lane. This experiment was performed twice with similar results. All results shown here were performed side by side in a single experiment.
(g) Quantitation of streptavidin blot data in f and from a 30-min labeling experiment shown in Supplementary Figure 4b. Quantitation excludes
self-biotinylation band. Sum intensity of each lane is divided by the sum intensity of the ligase expression band; ratios are normalized to that of
BioID/18 h, which is set to 1.0. Gray dots indicate quantitation of signal intensity from each replicate, colored bars indicate mean signal intensity
calculated from the two replicates.
advance online publication nature biotechnology

letters
labeling was carried out for 18 h. Cells were lysed and biotinylated miniTurbo-derived proteomes for the ERM (Supplementary Table 5),
proteins enriched with streptavidin beads. After on-bead digestion nucleus (Supplementary Table 6), and mitochondrial matrix
of proteins to peptides, we chemically labeled the peptides with iso- (Supplementary Table 7).
topically distinct TMT (tandem mass tag) labels. This enabled us TurboID- and miniTurbo-derived 10-min proteomes had similar
to quantify the relative abundance of each protein across samples. size and specificity as BioID-derived 18-h proteomes in all three com-
After liquid chromatography (LC)-MS/MS analysis of pooled pep- partments (Figure 2e–h). In particular, we note that TurboID was just
tides, we filtered the data via receiver operating characteristic (ROC) as effective as BioID in enriching secretory proteins over cytosolic
analysis (Supplementary Fig. 9c and Supplementary Fig. 10f,g), proteins when localized to the ERM (Fig. 2e–g), suggesting a similar
using true-positive and false-positive protein lists for each organelle labeling radius despite much faster labeling kinetics. Depth of cover-
(Supplementary Tables 2–4)2,33, to obtain BioID-, TurboID-, and age was similar in the mitochondrial matrix and ERM for the three
a b
Biotin Distal myc myc Retain
endogenous B Streptavidin-
ATP + B Ligase Ligase
proteins Aga2p Aga2p phycoerythrin
B library Biotin, ATP library
Aga1p 10 min–24 h & anti-myc
AMP B Aga1p
antibody, Discard
Promiscuous then FACS
Biotinylated Yeast cell Yeast cell
biotin ligase
proximal endogenous
protein
c d TurboID (35 kD) N-terminal domain miniTurbo (28 kD)

R118S R118S library G1
aa 1–63
5 0% 0% 0%
4
3 18 h I87V Q65P
I87V Q65P
2 Q2 Biotinol-5′-AMP
Streptavidin-phycoerythrin (log10)
Biotinol-5′-AMP M209V
1 Q4 M209V
R118S
5 1% 2% 26% R118S M241T
4 T192A
18 h T192A V160A
3 +TSA V160A K194I
2 K194I S150G
1 S150G
5 0% 0% 1% E140K L151P
E140K L151P
4 Omit Q141R
3 biotin; Q141R S263P
2 +TSA
1 I305V
I305V
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Anti-myc (AlexaFluor647, log10)
e R118S G1 G2 G3 G3∆ miniTurbo TurboID

5 0% 2% 15% 30% 24% 44% 54%
4
3 6h
phycoerythrin (log10)
biotin
2 Q2
Streptavidin-
1 Q4
5
4 Omit
3 biotin
2
1
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Anti-myc (AlexaFluor647, log10)
f g 2.5
0.8
BioID,18 h
30 min biotin
3h U S G1 G2 G3 G3∆ mTb TbID BioID 0.6
2.0
Relative biotinylation activity,
biotin: + – + – + – + – + – + – + – + – +
0.4
normalized to ligase
expression level
80
1.5 0.2
58
46 0.0
* * * * *
TbID
mTb
BioID
32
* 1.0
* *
25
0.5 Zoom
Streptavidin-HRP
46
32 0.0
Anti-V5 Biotin: – + – + – + – + – + – + – + – + – + – + – + – + – + – + – + – +
kD
G1
G2
G3
TbID
S
BioID
G3∆
mTb
S
G1
G2
G3
G3∆
mTb
TbID
BioID
30 min biotin 3 h biotin

letters
ligases, but slightly lower for TurboID and miniTurbo in the nucleus Supplementary Fig. 12a,b), and in all tissues at non-permissive
(Supplementary Figs. 9f and 10j). temperature (tub-Gal4/tub-Gal80ts driver, Supplementary Fig. 12c,d).
Given the extremely high activity of TurboID, we wondered Animals were raised on either biotin-containing food from early-
whether increasing the labeling time would produce a bigger and bet- embryo stages to adulthood (13 d) (Fig. 3f,g), or regular food to adult-
ter proteome. For the ERM, we found that 1-h labeling with TurboID hood (13 d), and then switched to biotin-supplemented food for 4 or
did increase proteome size by 46% compared to 10-min labeling, but 16 h (Supplementary Fig. 12). Streptavidin blotting of whole fly lysate
at the expense of specificity (Fig. 2e). With increased labeling time, showed extensive biotinylation in TurboID and miniTurbo flies, as early
proximal nucleophiles may become saturated with biotin, enabling as 4 h post-biotin addition (Supplementary Fig. 12), but no signal was
TurboID-generated biotin-AMP to travel farther and biotinylate dis- detectable in BioID flies, even after 13 d of biotin exposure (Fig. 3g). The
tal, non-specific proteins. absence of detectable BioID signal here, compared to the wing experi-
Despite the widespread application of BioID, there have been only ment (Fig. 3d), may have resulted from endogenous biotinylated proteins
two in vivo demonstrations to date24,34, which may be is related to drowning out specific signal in the streptavidin blot.
BioID’s low catalytic activity. We wondered whether TurboID and To test for possible toxicity of TurboID, miniTurbo, and BioID
miniTurbo’s increased activity might enable biotin-based PL in new expression in flies, we performed morphological and survival assays.
settings. We first tested these ligases in bacteria (E. coli) and yeast We observed no evidence of toxicity when any of the ligases were
(Saccharomyces cerevisiae). As in mammalian cells, TurboID and expressed tissue-specifically. However, we did find a decrease in fly
miniTurbo were considerably more active than BioID (Fig. 3a,b). viability and size when TurboID was expressed ubiquitously and
In particular, we and others25 observe that BioID activity was nearly constitutively, and exogenous biotin was withheld (Supplementary
undetectable in yeast, perhaps in part because yeast is cultured at Fig. 13, Supplementary Note 5, and Supplementary Fig. 14). Under
30 °C whereas BioID functions optimally at 37 °C 26. Because we these conditions, TurboID may consume all the biotin, effectively
carried out our directed evolution in yeast at 30 °C, TurboID and starving cells of biotin.
miniTurbo exhibit high activity at 30 °C. We also tested BioID, TurboID, and miniTurbo in C. elegans. We
BioID has not previously been reported in flies (Drosophila expressed the ligases early in the intestinal lineage (~150 min after
melanogaster) or worms (Caenorhabditis elegans), despite their appeal the first cleavage) and assessed biotinylation activity ~4 h later, at the
as highly genetically tractable model organisms. To test biotin-based PL embryonic bean stage (stage 1), ~5.5 h later at the embryonic comma
in flies, we expressed BioID, TurboID, or miniTurbo selectively in the stage (stage 2), or 3 d later in the adult worm (Fig. 3h). TurboID and
larval wing disc, which gives rise to the adult wing, and raised animals miniTurbo were significantly more active than BioID by both imag-
on biotin-containing food for 5 d from early embryo stages (Fig. 3c). ing and streptavidin blotting at all observed developmental stages
TurboID- and miniTurbo-catalyzed biotinylation were 22-fold and ten- (Fig. 3i,j and Supplementary Fig. 15). We also observed that
fold higher, respectively, than BioID-catalyzed biotinylation, as shown TurboID was expressed at higher levels than miniTurbo expression
by staining of dissected wing discs with streptavidin-fluorophore in adult worms, resulting in much stronger labeling (Fig. 3i and
(Fig. 3d,e). Consistent with our observations in HEK 293T cells, Supplementary Fig. 15g). TurboID and miniTurbo labeling yield
TurboID also gives some low biotinylation signal in flies fed regular, could be further increased by raising worms at higher temperatures
non-biotin supplemented, food. (25 °C vs. 20 °C; Supplementary Fig. 15g).
We also generated flies expressing BioID, TurboID, or miniTurbo While we observed background labeling by TurboID in adult
in all tissues (Act-Gal4 driver, Fig. 3f,g), in muscle (Mef2-Gal4 driver, biotin-depleted worms (Fig. 3i), similar to our observations in
Figure 2 Characterization of TurboID and miniTurbo in mammalian cells. (a) Comparison of TurboID and miniTurbo to three other promiscuous ligases
(BioID5, BioID226, and BASU27) in the cytosol of HEK 293T cells. Here, 500 µM exogenous biotin was used for labeling, whereas 50 µM was used in
Supplementary Figure 6c–e. Streptavidin-HRP blotting detects promiscuously biotinylated proteins, and anti-V5 blotting detects ligase expression.
U, untransfected. Asterisks denote ligase self-biotinylation bands. This experiment was performed twice with similar results. All results shown here
were performed side by side in a single experiment. (b) Quantitation of experiment in a. For shorter time points (–biotin and 10 min), we used a
longer-exposure image of the same blot, shown in Supplementary Figure 6a; for longer time points (1 h, 6 h, 18 h), we used a shorter-exposure image
of the blot in a, shown in Supplementary Figure 6b. Quantitation performed as in Figure 1g. Gray dots indicate quantitation of signal intensity from
each replicate, colored bars indicate mean signal tyramide signal amplification (TSA) intensity calculated from the two replicates. (c) Comparison of
promiscuous ligases in multiple HEK organelles. Each ligase was fused to a peptide targeting sequence (Supplementary Table 8) directing them to the
locations indicated in the scheme at right. BioID samples were treated with 50 µM biotin for 18 h. TurboID and miniTurbo samples were labeled for
10 min with 50 (+) or 500 (++) µM biotin. U, untransfected. Asterisks denote ligase self-biotinylation. This experiment was performed five times for
nuclear constructs, three times for mitochondrial constructs, four times for ER membrane constructs, and twice for ER lumen constructs with similar
results. (d) MS-based proteomic experiment comparing TurboID and BioID on the ER membrane (ERM), facing cytosol. Experimental design and
labeling conditions. Ligase fusion constructs were stably expressed in HEK 239T. BioID samples were treated with 50 µM biotin for 18 h, while TurboID
samples were treated with 500 µM biotin for 10 min or 1 h. After labeling, cells were lysed and biotinylated proteins were enriched with streptavidin
beads, digested to peptides, and conjugated to TMT (tandem mass tag) labels. All 11 samples were then combined and analyzed by LC-MS/MS.
Proteomic lists in Supplementary Table 5; further analysis of proteome data in Supplementary Figure 9. This experiment was performed once with two
replicates per condition. All results shown here are from two replicates performed within one experiment. (e) Specificity analysis for proteomic data
sets derived from experiment in d. Size of each ERM proteome at top. Bars show percentage of each proteome with prior secretory pathway annotation,
according to GOCC, Phobius, human protein atlas, human plasma proteome database, and literature (Supplementary Table 2). (f) Same as e, except for
each ERM proteome, we analyze the subset with ER, Golgi, or plasma membrane annotation. Annotations from GOCC were assigned in the priority order:
ER > Golgi > plasma membrane (Supplementary Table 2). (g) Breakdown of ER proteins enriched by TurboID and BioID, by transmembrane or soluble.
Soluble proteins were further divided into luminal or cytosol-facing. Annotations obtained from GOCC, UniProt, TMHMM, and literature (Supplementary
Table 2). (h) Characterization of nuclear and mitochondrial matrix proteomes obtained via BioID (18 h), TurboID (10 min), and miniTurbo (10 min)-
catalyzed labeling. Proteome sizes across top. Bars show fraction of each nuclear (left) or mitochondrial (right) proteome with prior nuclear or
mitochondrial annotation, according to GOCC, MitoCarta, or literature (Supplementary Tables 3 and 4). Design of proteomic experiment shown in
Supplementary Figure 10a, proteomic lists in Supplementary Tables 6 and 7; further analysis of proteome data in Supplementary Figure 10.

letters
flies and mammalian cell culture, we found that miniTurbo, but ligases in intestinal cells; however, developmental delay was evi-
not TurboID, gave some background labeling in biotin-depleted dent in worms expressing TurboID (Supplementary Fig. 16 and
worm embryos at stage 2 (Fig. 3k and Supplementary Fig. 15a). Supplementary Note 5).
We also assessed viability and developmental timing, and did not In summary, we have performed yeast-display-based directed evo-
observe decreased survival in worms expressing any of the three lution, incorporating TSA signal amplification, reductive removal of
a – Biotin 10 min 1h 6h 18 h
b 30
–Biotin 10 min 1h 6h 18 h
1.0
Relative biotinylation activity, normalized

BioID2
BioID2
BioID2
BioID2
BioID2
BASU
BASU
BASU
BASU
BASU
BioID
BioID
BioID
BioID
BioID
TbID
TbID
TbID
TbID
TbID
mTb
mTb
mTb
mTb
mTb
25 0.8
U
to ligase expression level

Streptavidin-HRP 0.6
20
80 – Biotin 10 min 0.4
58 BTm BTm
15 0.2
46
0.0
* * * * *
BioID
TbID
mTb
BioID2
BASU
BioID
TbID
mTb
BioID2
BASU
10
32 * * * * *
Longer exposure
* * 5
25 Zoom
46
0
BioID
TbID
mTb
BioID 2
BASU
BioID
TbID
mTb
BioID 2
BASU
BioID
TbID
mTb
BioID 2
BASU
BioID
TbID
mTb
BioID 2
BASU
BioID
TbID
mTb
BioID 2
BASU
32
kD Anti-V5
c Nucleus Mitochondrial matrix ER membrane ER lumen

BioID TbID mTb BioID TbID mTb BioID TbID mTb BioID TbID mTb
U 18 h 10 min 10 min U 18 h 10 min 10 min U 18 h 10 min 10 min U 18 h 10 min 10 min
+ + + + + + + +
Biotin: + – + − + + − + + Biotin: + − + − + + − + + Biotin: + − + − + + − + + Biotin: + − + − + + − + +
Nucleus
80 80 80
58 58 80 ER
58
58 lumen
46 46 46
* * ER
* * * * *
46
32 * 32 32 * * membrane
* 32 *
25 25 Streptavidin-HRP 25
Streptavidin-HRP Streptavidin-HRP 25 Streptavidin-HRP
46 46 46 46 Mito matrix
32 32 32 32
kD Anti-V5 kD Anti-V5 kD Anti-V5 kD Anti-V5
d e Secretory pathway specificity
TMT label: 127N 128N 129N 128C 130N 131N 19,081 707 808 1,180 Proteome
100
% with prior secretory annotation
size
97% 92%
ERM-BioID (1) ERM-BioID (2) ERM-TurboID (1) ERM-TurboID (2) ERM-TurboID (1) ERM-TurboID (2)
80 84%
18 h biotin 18 h biotin 10 min biotin 10 min biotin 1 h biotin 1 h biotin
TMT label: 127C 126C 129C 131C 130C
60 62%
BioID-NES Untransfected TurboID-NES ERM-TurboID TurboID-NES
18 h biotin 18 h biotin 10 min biotin Omit biotin 1 h biotin 40
Lyse cells
20
Streptavidin bead enrichment
On-bead trypsin digestion
TMT labeling 0
Entire BioID TbID TbID
human (10 m) (1 h)
Combine samples proteome ERM proteomes
LC-MS/MS
f Subsecretory specificity g ER specificity h Nuclear specificity Mitochondrial specificity

5,396 409 401 464 Subsecretory 195 186
197 ER-specific 19,081 1,512 1,455 1,362 19,081 362 314 359 Proteome
100
nuclear or mitochondrial annotation
100 100
Percentage of proteome with prior
proteome proteome size

prior nuclear or mitochondrial
Percentage of proteome with
7% 9% 12%
size size
Percentage of proteome
80 80 88% 87% 84% 80

79% 77% 76%
Plasma Soluble 75%
70%
annotation
60 membrane 60 (lumen) 60 67%

Golgi Soluble
40 48% 46% apparatus 40 (cytosol) 40
42%
35%
Membrane
20 ER 20 20
16%
8%
0 0 0
Secretory BioID TbID TbID BioID TbID TbID Entire BioID TbID mTb Entire BioID TbID mTb
proteome (10 m) (1 h) (10 m) (1 h) human human
Nuclear Mitochondrial
ERM proteomes ERM proteomes proteome proteomes proteome matrix proteomes

letters
ligases, and negative selections, to generate two new ligases for PL TurboID is expressed ubiquitously, it can sequester endogenous
applications: TurboID and miniTurbo. TurboID is the most active, and biotin and cause toxicity; the solution is to supplement animals with
should be used when the priority is to maximize biotinylation yield and exogenous biotin. Second, users should empirically optimize the
sensitivity and/or recovery. However, in many contexts, we observe a in vivo labeling time window, and use the shortest labeling time
small degree of labeling before exogenous biotin is supplied, indicating that produces sufficient biotinylated material for analysis. Longer-
that TurboID can utilize the low levels of biotin present in cells and/or than-necessary labeling can cause toxicity via chronic biotinylation
organisms grown in typical biotin-containing media or food. Hence, if of endogenous proteomes, and/or degrade spatial specificity due to
the priority is to precisely define the labeling time window, miniTurbo saturation of proximal labeling sites (Fig. 2e).
may be preferable to TurboID. Though 1.5- to twofold less active than
TurboID, miniTurbo gives much less background in the biotin-omitted Methods
condition, and it is also 20% smaller (28 vs. 35 kD), which may reduce Methods, including statements of data availability and any associated
interference with fusion protein trafficking and function. Yet another accession codes and references, are available in the online version of
factor to consider when choosing a ligase for PL is ligase stability. Our the paper.
results indicate that miniTurbo is less stable than TurboID (likely due
Note: Any Supplementary Information and Source Data files are available in the
to removal of its N-terminal domain), resulting in lower expression
online version of the paper.
levels in the adult worm intestine and adult fly, for example. miniTurbo
also exhibits biotin-dependent stability, similar to BioID (e.g., see anti-
Acknowledgments
V5 western blots in Fig. 2a). FACS was performed at the Koch Institute Flow Cytometry Core (MIT) and
Stanford Shared FACS Facility. S. Han (Stanford) synthesized neutravidin-
Up to now, in vivo applications of PL have required very long labe-
AlexaFluor647. S. Ax (Stanford) cloned the cell surface TurboID and miniTurbo
ling times24,34 or extensive genetic or manual manipulation35–37 to constructs. We are grateful to I. Droujinine (Harvard) for advice on biotin labeling
deliver chemical substrates to relevant cells. TurboID and miniTurbo in D. melanogaster. Biotin auxotrophic E. coli MG1655bioB:kan was kindly donated
offer facile substrate delivery and rapid labeling in vivo. In addition by J. Cronan (University of Illinois). This work was supported by NIH R01-
to increased catalytic efficiency, we believe that the temperature- CA186568 (to A.Y.T.), Howard Hughes Medical Institute Collaborative Innovation
Award (to A.Y.T., S.C., and N.P.), and NIH New Innovator Award DP2GM119136
activity profiles of TurboID and miniTurbo help to explain their (to J.L.F.). T.C.B. was supported by Dow Graduate Research and Lester Wolfe
superior performance to BioID in vivo. Whereas BioID is derived Fellowships. J.A.B. was supported by a Damon Runyon Post-Doctoral Fellowship.
from E. coli (37 °C), TurboID and miniTurbo were evolved in A.D.S. was supported by NIH Training Grant 2T32GM007276.
yeast (30 °C). Flies grow at 25 °C, while worms are typically grown
AUTHOR CONTRIBUTIONS
at 20 °C.
T.C.B. and A.Y.T. designed the research and analyzed all the data except those
Our toxicity analyses in flies, worms, and mammalian cell culture noted. T.C.B. performed all experiments except those noted. T.C.B., A.Y.T., N.D.U.,
(Supplementary Figs. 13, 14 and 16) do suggest some necessary and S.A.C. designed the proteomics experiments. T.C.B. prepared the proteomic
precautions when using TurboID and miniTurbo in vivo. First, if samples. N.D.U. and T.S. processed the proteomic samples and performed mass
Figure 3 TurboID and miniTurbo in flies, worms, and other species. (a) Comparison of ligases in yeast. EBY100 S. cerevisiae expressing BioID, TurboID,
or miniTurbo in the cytosol were treated with 50 µM biotin for 18 h. Whole cell lysates were blotted with streptavidin-HRP to visualize biotinylated
proteins, and anti-V5 antibody to visualize ligase expression. U, untransfected. Asterisks denote ligase self-biotinylation. Bands in untransfected lane
are endogenous, naturally biotinylated proteins. This experiment was performed twice with similar results. (b) Comparison of ligases in E. coli. Ligases,
fused at their N-terminal ends to His6-maltose binding protein, were expressed in the cytosol of BL21 E. coli and 50 µM exogenous biotin was added
for 18 h. Whole cell lysates were analyzed as in a. This experiment was performed twice with similar results. (c–g) Comparison of ligases in flies.
(c) Scheme for tissue-specific expression of ligases in the wing disc of D. melanogaster. ptc-Gal4 induces ligase expression in a strip of cells within
the wing imaginal disc that borders the anterior/posterior compartments. (d) Imaging of larval wing discs after 5 d of growth on biotin-containing food.
Biotinylated proteins are detected by staining with streptavidin-AlexaFluor555, and ligase expression is detected by anti-V5 staining. Panels show the
pouch region of the wing disc, indicated by the dashed line in c. Scale bar, 40 µm. Each experimental condition has at least three technical replicates;
one representative image is shown. This experiment was independently repeated two times with similar results. (e) Quantitation of streptavidin-
AlexaFluor555 signal intensities in d. Error bars, s.e.m. Average fold-change shown as text above bars. Sample size values (n) from left column to
right: 5, 6, 3. (f) Scheme for ubiquitous expression of ligases in flies, at all developmental time points, via the act-Gal4 driver. (g) Western blotting of
fly lysates prepared as in f. Biotinylated proteins detected by blotting with streptavidin-HRP, ligase expression detected by anti-V5 blotting. In control
sample, act-Gal4 drives expression of UAS-luciferase. Bands in control lanes are endogenous, naturally biotinylated proteins. This experiment was
performed twice with similar results. (h–k) Comparison of ligases in worms. (h) Scheme for tissue-specific expression of ligases in C. elegans intestine
via ges-1p promoter. Transgenic strains are fed either biotin-producing E. coli OP50 (biotin+), or biotin-auxotrophic E. coli MG1655bioB:kan
(biotin−). Promoter ges-1p drives ligase expression ~150 min after the first cell cleavage. (i) Adult worms prepared as in h were shifted to 25 °C for
one generation, then lysed and analyzed by western blotting. Control worms (N2) do not express ligase. Anti-HA antibody detects ligase expression.
Streptavidin-IRDye detects biotinylated proteins. This experiment was performed five times (n = 5). In biotin+ conditions, BioID biotinylation activity
was undetectable and TurboID gave robust biotinylation signal (n = 5/5). Despite high activity detected in embryos (see j,k), we only detected low
levels of biotinylation by miniTurbo in adults (n = 2/5), likely due to low ligase expression. (j) Representative images of bean stage (stage 1) worm
embryos from h. See Supplementary Figure 15a for representative images of comma-stage worm embryos (stage 2). Embryos were fixed and stained
with streptavidin-AF488 to detect biotinylated proteins, and anti-HA antibody to detect ligase expression. Intestine is outlined by a white dotted line.
Scale bar, 10 µm. (k) Quantitation of streptavidin-AF488 signal acquired from staining of embryonic stages 1 and 2 shown in j and Supplementary
Figure 15a. Mean streptavidin pixel intensities for each embryo assessed are plotted for BioID (B), TurboID (T), and miniTurbo (mT). Two independent
transgenic lines for BioID and TurboID and one for miniTurbo were assessed. Number of embryos imaged (n) from left to right: 26, 18, 11, 16, 25, 8,
19, 23, 14, 14, 23, 9. Statistical significance via Mann–Whitney U test (two-sided). ***P ≤ 0.0001, **P ≤ 0.001, *P ≤ 0.01. Pink asterisks indicate
significance of pairwise comparisons between biotin- and corresponding biotin+-treated embryos. Mean (reported in Supplementary Fig. 15b) is shown
as a black horizontal line for each condition, and error bars indicate s.e.m. Note that the streptavidin-AF488 pixel intensities for miniTurbo are an
underrepresentation of the signal as camera exposure settings were lowered to avoid pixel saturation (see Methods). See Supplementary Figure 15 for
more details.

letters
spectrometry. J.A.B. performed D. melanogaster experiments. J.A.B. and N.P. Reprints and permissions information is available online at http://www.nature.com/
analyzed D. melanogaster data. T.C.B., A.Y.T., A.D.S., and J.L.F. designed the reprints/index.html. Publisher’s note: Springer Nature remains neutral with regard to
C. elegans experiments. A.D.S. performed C. elegans experiments. A.D.S. and J.L.F. jurisdictional claims in published maps and institutional affiliations.
analyzed C. elegans data.
1. Kim, D.I. & Roux, K.J. Filling the void: proximity-based labeling of proteins in living
cells. Trends Cell Biol. 26, 804–817 (2016).
COMPETING INTERESTS 2. Rhee, H.-W. et al. Proteomic mapping of mitochondria in living cells via spatially
A.Y.T. and T.C.B. have filed a patent application covering some aspects of this work. restricted enzymatic tagging. Science 339, 1328–1331 (2013).
a S. cerevisiae (yeast)
b E. coli (bacteria)
c
Wing disc
U BioID TbID mTb U BioID TbID mTb 3rd instar larva
ptc-Gal4
Biotin: − + − + − + − + Biotin: − + − + − + − + 5d Dissect
X
UAS-ligase +100 µM Stain with
80 biotin in Ligase streptavidin-
80
58
* * * food expressed in AF555
58
46 wing disc
46
32
32 * *
25
f Adult fly
* Lyse flies
Streptavidin-HRP atc-Gal4 13 d
25 Streptavidin-HRP X SDS-PAGE
80 UAS-ligase +100 µM Blot with streptavidin-HRP
biotin in
46 Anti-His6 Ligase
32 food
kD expressed in
kD Anti-V5 all tissues
h Ligase expressed in
Worms fed:
d Streptavidin-555 e 40
intestinal cells 22x
Biotin+ bacteria (OP50) Lower Higher
Biotin Anti-V5 contrast contrast
Fold change signal

or
intensity vs BioID
30
Biotin- bacteria (MG1655(bioB:kan))
− 20
10x
Lyse worms
BioID
Isolate embryos
SDS-PAGE 10
Stage 1
Blot with streptavidin-IRDye
+ 1
0
i
D
bo
Control BioID TbID mTb
ol
ol
ur
Bi
rb
Stage 2
iT
Tu
Biotin: – + – + – + – +
in
g
m
250
150 − Control BioID TbID mTb
Biotin: – + – + – + – +
TurboID
100
75 Stain for Stain with
50 ligase streptavidin-
37 AF488
+ 180
25
Streptavidin-IRDye g 100
37 55
25 Anti-HA
− 35
kD
miniTurbo
25
j Biotin DAPI Anti-HA Streptavidin-AF488

15 Streptavidin-HRP
+ 40
30
− Anti-V5
kD
BioID
k 15
Biotin– Biotin+
*** ****
+
Average streptavidin-AF488 pixel intensity (a.u.)
** ** ** ***
BioID (B)
− 10 Line 1
Line 2
TurboID
TurboID (T)
+ Line 1
Line 2
5
** miniTurbo (mT)
*** Line 1
− *** *** *** *** ***
miniTurbo
0
B T mT B T mT B T mT B T mT
+
Stage 1 Stage 2 Stage 1 Stage 2

letters
3. Lam, S.S. et al. Directed evolution of APEX2 for electron microscopy and proximity 21. Gaji, R.Y. et al. Phosphorylation of a myosin motor by TgCDPK3 facilitates rapid
labeling. Nat. Methods 12, 51–54 (2015). initiation of motility during Toxoplasma gondii egress. PLoS Pathog. 11, e1005268
4. Choi-Rhee, E., Schulman, H. & Cronan, J.E. Promiscuous protein biotinylation by (2015).
Escherichia coli biotin protein ligase. Protein Sci. 13, 3043–3050 (2004). 22. Batsios, P., Ren, X., Baumann, O., Larochelle, D.A. & Gräf, R. Src1 is a protein of
5. Roux, K.J., Kim, D.I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion the inner nuclear membrane interacting with the Dictyostelium Lamin NE81. Cells
protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 5, 13 (2016).
196, 801–810 (2012). 23. Meyer, I. et al. CP39, CP75 and CP91 are major structural components of the
6. Paek, J. et al. Multidimensional tracking of GPCR signaling via peroxidase-catalyzed Dictyostelium centrosome’s core structure. Eur. J. Cell Biol. 96, 119–130
proximity labeling. Cell 169, 338–349.e11 (2017). (2017).
7. Lobingier, B.T. et al. An approach to spatiotemporally resolve protein interaction 24. Uezu, A. et al. Identification of an elaborate complex mediating postsynaptic
networks in living cells. Cell 169, 350–360.e12 (2017). inhibition. Science 353, 1123–1129 (2016).
8. Kaewsapsak, P., Shechner, D.M., Mallard, W., Rinn, J.L. & Ting, A.Y. Live-cell 25. Opitz, N. et al. Capturing the Asc1p/receptor for activated C kinase 1 (RACK1)
mapping of organelle-associated RNAs via proximity biotinylation combined with microenvironment at the head region of the 40S ribosome with quantitative BioID
protein-RNA crosslinking. eLife 6, e29224 (2017). in yeast. Mol. Cell. Proteomics 16, 2199–2218 (2017).
9. Martell, J.D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter 26. Kim, D.I. et al. An improved smaller biotin ligase for BioID proximity labeling. Mol.
for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012). Biol. Cell 27, 1188–1196 (2016).
10. Gupta, G.D. et al. a dynamic protein interaction landscape of the human centrosome- 27. Ramanathan, M. et al. RNA-protein interaction detection in living cells. Nat.
cilium interface. Cell 163, 1484–1499 (2015). Methods 15, 207–212 (2018).
11. Kim, D.I. et al. Probing nuclear pore complex architecture with proximity-dependent 28. Birendra, K.C. et al. VRK2A is an A-type lamin-dependent nuclear envelope kinase
biotinylation. Proc. Natl. Acad. Sci. USA 111, E2453–E2461 (2014). that phosphorylates BAF. Mol. Biol. Cell 28, 2241–2250 (2017).
12. Lin, Q. et al. Screening of proximal and interacting proteins in rice protoplasts by 29. Redwine, W.B. et al. The human cytoplasmic dynein interactome reveals novel
proximity-dependent biotinylation. Front. Plant Sci. 8, 749 (2017). activators of motility. eLife 6, e28257 (2017).
13. Morriswood, B. et al. Novel bilobe components in Trypanosoma brucei identified 30. Jung, E.M. et al. Arid1b haploinsufficiency disrupts cortical interneuron development
using proximity-dependent biotinylation. Eukaryot. Cell 12, 356–367 (2013). and mouse behavior. Nat. Neurosci. 20, 1694–1707 (2017).
14. Chen, A.L. et al. Novel components of the Toxoplasma inner membrane complex 31. Martell, J.D. et al. A split horseradish peroxidase for the detection of intercellular
revealed by BioID. MBio 6, e02357–e14 (2015). protein-protein interactions and sensitive visualization of synapses. Nat. Biotechnol.
15. Nadipuram, S.M. et al. In vivo biotinylation of the toxoplasma parasitophorous 34, 774–780 (2016).
vacuole reveals novel dense granule proteins important for parasite growth and 32. Bobrow, M.N., Harris, T.D., Shaughnessy, K.J. & Litt, G.J. Catalyzed reporter
pathogenesis. MBio 7, e00808–16 (2016). deposition, a novel method of signal amplification. Application to immunoassays.
16. Chen, A.L. et al. Novel insights into the composition and function of the Toxoplasma J. Immunol. Methods 125, 279–285 (1989).
IMC sutures. Cell. Microbiol. 19, e12678 (2017). 33. Hung, V. et al. Proteomic mapping of cytosol-facing outer mitochondrial and ER
17. Long, S. et al. Calmodulin-like proteins localized to the conoid regulate motility membranes in living human cells by proximity biotinylation. eLife 6, e24463
and cell invasion by Toxoplasma gondii. PLoS Pathog. 13, e1006379 (2017). (2017).
18. Zhou, Q., Hu, H. & Li, Z. An EF-hand-containing protein in Trypanosoma brucei 34. Dingar, D. et al. BioID identifies novel c-MYC interacting partners in cultured cells
regulates cytokinesis initiation by maintaining the stability of the cytokinesis and xenograft tumors. J. Proteomics 118, 95–111 (2015).
initiation factor CIF1. J. Biol. Chem. 291, 14395–14409 (2016). 35. Reinke, A.W., Balla, K.M., Bennett, E.J. & Troemel, E.R. Identification of
19. Dang, H.Q. et al. Proximity interactions among basal body components in microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins.
Trypanosoma brucei identify novel regulators of basal body biogenesis and Nat. Commun. 8, 14023 (2017).
inheritance. MBio 8, e02120–16 (2017). 36. Reinke, A.W., Mak, R., Troemel, E.R. & Bennett, E.J. In vivo mapping of tissue- and
20. Kehrer, J., Frischknecht, F. & Mair, G.R. Proteomic analysis of the Plasmodium subcellular-specific proteomes in Caenorhabditis elegans. Sci. Adv. 3, e1602426
berghei gametocyte egressome and vesicular bioID of osmiophilic body proteins (2017).
identifies merozoite TRAP-like protein (MTRAP) as an essential factor for parasite 37. Chen, C.-L. Proteomic mapping in live Drosophila tissues using an engineered
transmission. Mol. Cell. Proteomics 15, 2852–2862 (2016). ascorbate peroxidase. Proc. Natl. Acad. Sci. USA 112, 12093–12098 (2015).

ONLINE Methods yeast cultures were diluted at as high as 1:100 dilution into the appropriate
Cloning. See Supplementary Table 8 for a list of genetic constructs used induction media in order to conserve media.
in this study, with detailed description of construct designs, linker orienta- For samples biotin labeled for 18 h, yeast were induced in 10% SD/GCAA or
tions, epitope tags, and signal sequence identities. All ligase variants were biotin-depleted medium supplemented with 50 µM biotin, 1 mM ATP, and 5 mM
derived from E. coli biotin protein ligase, have the residue A146 deleted to MgCl2 at 30 °C for 20-24 h (to allow time for protein expression and ensure labe-
suppress dimerization38, and are codon optimized for expression in mam- ling occurs for at least 18 h). For samples labeled for shorter time periods, yeast
malian cells. For cloning, PCR fragments were amplified using Q5 polymer- were induced in 10% SD/GCAA or biotin-depleted medium for 18 h at 30 °C
ase (New England BioLabs (NEB)). The vectors were double-digested using (no biotin), then supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2
standard enzymatic restriction digest and ligated to gel purified PCR products for the labeling times indicated. For ‘omit biotin’ samples, yeast were induced in
by T4 DNA ligation or Gibson assembly. Ligated plasmid products were intro- 10% SD/GCAA or biotin-depleted medium at 30 °C for 18-24 h. After labeling,
duced by heat shock transformation into competent XL1-Blue bacteria. Ligase approximately 5 million cells (assuming 1 OD600 ≈ 3 × 107 cells42) were pelleted at
mutants were either generated using QuikChange mutagenesis (Stratagene) or 5000×g for 30 s at 4 °C and washed five times with 1 mL PBS (phosphate buffered
isolated from individual yeast clones and transferred to mammalian expression saline) + 0.5% bovine serum albumin (BSA; 1 mg/mL) (PBS-B).
vectors using standard cloning techniques. For tyramide signal amplification32 (TSA, Supplementary Fig. 3b), yeast
cells were incubated in 50 µL PBS-B + 1:100 streptavidin-horseradish peroxi-
Yeast cell culture. For yeast-display (Fig. 1c,e and Supplementary Figs. 3 dase (HRP) for 1 h at 4 °C, then washed three times with 1 mL PBS-B. HRP
and 4a), S. cerevisiae strain EBY100 was cultured according to previously labeling was performed by incubating yeast in 750 µL PBS-B with 50 µM
published protocols39. Cells were propagated at 30 °C in synthetic dextrose biotin-phenol and 1 mM H2O2 for 1 min at room temperature. The reac-
plus casein amino acid (SDCAA, ‘regular’) medium supplemented with tryp- tion was quenched by adding 750 µL PBS-B + 20 mM sodium ascorbate and
tophan (20 mg/L). Yeast cells were transformed with the yeast-display plasmid 10 mM Trolox followed by rapid mixing via inversion. Cells were then washed
pCTCON239 using the Frozen E-Z Yeast Transformation II kit (Zymoprep) two times with 1 mL PBS-B + 10 mM sodium ascorbate and 5 mM Trolox,
according to manufacturer protocols. Transformed cells containing the TRP1 and once with 1 mL PBS-B. For removal of ligase proteins via TCEP reduction
gene were selected on SDCAA plates and propagated in SDCAA medium at (Supplementary Fig. 3c), yeast were incubated in 500 µL PBS-B + 2 mM TCEP
30 °C. Protein expression was induced by inoculating saturated yeast culture at 30 °C for 90 min, then washed four times with 1 mL PBS-B.
into 10% SD/GCAA (SDCAA medium with 90% of dextrose replaced with For biotin and epitope tag detection, yeast cells were then incubated in 50 µL
galactose), or into “biotin-depleted” medium40 (1.7 g/L YNB-Biotin (Sunrise PBS-B + 1:400 chicken anti-myc and 1:50 rabbit anti-biotin (when detecting
Science Products), 5 g/L ammonium sulfate, 2 g/L dextrose, 18 g/L galactose, biotinylated proteins with anti-biotin antibody) for 1 h at 4 °C, then washed
complete amino acids, 0.125 ng/mL d-biotin), at a 1:100-1000 dilution and three times with 1 mL PBS-B. Yeast cells were then incubated in 50 µL PBS-B +
incubating at 30 °C for 18 – 24 h. 1:200 Alexa Fluor 647 (AF647) goat anti-chicken IgG and 1:50 phycoerythrin
(PE) goat anti-rabbit IgG (when detecting biotinylated proteins with anti-biotin
Generation of ligase libraries for yeast display. Libraries of ligase mutants antibody) or streptavidin-PE (when detecting biotinylated proteins with strepta-
were generated by error-prone PCR according to published protocols41. 150 ng vidin) for 1 h at 4 °C, then washed three times with 1 mL PBS-B for FACS analy-
of the template ligase in vector pCTCON2 (ref. 41) was amplified for 10 – 20 sis. Refer to Supplementary Table 9 for a list of antibodies used in this study.
rounds with 0.4 µM forward and reverse primers: For two-dimensional FACS sorting, samples were resuspended in PBS-B at a
F: 5′-CTAGTGGTGGAGGAGGCTCTGGTGGAGGCGGTAGCGGAG maximal concentration of 100 million cells/mL and sorted on a BD FACS Aria
GCGGAGGGTCGGCTAGC-3′ II cell sorter (BD Biosciences) with the appropriate lasers and emission filters
R: 5′-TATCAGATCTCGAGCTATTACAAGTCCTCTTCAGAAATAAGC (561 nm excitation laser and 582/15 emission filter for PE, 640 nm excitation
TTTTGTTCGGATCC-3′ and 2 mM MgCl2, 5 units of Taq polymerase (NEB), laser and 670/30 emission filter for AF647). To analyze and sort single yeast
and 2 – 20 µM each of the mutagenic nucleotide analogs 8-oxo-2′-deoxygua- cells, cells were plotted by a forward-scatter area (FSC-A) and side-scatter
nosine-5′-triphosphate (8-oxo-dGTP) and 2′-deoxy-P-nucleoside-5′-triphos- area (SSC-A) and a gate was drawn around cells clustered between 10 4 – 105
phate (dPTP). The PCR products were then gel purified and reamplified for FSC-A, 103 – 105 SSC-A to give population P1 (Supplementary Fig. 3i). Cells
another 30 cycles under normal PCR conditions using: from population P1 were then plotted by side-scatter width (SSC-W) and side-
F: 5′ CAAGGTCTGCAGGCTAGTGGTGGAGGAGGCTCTGGTG-3′ scatter height (SSC-H) and a gate was drawn around cells clustered between
R: 5′ - CTACACTGTTGTTATCAGATCTCGAGCTATTACAAGTC-3′. 10 – 100 SSC-W and 103 – 105 SSC-H to give population P2 (Supplementary
The inserts were then electroporated into electrocompetent S. cerevisiae Fig. 3i). Cells from population P2 were then plotted by forward-scatter width
EBY100 (ref. 41) with the BamHI-NheI linearized pCTCON2 vector (10 µg (FSC-W) and forward-scatter height (FSC-H) and a gate was drawn around
insert/1 µg vector) backbone. The electroporated cultures were rescued cells clustered between 10 – 100 FSC-W and 103 – 105 FSC-H to give popula-
in 2 mL yeast extract peptone dextrose (YPD) complete medium 41 for tion P3 (Supplementary Fig. 3i). Population P3 often represented >90% of
1 h at 30 °C with no shaking. Cells were vortexed briefly, and 1.99 mL of the total population analyzed.
the rescued cell suspension was transferred to 100 mL of SDCAA medium From population P3, gates were drawn to collect yeast with the highest
supplemented with 50 units/mL penicillin and 50 µg/mL streptomycin and activity/expression ratio, i.e., positive for AF647 signal that also had high PE
grown for 2 days at 30 °C. The remaining 10 µL of the rescued cell suspen- signal (Supplementary Fig. 3i). For TCEP treated samples, gates were drawn
sion was diluted 100×, 1000×, 10000×, and 100000×; 20 µL of each dilution to collect yeast with high PE signal and no AF647 signal above background
was plated on SDCAA plates and incubated at 30 °C for 3 days. After 3 days, (Supplementary Fig. 3i). For negative selections (Supplementary Fig. 3f),
each colony observed in the 100×, 1000×, 10000×, or 100000× segments gates were drawn to collect yeast with AF647 signal and no PE signal above
of plates will correspond to 104, 105, 106, or 107 transformants in the background (Supplementary Fig. 3i). After sorting, yeast were collected in
library, respectively. SDCAA medium containing 1% penicillin-streptomycin and incubated at
30 °C for 24 h. 1 mL of the growing culture was removed for DNA extraction
General methods for yeast display-based directed evolution. For each round using the Zymoprep yeast Plasmid Miniprep II (Zymo Research) kit accord-
of evolution (Supplementary Fig. 3), we input 10-fold more yeast cells than ing to manufacturer protocols (using 6 µL zymolyase, vigorously vortex
the estimated library size. For the first round, library size was estimated by the after lysis), and at least ten-fold excess of the number of cells retained dur-
transformation efficiency of the initial ligase library. For subsequent rounds, ing sorting were propagated in SDCAA + 1% pen-strep to ensure oversam-
library size was taken to be the number of yeast cells collected during the pling (yeast cells were passaged in this manner at least two times prior to the
previous sort. Ligase protein expression was induced by inoculating saturated next round of selection). To analyze yeast populations and clones by FACS
yeast culture into 10% SD/GCAA or biotin-depleted medium at a 1:1000 dilu- (Fig. 1c,e; Supplementary Figs. 3 and 4a), yeast samples were prepared on a
tion and incubating at 30 °C for 18 – 24 h. However, when inducing yeast small scale (1 mL cultures) as described above and analyzed on a BD Accuri
populations with high diversity, for example the initial libraries, saturated flow cytometer (BD Biosciences). BD FACSDIVA software was used to analyze
nature biotechnology doi:10.1038/nbt.4201

all data from FACS sorting and analysis. Summaries of all yeast-display directed Population E1-R6 was passaged twice, and analyzed by FACS side-by-side
evolution and resulting mutants are shown in Figure 1e; Supplementary with previous rounds and BirA-R118S. Sequencing of E1-R6 revealed sev-
Figures 3 and 4, and Supplementary Table 1, and are described in detail in eral mutants with the mutation E313K. Several mutants with and without this
the ‘Directed evolution of TurboID and miniTurbo’ sections below. mutation were assayed as single clones on the yeast surface, and the most
promising mutants, including two with the E313K mutation, were assayed
Directed evolution of TurboID and miniTurbo: generation 1. For the first in the mammalian cell cytosol. While neither of the E313K mutants showed
round of evolution (Supplementary Fig. 3b), three libraries were generated significant difference in activity to R118S over 24 h, they both showed very
using BirA-R118S (Supplementary Table 8) as the starting template. The three strong self-labeling at shorter time points, e.g. 1 h. The crystal structure of
libraries were generated using error prone PCR as described above, using the BirA43 shows that this residue points directly into the active site, where a lysine
following conditions to produce varying levels of mutagenesis: mutation could easily react with the phosphate group of biotin-5’-AMP. We
Library 1: 2 µM 8-oxo-dGTP, 2 µM dPTP, 10 PCR cycles removed this mutation from the two promising clones bearing it and assayed
Library 2: 2 µM 8-oxo-dGTP, 2 µM dPTP, 20 PCR cycles again in the mammalian cell cytosol. One of the mutants, denoted in this study
Library 3: 20 µM 8-oxo-dGTP, 20 µM dPTP, 10 PCR cycles as G1 (Supplementary Table 1), displayed significantly higher promiscuous
The library sizes (approximated by number of transformants as described activity than R118S after 24 hours of labeling. Another mutant from the mam-
above), were 1.4 × 107 for Library 1, 1.7 × 107 for Library 2, and 8 × 106 for malian cell screen, denoted in this study as R6-1 (Supplementary Table 1),
Library 3. FACS analysis of the three libraries showed robust expression and also displayed significantly higher promiscuous activity than R118S after 24 h
wide range of activities for Library 1 and Library 2, however Library 3 showed of labeling. Both of these mutants, with 4 mutations each, had each of their
poor expression and no activity. Sequencing of 24 clones in Library 1 revealed mutations removed individually and in different combinations. Analysis of the
an average of 1.5 amino acid changes per ligase gene. Sequencing of 24 clones resulting mutants in mammalian cells showed that each mutation was contrib-
in Library 2 revealed an average of 2.4 amino acid changes per ligase gene. uting to increased activity relative to R118S observed for R6-1 and G1.
Library 1 and Library 2 were combined and used as the initial population for
the first round of selections. This combined library was induced as described Directed evolution of TurboID and miniTurbo: generation 2. For the second
above, supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2, for round of evolution (Supplementary Fig. 3c), six libraries were generated.
24 h. From this culture, approximately 5 × 108 cells were prepared for sorting Three libraries were made using R6-1 (Supplementary Tables 1 and 8) as the
(assuming 1 OD600 ≈ 3 × 107 cells42) as described above with TSA treatment starting template, and the three libraries were made using G1 (Supplementary
(Supplementary Fig. 3b). 6.24 × 107 cells were sorted by FACS. A square gate Tables 1 and 8) as the starting template, both using error prone PCR with the
that collected cells positive for both anti-myc and streptavidin (conjugated following conditions:
to fluorophores, see Supplementary Table 9) was drawn, and approximately Library 1: R6-1, 2 µM 8-oxo-dGTP, 2 µM dPTP, 10 PCR cycles
2.5 × 106 cells were collected (4%) to give population E1-R1. Library 2: R6-1, 2 µM 8-oxo-dGTP, 2 µM dPTP, 20 PCR cycles
Population E1-R1 was passaged twice, and analyzed by FACS side-by-side Library 3: R6-1, 20 µM 8-oxo-dGTP, 20 µM dPTP, 10 PCR cycles
with the original combined library and BirA-R118S to ensure the sort was Library 4: G1, 2 µM 8-oxo-dGTP, 2 µM dPTP, 10 PCR cycles
successful (resulting population still had expression and had higher or equiva- Library 5: G1, 2 µM 8-oxo-dGTP, 2 µM dPTP, 20 PCR cycles
lent activity). Sequencing of 24 clones from E1-R1 revealed an average of Library 6: G1, 20 µM 8-oxo-dGTP, 20 µM dPTP, 10 PCR cycles
1.5 mutations per ligase gene. Population E1-R1 was induced, supplemented The library sizes, as calculated by transformation efficiency, were 3.8 ×
with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2, for 24 h. From this culture, 107 for Library 1, 1.9 × 107 for Library 2, 1.6 × 107 for Library 3, 8 × 107 for
approximately 10-fold excess (i.e. >2.5 × 107) cells were prepared for sort- Library 4, 3.9 × 107 for Library 5, and 3.9 × 107 for Library 6. FACS analysis
ing with TSA treatment. A square gate that collected cells positive for both of the three libraries showed robust expression and wide range of activities
anti-myc and streptavidin was drawn, and approximately 3.8% of cells were for Libraries 1, 2, 4, and 5, however Libraries 3 and 6 showed poor expression
collected to give population E1-R2. and no activity.
Population E1-R2 was passaged twice, and analyzed by FACS side-by-side Libraries 1, 2, 4, and 5 were combined and used as the initial population
with previous rounds and BirA-R118S. Sequencing of 24 clones from E1-R2 for the first round of selections. This combined library was induced, sup-
revealed an average of 1.5 mutations per ligase gene. Population E1-R2 was plemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2, for 24 h. From
induced for ~18 h then supplemented with 50 µM biotin, 1 mM ATP, and this culture, approximately 10-fold excess cells were prepared for sorting with
5 mM MgCl2 for 6 hours. From this culture, approximately 10-fold excess cells TSA treatment. A square gate that collected cells positive for both anti-myc
were prepared for sorting with TSA treatment. A square gate that collected and streptavidin was drawn, and approximately 8.4% of cells were collected
cells positive for both anti-myc and streptavidin was drawn, and approximately to give population E2-R1.
0.7% of cells were collected to give population E1-R3. Population E2-R1 was passaged twice, and analyzed by FACS side-by-side
Population E1-R3 was passaged twice, and analyzed by FACS side-by-side with the combined library template. Population E2-R1 was induced, supple-
with previous rounds and BirA-R118S. Population E1-R3 was induced for ~18 h mented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2, for 24 h. From
then supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 for this culture, approximately 10-fold excess cells were prepared for sorting
6 hours. From this culture, approximately 10-fold excess cells were prepared with TCEP treatment (Supplementary Fig. 3c) followed by TSA treatment.
for sorting with TSA treatment. A square gate that collected cells positive for A square gate that collected cells positive for streptavidin but negative for
both anti-myc and streptavidin was drawn, and approximately 2.4% of cells anti-myc was drawn, and approximately 1.2% of cells were collected to give
were collected to give population E1-R4. population E2-R2.
Population E1-R4 was passaged twice, and analyzed by FACS side-by-side Population E2-R2 was passaged twice, and analyzed by FACS side-by-side
with previous rounds and BirA-R118S. Population E1-R4 was induced for ~18 h with the combined library template and previous rounds. Population E2-R2
then supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 for induced, supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2, for
3 hours. From this culture, approximately 10-fold excess cells were prepared 24 h. From this culture, approximately 10-fold excess cells were prepared for
for sorting. A square gate that collected cells positive for both anti-myc and sorting with TSA treatment. A square gate that collected cells positive for both
streptavidin was drawn, and approximately 2.6% of cells were collected to anti-myc and streptavidin was drawn, and approximately 19% of cells were
give population E1-R5. collected to give population E2-R3.
Population E1-R5 was passaged twice, and analyzed by FACS side-by-side with Population E2-R3 was passaged twice, and analyzed by FACS side-by-side
previous rounds and BirA-R118S. Population E1-R5 was induced for ~18 h then with previous rounds. Population E2-R3 was induced, supplemented with
supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 for 1 h. From this 50 µM biotin, 1 mM ATP, and 5 mM MgCl2, for 24 h. From this culture,
culture, approximately 10-fold excess cells were prepared for sorting. A square approximately 10-fold excess cells were prepared for sorting. A trapezoidal
gate that collected cells positive for both anti-myc and streptavidin was drawn, gate that collected cells positive for both anti-myc and streptavidin, but with
and approximately 0.9% of cells were collected to give population E1-R6. high streptavidin/anti-myc ratios, was drawn, and approximately 1.4% of cells
doi:10.1038/nbt.4201 nature biotechnology

were collected to give population E2-R4. From here on, only trapezoidal gates in decreased activity (5-fold less than E3-R3). The library was ‘cleaned’ by
as described here were used for double-positive selections. removing this insertion via PCR with primers that restored the wild-type
Population E2-R4 was passaged twice, and analyzed by FACS side-by-side N-terminal sequence, and subjected to one additional round of double-posi-
with previous rounds. Population E2-R4 was induced for ~18 h, then sup- tive selection with 10 minute labeling and 0.1% cells collected. The resulting
plemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 for 1 h. From population was E3-R4.
this culture, approximately 10-fold excess cells were prepared for sorting. Population E3-R4 was passaged twice, and analyzed by FACS side-by-side
A trapezoidal gate that collected cells positive for both anti-myc and strepta- with previous rounds. Sequencing of E3-R4 revealed several mutations that
vidin was drawn, and approximately 1.1% of cells were collected to give appeared in multiple clones. Several of these mutants were assayed as single
population E2-R5. clones on the yeast surface, the most promising mutants were assayed in the
Population E2-R5 was passaged twice, and analyzed by FACS side-by-side mammalian cell cytosol. Two mutants had significantly higher activity than
with the combined library template and previous rounds. Population E2-R5 the template G2 or any other mutants. The mutations from these mutants were
was induced for ~18 h, then supplemented with 50 µM biotin, 1 mM ATP, and combined in various combinations, resulting in the highest activity mutant,
5 mM MgCl2 for 6 h. From this culture, approximately 10-fold excess cells were denoted in this study as G3 (Supplementary Table 1).
prepared for sorting with TCEP treatment followed by TSA. A square gate that
collected cells positive for streptavidin and negative for anti-myc was drawn, Directed evolution of TurboID and miniTurbo: Generations 4 and 5. G3 was
and approximately 1.5% of cells were collected to give population E2-R6. the highest activity mutant found to date, but it also appeared to give labeling
Population E2-R6 was passaged twice, and analyzed by FACS side-by-side even without the addition of exogenous biotin. This was observed in yeast,
with previous rounds and the combined library template. Sequencing of where this signal proved to be biotin-dependent (Supplementary Fig. 3e),
E2-R6 revealed several mutations that appeared in multiple clones. Several of and also in the mammalian cytosol (Fig. 1f,g, Supplementary Fig. 4b). From
these mutants were assayed as single clones on the yeast surface, however it this point, we continued with two evolutionary paths as follows:
was found after re-sequencing that many of the most promising mutants had In one path, we truncated the N-terminal domain (aa 1-63) of G3 to give
mutated stop codons. After mutating back the stop codons, the mutants were G3∆ (Supplementary Table 1). Consistent with literature44,45, this truncation
re-assayed on the yeast surface, and the mutants that remained promising resulted in reduced streptavidin signal when exogenous biotin was omitted
were assayed in the mammalian cell cytosol. One of the mutants, denoted (Supplementary Fig. 3e). Using G3∆ as the starting template (Supplementary
in this study as G2 (Supplementary Table 1), displayed significantly higher Tables 1 and 8) for another round of evolution (Supplementary Fig. 3g), we
promiscuous activity than R118S, G1 (its template), or any other mutant tested generated three libraries using error prone PCR with the following conditions:
after 1 hour of labeling. G1, with 2 additional mutations relative to G1, had Library 1: 2 µM 8-oxo-dGTP, 2 µM dPTP, 10 PCR cycles
each or both of its mutations removed. Analysis of the resulting mutants in Library 2: 2 µM 8-oxo-dGTP, 2 µM dPTP, 20 PCR cycles
mammalian cells showed that each mutation was contributing to activity boost Library 3: 4 µM 8-oxo-dGTP, 2 µM dPTP, 20 PCR cycles
observed for G2. The library sizes, as calculated by transformation efficiency, were 4.9 ×
108 for Library 1, 4.6 × 108 for Library 2, and 3.7 × 108 for Library 3. FACS
Directed evolution of TurboID and miniTurbo: generation 3. For the third analysis of the three libraries showed robust expression and wide range of
round of evolution (Supplementary Fig. 3d), three libraries were made using activities for all libraries, therefore all were combined and used for the first
G2 as the starting template (Supplementary Tables 1 and 8) using error prone round of selections.
PCR with the following conditions: This combined library was induced in biotin-depleted media, supplemented
Library 1: 2 µM 8-oxo-dGTP, 2 µM dPTP, 10 PCR cycles with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2, for 18 h. From this culture,
Library 2: 2 µM 8-oxo-dGTP, 2 µM dPTP, 20 PCR cycles approximately 10-fold excess cells were prepared for sorting with streptavidin.
Library 3: 10 µM 8-oxo-dGTP, 20 µM dPTP, 10 PCR cycles A trapezoidal gate that collected cells positive for both anti-myc and streptavi-
The library sizes, as calculated by transformation efficiency, were 3.5 × 108 din was drawn, and 0.1% of cells were collected to give population E4-R1.
for Library 1, 3.6 × 107 for Library 2, and 6.8 × 106 for Library 3. FACS analysis Population E4-R1 was passaged twice, and analyzed by FACS side-by-side
of the three libraries showed robust expression and wide range of activities with G3∆ and the combined library template. Population E4-R1 was induced
for Library 1 and Library 2, however Library 3 showed weak expression and for ~18 h in biotin-depleted media, then supplemented with 50 µM biotin,
no activity. 1 mM ATP, and 5 mM MgCl2 for 3.5 h. From this culture, approximately
Libraries 1 and 2 were combined and used as the initial population for the 10-fold excess cells were prepared for sorting with anti-biotin antibody.
first round of selections. This combined library was induced for ~18 h, then A trapezoidal gate that collected cells positive for both anti-myc and anti-
supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 for 1 h. From biotin was drawn, and 1% of cells were collected to give population E4-R2.
this culture, approximately 10-fold excess cells were prepared for sorting. A trap- Population E4-R2 was passaged twice, and analyzed by FACS side-by-side
ezoidal gate that collected cells positive for both anti-myc and streptavidin was with G3∆, the combined library template, and previous rounds. Population
drawn, and less than 0.1% of cells were collected to give population E3-R1. E4-R2 was induced for ~18 h in biotin-depleted media, then supplemented
Population E3-R1 was passaged twice, and analyzed by FACS side-by-side with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 for 1 h. From this culture,
with G2 and the combined library template. Population E3-R1 was induced for approximately 10-fold excess cells were prepared for sorting with streptavidin.
~18 h, then supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 A trapezoidal gate that collected cells positive for both anti-myc and streptavi-
for 1 h. From this culture, approximately 10-fold excess cells were prepared din was drawn, and 0.2% of cells were collected to give population E4-R3.
for sorting. A trapezoidal gate that collected cells positive for both anti-myc Population E4-R3 was passaged twice, and analyzed by FACS side-by-side
and streptavidin was drawn, and 0.15% of cells were collected to give popula- with G3∆, the combined library template, and previous rounds. Population
tion E3-R2. E4-R3 was induced for ~18 h in biotin-depleted media, then supplemented with
Population E3-R2 was passaged twice, and analyzed by FACS side-by-side 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 for 1 h. From this culture, approxi-
with G2, the combined library template, and previous rounds. Population mately 10-fold excess cells were prepared for sorting with anti-biotin antibody.
E3-R2 was induced for ~18 h, then supplemented with 50 µM biotin, 1 mM A trapezoidal gate that collected cells positive for both anti-myc and anti-biotin
ATP, and 5 mM MgCl2 for 10 min. From this culture, approximately 10-fold was drawn, and 0.1% of cells were collected to give population E4-R4.
excess cells were prepared for sorting. A trapezoidal gate that collected cells Population E4-R4 was passaged twice, and analyzed by FACS side-by-side
positive for both anti-myc and streptavidin was drawn, and less than 0.1% of with G3∆, the combined library template, and previous rounds. Population
cells were collected to give population E3-R3. E4-R4 was induced for ~18 h in biotin-depleted media, labeling was omitted for
At E3-R3, it was noted that the population had strong streptavidin signal negative selection (Supplementary Fig. 3f). From this culture, approximately
in the absence of exogenous biotin addition. Sequencing of population E3-R3 10-fold excess cells were prepared for sorting with streptavidin. A square gate
revealed that the majority of clones had a large insertion at the 5’ of the ligase that collected cells positive for anti-myc and negative for streptavidin was
gene. Removal of this insertion restored biotin dependence, but also resulted drawn, and 50% of cells were collected to give population E4-R5.

Population E4-R5 was passaged twice, and analyzed by FACS side-by-side negative selection that resulted E5-R4 reduced overall activity of the popula-
with G3∆, the combined library template, and previous rounds. Two selection. Population E5-R4 was induced for ~18 h in biotin depleted media, then
tions were performed on E4-R5. In the first selection, population E4-R5 was supplemented with 50 µM biotin, 1 mM ATP, and 5 mM MgCl2 for 10 min.
induced for ~18 h in biotin-depleted media, labeling was omitted for negative From this culture, approximately 10-fold excess cells were prepared for sort-
selection. From this culture, approximately 10-fold excess cells were prepared ing with streptavidin. A trapezoidal gate that collected cells positive for both
for sorting with anti-biotin antibody. A square gate that collected cells positive anti-myc and streptavidin was drawn, and 0.8% of cells were collected to give
for both anti-myc and anti-biotin was drawn, and 45% of cells were collected population E5-R5.
to give population E4-R6.1. Population E5-R5 was passaged twice, and analyzed by FACS side-by-side
In the second selection, population E4-R5 was induced for ~18 h in biotin- with G3, the combined library template, and previous rounds. Population
depleted media, then supplemented with 50 µM biotin, 1 mM ATP, and 5 mM E5-R5 was induced for ~18 h in regular media, labeling was omitted for nega-
MgCl2 for 20 min. From this culture, approximately 10-fold excess cells were tive selection. From this culture, approximately 10-fold excess cells were pre-
prepared for sorting with streptavidin. A trapezoidal gate that collected cells pared for sorting with anti-biotin antibody. A square gate that collected cells
positive for both anti-myc and streptavidin was drawn, and 0.1% of cells were positive for anti-myc and negative for anti-biotin was drawn, and 11.6% of
collected to give population E4-R6.2. cells were collected to give population E5-R6.
One more round of selections was performed on E4-R6.1, which was Population E5-R6 was passaged twice, and analyzed by FACS side-by-side
induced for ~18 h in biotin-depleted media, then supplemented with 50 µM with previous rounds. Sequencing of E5-R6 revealed several mutations that
biotin, 1 mM ATP, and 5 mM MgCl2 for 1 h. From this culture, approximately appeared in multiple clones. Several of these mutations were assayed as single
10-fold excess cells were prepared for sorting with streptavidin. A trapezoidal mutations and in various combinations in the mammalian cytosol. None of
gate that collected cells positive for both anti-myc and streptavidin was drawn, the mutations gave dramatic increases in activity, but one mutation M241T,
and 0.2% of cells were collected to give population E4-R7. appeared to impart benefits to activity.
Population E4-R7 was passaged twice, and analyzed by FACS side-by-side Screening of mutations present in E4-R6.2 in the mammalian cell cytosol
with previous rounds. Sequencing of E4-R7 revealed several mutations that revealed one mutation, S263P, which boosted activity, but also increased signal
appeared in multiple clones. Several of these mutations were assayed as single when biotin was omitted. This mutation, along with K194I from E4-R7 and
mutations and in various combinations in the mammalian cytosol. One muta- M241T from E5-R6, were introduced into G3 to give TurboID (Supplementary
tion, K194I, was found to significantly increase activity while not increasing Table 1). We also tested M241T in miniTurbo, however it was not added
signal exogenous when biotin is omitted. Introducing K194I into G3∆ resulted because it increased background signal when biotin was omitted.
in miniTurbo (Supplementary Table 1).
In a second evolutionary path, we continued with evolving G3 Mammalian cell culture, transfection, and stable cell line generation. HEK
(Supplementary Fig. 3h). Two libraries were made using G3 as the starting 293T cells from ATCC (passage number <25) were cultured as a monolayer in
template (Supplementary Tables 1 and 8) using error prone PCR with the growth media (either MEM (Cellgro) or a 1:1 DMEM:MEM mixture (Cellgro)
following conditions: supplemented with 10% (w/v) fetal bovine serum (VWR)), 50 units/mL peni-
Library 1: 2 µM 8-oxo-dGTP, 2 µM dPTP, 10 PCR cycles cillin, and 50 µg/mL streptomycin at 37 °C under 5% CO2. Mycoplasma testing
Library 2: 2 µM 8-oxo-dGTP, 2 µM dPTP, 20 PCR cycles was not performed before experiments. For confocal imaging experiments,
The library sizes, as calculated by transformation efficiency, were 2 × 107 cells were grown on 7 × 7 mm glass coverslips in 48-well plates with 250 µL
for Library 1 and 1.1 × 107 for Library 2. FACS analysis of the libraries showed growth medium. To improve adherence of HEK 293T cells, glass coverslips
robust expression and wide range of activities for Library 1 and Library 2. were pretreated with 50 µg/mL fibronectin (Millipore) in MEM for at least
Libraries 1 and 2 were combined and used as the initial population for 20 min at 37 °C before cell plating. For Western blotting, cells were grown on
the first round of selections. This combined library was induced for ~18 h in polystyrene 6-well plates (Greiner) with 2.5 mL growth medium.
biotin-depleted media, then supplemented with 50 µM biotin, 1 mM ATP, and For transient expression (Fig. 1f, 2c and Supplementary Figs. 1, 4b, 5, 10b,
5 mM MgCl2 for 10 min. From this culture, approximately 10-fold excess cells and 14c,d), cells were typically transfected at approximately 60% confluency
were prepared for sorting with anti-biotin antibody (Supplementary Table 9) using 3.2 µL/mL Lipofectamine2000 (Life Technologies) and 800 ng/mL plas-
in place of streptavidin. A trapezoidal gate that collected cells positive for mid in serum-free media (250 µL total volume for 48-wells, 2.5 mL total vol-
both anti-myc and anti-biotin was drawn, and 0.1% of cells were collected to ume for 6-wells) for 3-4 h, after which time Lipofectamine-containing media
give population E5-R1. was replaced with fresh serum-containing media.
Population E5-R1 was passaged twice, and analyzed by FACS side-by-side In an attempt to achieve similar expression levels of ligase in the experiment
with G3 and the combined library template. Population E5-R1 was induced for presented in Figure 2a, b, Supplementary Figure 6a, b, and Supplementary
~18 h in biotin-depleted media, then supplemented with 50 µM biotin, 1 mM Figure 7, cells were transfected at approximately 60% confluency using 1.6 µL/mL
ATP, and 5 mM MgCl2 for 10 min. From this culture, approximately 10-fold Lipofectamine2000 (Life Technologies) in serum-free media with the follow-
excess cells were prepared for sorting with anti-biotin antibody. A trapezoidal ing amounts of each plasmid (250 µL total volume for 48-wells, 2.5 mL total
gate that collected cells positive for both anti-myc and anti-biotin was drawn, volume for 6-wells): 160 ng/mL V5-BioID-NES, 80 ng/mL V5-TurboID-NES,
and 0.1% of cells were collected to give population E5-R2. 200 ng/mL V5-miniTurbo-NES, 30 ng/mL V5-BioID2-NES, and 1000 ng/mL
Population E5-R2 was passaged twice, and analyzed by FACS side-by-side V5-BASU-NES (Supplementary Table 8). After 3-4 h, the Lipofectamine-
with G3, the combined library template, and previous rounds. Population E5-R2 containing media was replaced with fresh serum-containing media.
was induced for ~18 h in biotin-depleted media, then supplemented with 50 µM In an attempt to achieve similar expression levels of ligase in the experi-
biotin, 1 mM ATP, and 5 mM MgCl2 for 10 min. From this culture, approxi- ment presented in Supplementary Figure 6c–e, cells were transfected at
mately 10-fold excess cells were prepared for sorting with anti-biotin antibody. approximately 60% confluency using 1.6 µL/mL Lipofectamine2000 (Life
A trapezoidal gate that collected cells positive for both anti-myc and anti-biotin Technologies) in serum-free media with the following amounts of each
was drawn, and 1.7% of cells were collected to give population E5-R3. plasmid (250 µL total volume for 48-wells, 2.5 mL total volume for 6-wells):
Population E5-R3 was passaged twice, and analyzed by FACS side-by-side 320 ng/mL V5-BioID-NES, 160 ng/mL V5-TurboID-NES, 400 ng/mL V5-
with G3, the combined library template, and previous rounds. Population miniTurbo-NES, 60 ng/mL V5-BioID2-NES, and 1000 ng/mL V5-BASU-NES
E5-R3 was induced for ~18 h in regular media, labeling was omitted for nega- (Supplementary Table 8). After 3-4 h, the Lipofectamine-containing media
tive selection. From this culture, approximately 10-fold excess cells were pre- was replaced with fresh serum-containing media.
pared for sorting with anti-biotin antibody. A square gate that collected cells For preparation of lentiviruses, HEK 293T cells in T25 flasks (BioBasic)
positive for anti-myc and negative for anti-biotin was drawn, and 34% of cells were transfected at ~60-70% confluency with the lentiviral vector pLX304
were collected to give population E5-R4. containing the gene of interest (2500 ng; Supplementary Table 8), and the
Population E5-R4 was passaged twice. FACS analysis side-by-side with lentiviral packaging plasmids pVSVG (250 ng; Supplementary Table 8) and
G3, the combined library template, and previous rounds showed that the ∆8.9 (2250 ng; Supplementary Table 8) with 30 µL Lipofectamine2000 in

serum-free media for 3 h, after which time the Lipofectamine-containing 6× protein loading buffer (0.33 M Tris-HCl pH 8, 34% glycerol, 94 mg/mL
media was replaced with fresh serum-containing media. Approximately 60 h SDS, 88 mg/mL DTT, 113 µg/mL bromophenol blue). The protein was boiled
after transfection, the cell medium containing the lentivirus was harvested for 5 min, diluted to 1×, and then separated on a 9% SDS-PAGE gel.
and filtered through a 0.45-µm filter. For all Western blots in Figures 1f, 2a,c and 3a,b and Supplementary
To generate stable cell lines, HEK cells were then infected at ~50% conflu- Figures 1, 4b, 5, 6, 8b–d, 9a and 10b,c, proteins separated on SDS-PAGE gels
ency, followed by selection with 8 µg/mL blasticidin in growth medium for were transferred to nitrocellulose membrane, and then stained by Ponceau S
at least 7 days before further analysis (Fig. 2c,d and Supplementary Figs. 8, (5 min in 0.1% (w/v) Ponceau S in 5% acetic acid/water). The blots were then
9a,b, 10c,e and 14a,b). blocked in 5% (w/v) milk (LabScientific) in TBS-T (Tris-buffered saline, 0.1%
Tween 20) for at least 30 min at room temperature, or as long as overnight at
Biotin labeling with TurboID and miniTurbo in live mammalian cells. 4 °C. Blots were then stained with primary antibodies (Supplementary Table 9)
For labeling of transiently transfected cells, we initiated biotin labeling 18 to in 3% BSA (w/v) in TBS-T for 1 - 16 h at 4 °C, washed four times with TBS-T
36 h following transfection. From a 100 mM biotin stock in dimethyl sulfox- for 5 min each, then stained with secondary antibodies or 0.3 µg/mL strepta-
ide (DMSO), we diluted biotin directly into serum-containing cell culture vidin-HRP (Supplementary Table 9) in 3% BSA (w/v) in TBS-T for 1 at
medium, to the desired final concentration. For BioID, we used a final con- 4 °C. The blots were washed four times with TBS-T for 5 min each time before to
centration of 50 µM biotin. For TurboID and miniTurbo, we typically used a development with Clarity Western ECL Blotting Substrates (Bio-Rad) and imag-
final concentration of 500 µM biotin (unless indicated otherwise). Labeling ing on a UVP BioSpectrum Imaging System, except for blots in Figures 1f, 2c
was stopped after the desired time period by transferring the cells to ice and and 3a,b, and Supplementary Figures 5a (miniTurbo) and 10b,c,which were
washing five times with ice-cold PBS. Supplementary Figure 5 shows that imaged on a Bio-Rad Gel Doc 2000. Quantitation of Western blots was per-
cooling samples to 4 °C terminates the biotinylation reaction. For negative formed using ImageJ on raw images under non-saturating conditions.
controls, we omitted exogenous biotin, or omitted the ligase.
Confocal fluorescence imaging of cultured cells. For fluorescence imag-
Gels and Western blots. For gels and Western blots shown in Figures 1f and ing experiments in Supplementary Figures 7, 8e, 9b, 10d,e and 14e,f, HEK
2a,c and Supplementary Figures 1, 2, 4b, 5, 6, 8b–d, 9a and 10b,c, HEK 293T 293T cells expressing the indicated constructs were plated, transfected, and
cells expressing the indicated constructs were plated, transfected, and labeled labeled with biotin as described above, and subsequently fixed with 4% (v/v)
with biotin as described above, and subsequently detached from the flask by paraformaldehyde in PBS at 4 °C for 45 min. Cells were then washed three
gently pipetting a stream of ice-cold PBS directly onto the cells. Pellets were times with PBS and permeabilized with cold methanol at -20 °C for 5 min.
collected by centrifuging the resulting cell suspension at 1,500 r.p.m. for 3 min. Cells were then washed three times with PBS, and then incubated with pri-
The supernatant was removed, and the pellet was lysed by resuspending in mary antibody (Supplementary Table 9) in PBS supplemented with 3% (w/v)
RIPA lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 0.1% SDS, 0.5% sodium BSA for 1 h at 4 °C. After washing three times with PBS, cells were then
deoxycholate, 1% Triton X-100, 1× protease inhibitor cocktail (Sigma-Aldrich), incubated with DAPI/secondary antibody, and neutravidin-Alexa Fluor647
and 1 mM PMSF) by gentle pipetting and incubating for 5 min at 4 °C. Lysates (Supplementary Table 9) in PBS supplemented with 3% (w/v) BSA for 1 h
were clarified by centrifugation at 10,000 r.p.m. for 10 min at 4 °C. Protein at 4 °C. Cells were then washed three times with PBS and imaged by confocal
concentration in clarified lysate was estimated with Pierce BCA Protein Assay fluorescence microscopy.
Kit (ThermoFisher) prior to separation on a 9% SDS-PAGE gel. Silver-stained Confocal imaging was performed using a Zeiss AxioObserver.Z1 micro-
gels (Supplementary Figures 9a, 10b–c) were generated using Pierce Silver scope, outfitted with a Yokogawa spinning disk confocal head, a Cascade II:512
Stain Kit (ThermoFisher). camera, a Quad-band notch dichroic mirror (405/488/568/647), 405 (diode),
For the Western blot experiment in Figure 3a comparing ligase variants in 491 (DPSS), 561 (DPSS), and 640 (diode) nm lasers (all 50 mW). DAPI (405
the yeast cytosol, S. cerevisiae strain BY4741 cells were propagated at 30 °C in laser excitation, 445/40 emission), Alexa Fluor568 (561 laser excitation, 617/73
supplemented minimal medium (SMM; 6.7 g/L Difco nitrogen base without emission), and Alexa Fluor647 (640 laser excitation, 700/75 emission), and dif-
amino acids, 20 g/L dextrose, 0.54 g/L CSM –Ade –His –Leu –Lys –Trp –Ura ferential intereference contrast (DIC) images were acquired through a 63× oil-
(Sunrise Science Products), 20 mg/L adenine, 20 mg/L uracil, 20 mg/L histi- immersive objective; Acquisition times ranged from 50 to 100 ms. All images
dine, 30 mg/L lysine) supplemented with leucine (100 mg/L). Yeast cells were were collected and processed using SlideBook 6.0 software (Intelligent Imaging
transformed with pRS415 plasmids using the Frozen E-Z Yeast Transformation Innovations). The data in Supplementary Figures 7, 8e, 9b, 10d,e and 14e,f
II kit (Zymoprep) according to manufacturer protocols. Transformed cells are representative of at least 10 fields of view.
containing the LEU2 gene were selected on SMM plates (SMM with 20 g/L
agar) and propagated in SMM at 30 °C. Ligase expression was induced by Conjugation of AlexaFlour647 to neutravidin. A reaction mixture was
inoculating saturated yeast culture into 10% D/G SMM (SMM medium with assembled in a 1.5 mL Eppendorf tube with the following components
90% of dextrose replaced with galactose) supplemented with 50 µM biotin at (added in this order): 200 µL of 5 mg/mL Neutravidin (Life Technologies)
a 1:100 dilution and incubating at 30 °C. After ~ 12 h, the saturated induced in PBS, 20 µL of 1 M sodium bicarbonate in water, and 10 µL of 10 mg/mL
culture was diluted 1:30 in fresh induction media supplemented with 50 µM AlexaFluor647-NHS Ester (Life Technologies) in anhydrous DMSO. The tube
biotin and allowed to grow for approximately 6 h more until reaching OD600 was incubated at room temperature with rotation in the dark for 3 h. The
~1. Three milliliters of this culture was pelleted (normalized across samples neutravidin-AlexaFluor647 conjugate was purified from unreacted dye using
so that the same approximate amount of cells are collected for each sample), a NAP-5 size-exclusion column (GE Healthcare Life Sciences) according to
and lysed on ice in 50 µL 1.85 M NaOH + 300 mM β-mercaptoethanol for the manufacturer’s instructions. The conjugate was typically eluted from the
10 min on ice. The protein in the lysate was then precipitated by adding 50 µL column in 500 µL cold PBS. Absorbance values, determined using a Nanodrop
50% (w/v) TCA and incubating on ice for 15 min. The protein was pelleted at 2000c UV-vis spectrophotometer (Thermo Scientific), were typically as fol-
12000g for 5 min, then dissolved in 120 µL urea/SDS buffer (0.48 g/mL urea, lows: A280 = ~0.284 and A647 = ~1.625. The conjugate was stable at 4 °C in
50 mg/mL SDS, 29.2 mg/mL EDTA, 15.4 mg/mL DTT, 1 mg/mL bromophenol the dark for at least 4 months and was flash frozen and stored at 80 °C for
blue, 12 mg/mL Tris base, 0.2 mL/mL 1M Tris pH 6.8). Proteins were boiled longer term storage. For mammalian cell labeling experiments, the conjugate
for 10 min prior to separation on a 9% SDS-PAGE gel. was diluted 1,000-fold in PBS containing 1% BSA.
For the Western blot experiment in Figure 3b comparing ligase vari-
ants in bacteria, BL21 E. coli bacteria expressing the indicated constructs Sample preparation for proteomics and for the Western blot experiment
(Supplementary Table 8) were induced overnight (18 h) at 37 °C in Lysogeny in Supplementary Figure 8. For each sample, HEK 293T cells were grown
Broth (LB) supplemented with 100 µg/mL ampicillin, 100 µg/mL IPTG, and as a monolayer in 1:1 DMEM:MEM mixture (Cellgro) supplemented with
with or without 50 µM biotin. Grown to approximately OD600 = 0.6, 100 µL of 10% (w/v) fetal bovine serum (VWR), 50 µg/mL penicillin, and 50 µg/mL
each culture was pelleted (normalized across samples so that the same approxi- streptomycin in T150 flasks at 37 °C under 5% CO2. Nuclear proteomic sam-
mate amount of cells are collected for each sample) and resuspended in 15 µL ples were generated by transfecting cells at approximately 60% confluency with

30 µg DNA using 150 µL Lipofectamine 2000 for 4 h. Mitochondrial matrix, TMT labeling and fractionation of peptides. Desalted peptides were labeled
ER membrane, and outer mitochondrial membrane samples were generated with TMT (6-plex or 11-plex) reagents. Peptides were reconstituted in 100 µL
using HEK 293T cell lines that stably express the respective ligase. BioID of 50 mM HEPES. Each 0.8 mg vial of TMT reagent was reconstituted in
samples were labeled using 50 µM biotin for 18 h; TurboID and miniTurbo 41 µL of anhydrous acetonitrile and added to the corresponding peptide sample
samples were labeled using 500 µM biotin for 10 min. Labeling was stopped by for 1 h at room temperature. Labeling of samples with TMT reagents was com-
placing cells on ice and washing five times with ice-cold PBS (Supplementary pleted with the design shown in Figure 2d and Supplementary Figure 10a.
Fig. 5). Cells were detached from the flask by gently pipetting a stream of PBS TMT labeling reactions were quenched with 8 µL of 5% hydroxylamine at
directly onto the cells, then pellets were collected by centrifuging the result- room temperature for 15 min with shaking, evaporated to dryness in a vacuum
ing cell suspension at 1,500 r.p.m. for 3 min. The supernatant was removed, concentrator, and desalted on C18 StageTips. For each TMT 6-plex cassette
and the pellet was lysed in ~ 1.5 mL RIPA lysis buffer by gentle pipetting and and the TMT 11-plex cassette, 50% of the sample was fractionated by basic
incubating for 5 min at 4 °C. Lysates were clarified by centrifugation at 10,000 pH reversed phase using StageTips while the other 50% of each sample was
r.p.m. for 10 min at 4 °C. reserved for LC-MS analysis by a single-shot, long gradient. One StageTip
To enrich biotinylated material from proteomic samples, 350 µL strepta- was prepared per sample using 2 plugs of Styrene Divinylbenzene (SDB) (3M)
vidin-coated magnetic beads (Pierce) were washed twice with RIPA buffer, material. The StageTips were conditioned two times with 50 µL of 100% meth-
incubated with clarified lysates containing ~ 3 mg protein for each sample anol, followed by 50 µL of 50%MeCN/0.1% FA, and two times with 75 µL of
with rotation for 1 h at room temperature, then moved to 4 °C and incubated 0.1% FA. Sample, resuspended in 100 µL of 0.1% FA, was loaded onto the stage
overnight with rotation. The beads were subsequently washed twice with tips and washed with 100 µL of 0.1% FA. Following this, sample was washed
1 mL of RIPA lysis buffer, once with 1 mL of 1 M KCl, once with 1 mL of 0.1 M with 60 µL of 20mM NH4HCO2 /2% MeCN, this wash was saved and added
Na2CO3, once with 1 mL of 2 M urea in 10 mM Tris-HCl (pH 8.0), and twice to fraction 1. Next, sample was eluted from StageTip using the following con-
with 1 mL RIPA lysis buffer. The beads were then resuspended in 1 mL fresh centrations of MeCN in 20 mM NH4HCO2: 10%, 15%, 20%, 25%, 30%, 40%,
RIPA lysis buffer, transferred to a new Eppendorf tube, and shipped to Steve and 50%. For a total of 6 fractions, 10 and 40% (fractions 2 and 7) elutions were
Carr’s laboratory (Broad Institute) on ice for further processing and prepara- combined, as well as 15 and 50% elutions (fractions 3 and 8). The six fractions
tion for LC-MS/MS analysis. were dried by vacuum centrifugation.
For proteomic samples, 2.5% of the lysate was removed prior to enrichment
used to estimate the protein concentration in clarified lysates using Pierce Liquid chromatography and mass spectrometry. Desalted peptides were
BCA Protein Assay Kit (ThermoFisher), as well as to verify ligase expres- resuspended in 9 µL of 3% MeCN/0.1% FA and analyzed by online nanoflow
sion and confirm successful biotinylation by Western blotting as shown in liquid chromatography tandem mass spectrometry (LC-MS/MS) using an
Supplementary Figures 9a and 10b,c. After enrichment, 5% of beads were Orbirtrap Fusion Lumos Tribrid MS (ThermoFisher Scientific) coupled on-
removed and biotinylated proteins were eluted by boiling the beads in 75 µL line to a Proxeon Easy-nLC 1200 (ThermoFisher Scientific). Four microliters
of 3× protein loading buffer supplemented with 20 mM DTT and 2 mM biotin. of each sample was loaded onto a microcapillary column (360 µm outer diam-
The eluted proteins were separated on an SDS-PAGE gel and stained using eter × 75 µm inner diameter) containing an integrated electrospray emitter tip
Pierce Silver Stain Kit to ensure that enrichment of biotinylated material was (10 µm), packed to approximately 24 cm with ReproSil-Pur C18-AQ 1.9 µm
successful (Supplementary Figures 9a and 10b,c). beads (Dr. Maisch GmbH) and heated to 50 °C. The HPLC solvent A was 3%
To enrich biotinylated material from samples prepared for Supplementary MeCN, 0.1% FA, and the solvent B was 90% MeCN, 0.1% FA. The SDB frac-
Figure 8, 240 µL streptavidin-coated magnetic beads (Pierce) were washed tions were measured using a 110 min MS method, which used the following
twice with RIPA buffer, incubated with clarified lysates containing approxi- gradient profile: (min:%B) 0:2; 1:6; 85:30; 94:60; 95:90; 100:90; 101:50; 110:50
mately 3 mg protein for each sample with rotation for 1 h at room tempera- (the last two steps at 500 nL/min flow rate). Non-fractionated samples were
ture. The beads were subsequently washed twice with 1 mL of RIPA lysis analyzed using a 260 min MS method with the following gradient profile:
buffer, once with 1 mL of 1 M KCl, once with 1 mL of 0.1 M Na 2CO3, once (min:%B) 0:2; 1:6; 235:30; 244:60; 245:90; 250:90; 251:50; 260:50 (the last two
with 1 mL of 2 M urea in 10 mM Tris-HCl (pH 8.0), and twice with 1 mL steps at 500 nL/min flow rate).
RIPA lysis buffer. Biotinylated proteins were then eluted from the beads The Orbitrap Fusion Lumos Tribrid was operated in the data-dependent
by boiling the beads in 300 µL of 3× protein loading buffer supplemented mode acquiring HCD MS/MS scans (resolution = 15,000 for TMT6-plex,
with 20 mM DTT and 2 mM biotin. For each sample, 25 µL of this elu- or resolution = 50,000 for TMT11-plex ) after each MS1 scan (resolution =
ate was then separated on SDS-PAGE gel alongside 35 µg protein from the 60,000) on the most abundant ions within a 2 s cycle time using an MS1 target
corresponding clarified lysate prior to enrichment, and then transferred to of 3 × 106 and an MS2 target of 5 × 104 . The maximum ion time utilized for
nitrocellulose for Western blotting as described above with antibodies against MS/MS scans was 50 ms for TMT6-plex experiments and 105 ms for the TMT
the endogenous proteins indicated in Supplementary Figure 8 (also see 11-plex experiment; the HCD normalized collision energy was set to 34 for
Supplementary Table 9). TMT6 and 38 for TMT11; the dynamic exclusion time was set to 45 s, and the
peptide match and isotope exclusion functions were enabled. Charge exclusion
On-bead trypsin digestion of biotinylated proteins. To prepare samples for was enabled for charge states that were unassigned, 1 and >6.
mass spectrometry analysis, proteins bound to streptavidin beads (~300 µL
of slurry) were washed twice with 200 µL of 50 mM Tris HCl buffer (pH Analysis of mass spectrometry data. Collected data were analyzed using
7.5) followed by two washes with 2 M urea/50 mM Tris (pH 7.5) buffer. Spectrum Mill software package v6.1pre-release (Agilent Technologies).
The final volume of 2 M urea/50 mM Tris buffer (pH 7.5) was removed Nearby MS scans with the similar precursor m/z were merged if they were
and beads were incubated with 80 µL of 2 M urea/50 mM Tris containing within ± 60 s retention time and ±1.4 m/z tolerance. MS/MS spectra were
1 mM DTT and 0.4 µg trypsin for 1 h at 25 °C with shaking. After 1 h, the excluded from searching if they failed the quality filter by not having a sequence
supernatant was removed and transferred to a fresh tube. The streptavidin tag length 0 or did not have a precursor MH+ in the range of 750 – 4000.
beads were washed twice with 60 µL of 2 M urea/50 mM Tris buffer (pH 7.5) All extracted spectra were searched against a UniProt47 database containing
and the washes were combined with the on-bead digest supernatant. The human reference proteome sequences. Search parameters included: parent and
eluate was reduced with 4 mM DTT for 30 min at 25 °C with shaking. The fragment mass tolerance of 20 p.p.m., 30% minimum matched peak intensity,
samples were alkylated with 10 mM iodoacetamide for 45 min in the dark at and ‘calculate reversed database scores’ enabled. The digestion enzyme search
25 °C with shaking. An additional 0.5 µg of trypsin was added to the sample parameter used was Trypsin Allow P, which allows K-P and R-P cleavages. The
and the digestion was completed overnight at 25 °C with shaking. After missed cleavage allowance was set to 4. Fixed modifications were carbami-
overnight digestion, samples were acidified (to pH < 3) by adding formic domethylation at cysteine. TMT labeling was required at lysine, but peptide
acid (FA) such that the sample contained ~1% FA. Samples were desalted N termini were allowed to be either labeled or unlabeled. Allowed variable
on C18 StageTips and evaporated to dryness in a vacuum concentrator, as modifications were protein N-terminal acetylation and oxidized methionine.
previously described46. Individual spectra were automatically assigned a confidence score using the

Spectrum Mill autovalidation module. Score at the peptide mode was based on fraction of class (1) proteins above the TMT ratio in question, and FPR as
target-decoy false discovery rate (FDR) of 1%. Protein polishing autovalidation the fraction of class (2) above the TMT ratio in question. We selected TMT
was then applied using an auto thresholding strategy. Relative abundances of ratios that maximize the difference between TPR and FPR as our cutoffs
proteins were determined using TMT reporter ion intensity ratios from each (Supplementary Fig. 9c).
MS/MS spectrum and the mean ratio is calculated from all MS/MS spectra After applying both cutoffs to each experimental replicate, we then intersected
contributing to a protein subgroup. Proteins identified by 2 or more distinct both filtered lists to produce the final proteomes (Supplementary Table 5).
peptides were considered for the dataset. Overlap of proteins between proteomes obtained with BioID, TurboID 10
minute labeling, and TurboID 1 hour labeling are shown in Supplementary
Generation of proteomic lists for the ER membrane. Complete mass spec- Figure 9g, Supplementary Table 5.
trometry data for the ER membrane (ERM) proteomic experiment are shown To assess the specificity of our proteomes (Fig. 2e), we report the percentage
in Supplementary Table 5. To select cutoffs for proteins biotinylated by the of proteins present in Supplementary Table 2, a list of 11,838 human proteins
indicated ligase over non-specific bead binders, we classified the detected with secretory annotation according to Phobius48, the Human Protein Atlas51
proteins into three groups: (protein localized to endoplasmic reticulum, Golgi apparatus, plasma membrane,
(1) ERM proteins (Supplementary Table 2; true positive list of 90 well- vesicles, nuclear membrane, cell junctions; or predicted membrane proteins and
established ERM proteins33). predicted secreted proteins) , the Plasma Proteome Database52, literature (refer-
(2) Soluble matrix proteins (Supplementary Table 2; false positive list of ence cited in table), or are annotated with the following Gene Ontology49,50 terms:
173 soluble mitochondrial matrix proteins2). GO:0005783, GO:0005789, GO:0007029, GO:0030867, GO:0048237, GO:0061163,
(3) All other proteins GO:0016320, GO:0030868, GO:0006983, GO:0000139, GO:0051645, GO:0031985,
We then normalized the TMT ratios in order to account for differences in GO:0005796, GO:0005795, GO:0005794, GO:0007030, GO:0090168, GO:0005886,
total protein quantity between samples within the TMT 11-plex experiment. GO:0007009, GO:1903561, GO:0070062, GO:0005576, GO:0031012, GO:0005615,
To do this, the Log2(TMT ratios) corresponding to ERM-ligase/untransfected GO:0005769, GO:0035646, GO:0005765, GO:0090341, GO:0090340, GO:0005635,
(Log2(127N/126C), Log2(128N/126C), Log2(129N/126C), (Log2(128C/126C), GO:0007084, GO:0007077, GO:0006998, GO:0051081, GO:0005641, GO:0031965,
Log2(130N/126C), Log2(131N/126C), Log2(131C/126C)) were normalized to GO:0005637, GO:0071765, GO:0048471, GO:1905719, GO:0031982, GO:0006906,
the median for class (2) proteins, which was set to 0 (i.e. TMT ratios set to 1). GO:0048278, GO:0032587, GO:0016021, GO:0005887, GO:0005768, GO:0071816,
To calculate optimal cut-offs, we then calculated the true positive rate (TPR) GO:0031526, GO:0005913, GO:0072546, GO:1990440, GO:0030968, GO:1902236,
and false positive rate (FPR) we would obtain if we retained only proteins GO:1990441, GO:0034976, GO:0005788, GO:0005790, GO:1902237, GO:0070059,
above that TMT ratio. We defined TPR as the fraction of class (1) proteins GO:0005786, GO:0005793, GO:0044322, GO:0098554, GO:0005791, GO:1902010,
above the TMT ratio in question, and FPR as the fraction of class (2) above the GO:0043001, GO:0005802, GO:0006888, GO:0006890, GO:0005801, GO:0012510,
TMT ratio in question. We selected TMT ratios that maximize the difference GO:0006892, GO:0042147, GO:0034499, GO:0032588, GO:0006895, GO:0030140,
between TPR and FPR as our cutoffs (Supplementary Fig. 9c). GO:0051684, GO:0000042, GO:0032580, GO:0030173, GO:0006891, GO:0030198,
To select cutoffs for proteins biotinylated by the indicated ERM-ligase over GO:0031668, GO:0010715, GO:0035426, GO:1903053, GO:1903551, GO:0005578,
proteins biotinylated by the corresponding cytosol-targeted ligase, we classi- GO:1903055, GO:0001560, GO:0022617, GO:0006887, GO:0012505.
fied the detected proteins into three groups: The specificity of the ‘entire human proteome’ reported in Figure 2e was
(1) ERM proteins (Supplementary Table 2; true positive list of 90 well- calculated as the percentage of human proteins that are not present in cat-
established ERM proteins33). egory (2) non-secretory proteins, as defined above; i.e., the proteins that are
(2) Non-secretory proteins (Supplementary Table 2; false positive list of present in the list of 11,838 human proteins with secretory annotation in
7421 human proteins that are not predicted to be secretory by Phobius48 or Supplementary Table 2.
are not annotated with the following Gene Ontology49,50 terms: GO:0005783, To calculate subsecretory specificity, we took a subset of proteins with the
GO:0005789, GO:0007029, GO:0030867, GO:0048237, GO:0061163, following Gene Ontology49,50 terms: GO:0005783 for endoplasmic reticulum,
GO:0016320, GO:0030868, GO:0006983, GO:0000139, GO:0051645, GO:0005794 for Golgi apparatus, and GO:0005886 for plasma membrane
GO:0031985, GO:0005796, GO:0005795, GO:0005794, GO:0007030, and classified them according to this priority: endoplasmic reticulum>Golgi
GO:0090168, GO:0005886, GO:0007009, GO:1903561, GO:0070062, apparatus>plasma membrane (Supplementary Table 2). We then took the
GO:0005576, GO:0031012, GO:0005615, GO:0005769, GO:0035646, subset of proteins in the ERM proteomes with these GO terms and plotted their
GO:0005765, GO:0090341, GO:0090340, GO:0005635, GO:0007084, percentages in Figure 2f. To calculate ER specificity, the subset of proteins
GO:0007077, GO:0006998, GO:0051081, GO:0005641, GO:0031965, with GOCC49,50 annotation for endoplasmic reticulum (GO:0005783) were
GO:0005637, GO:0071765, GO:0048471, GO:1905719, GO:0031982, subdivided into those with membrane annotation, soluble cytosolic anno-
GO:0006906, GO:0048278, GO:0032587, GO:0016021, GO:0005887, tation, or soluble luminal annotation according to GOCC49,50, UniProt47,
GO:0005768, GO:0071816, GO:0031526, GO:0005913, GO:0072546, TMHMM53, or literature (Supplementary Table 2); these percentages are
GO:1990440, GO:0030968, GO:1902236, GO:1990441, GO:0034976, reported in Figure 2g.
GO:0005788, GO:0005790, GO:1902237, GO:0070059, GO:0005786, To assess the recall/sensitivity of our ERM proteomes, we utilized a list
GO:0005793, GO:0044322, GO:0098554, GO:0005791, GO:1902010, of true positive ERM proteins (Supplementary Fig. 9f, Supplementary
GO:0043001, GO:0005802, GO:0006888, GO:0006890, GO:0005801, Table 2). In the scatter plots shown in Supplementary Figure 9e, true posi-
GO:0012510, GO:0006892, GO:0042147, GO:0034499, GO:0032588, tive ERM proteins (Supplementary Table 2) are shown in green, cytosolic
GO:0006895, GO:0030140, GO:0051684, GO:0000042, GO:0032580, proteins (Supplementary Table 2; human proteins with Gene Ontology49,50
GO:0030173, GO:0006891, GO:0030198, GO:0031668, GO:0010715, term GO:0005829 that lack annotated or predicted transmembrane domains
GO:0035426, GO:1903053, GO:1903551, GO:0005578, GO:1903055, according to UniProt47 or TMHMM53) are shown in red, and all other proteins
GO:0001560, GO:0022617, GO:0006887, GO:0012505) are shown in black.
(3) All other proteins
We then normalized the TMT ratios in order to account for differences in Generation of mitochondrial matrix and nuclear proteomic lists. Complete
total protein quantity between samples within the TMT 11-plex experiment. mass spectrometry data for both the nucleus and mitochondrial matrix are
To do this, the Log2(TMT ratios) corresponding to ERM-ligase/ligase-NES shown in Supplementary Tables 6 and 7 respectively. Each of the two rep-
(Log 2(127N/127C), Log2(128N/127C), Log2(129N/129C), (Log2(128C/ licates for each proteomic experiment (mitochondrial matrix and nucleus)
129C), Log 2(130N/130C), Log 2(131N/130C), Log 2(131C/129C)) were were analyzed separately. To select cutoffs for proteins biotinylated by the
normalized to the median for class (2) proteins, which was set to 0 (i.e. indicated ligase over non-specific bead binders, we classified the detected
TMT ratios set to 1). To calculate optimal cut-offs, we then calculated the proteins into five groups:
true positive rate (TPR) and false positive rate (FPR) we would obtain if (1) nuclear annotated proteins (Supplementary Table 3; true positive list
we retained only proteins above that TMT ratio. We defined TPR as the of 6710 human proteins annotated with the following Gene Ontology49,50

terms: GO:0016604, GO:0031965, GO:0016607, GO:0005730, GO:0001650, generated using PhiC31 integration by injecting pWalium10-V5-ligase plas-
GO:0005654, GO:0005634). mids into flies carrying an attP docking site on chromosome III (attP2)58.
(2) mitochondrial annotated proteins (Supplementary Table 4; true posi- Final fly strains are referred to as UAS-V5-BioID, UAS-V5-TurboID, and UAS-
tive list of 1555 human proteins present in MitoCarta2.0 (ref. 54) or annotated V5-miniTurboID.
with the following Gene Ontology49,50 term: GO:0005739, but excluding any Drosophila culture and genetics. Experiments on flies were performed with
proteins also present in category 2 (Supplementary Table 4). wild type or transgenic strains of Drosophila melanogaster. The age and sex of
(3) proteins with non-nuclear annotation (Supplementary Table 3; false animals involved in experiments are indicated in figure legends and methods
positive list of 6815 human proteins annotated wih the following Gene below. The Harvard Medical School Standing Committee on Animals (through
Ontology49,50 terms: GO:0015629, GO:0016235, GO:0030054, GO:0005813, the Office of the Institutional Animal Care and Use Committee (IACUC))
GO:0045171, GO:0000932, GO:0005829, GO:0005783, GO:0005768, deems flies as invertebrates with limited sentience and therefore not subject
GO:0005929, GO:0005794, GO:0045111, GO:0005811, GO:0005764, to formal review and approval by the committee.
GO:0005815, GO:0015630, GO:0030496, GO:0070938, GO:0005739, Crosses were maintained on standard fly food at 25 °C. For temporal expres-
GO:0072686, GO:0005777, GO:0005886, GO:0043231; and are not anno- sion experiments using tub-Gal4, tubGal80ts, animals were kept at 18 °C during
tated with the following Gene Ontology49,50 terms: GO:0016604, GO:0031965, all developmental stages until transferred to 29 °C to induce gene expression.
GO:0016607, GO:0005730, GO:0001650, GO:0005654, GO:0005634, ‘nucleus Biotin food was prepared by microwaving standard fly food until liquid and
localization’, ‘nuclear envelope’, ‘nuclear matrix’, ‘nuclear chromatin’, ‘nuclear adding 1 mM biotin dissolved in H2O to a final concentration of 100 µM.
pore’, ‘nuclear inner membrane’, ‘nuclear chromosome’, ‘nuclear heterochro- Unless otherwise noted, fly stocks were obtained from the Bloomington
matin’, ‘nuclear euchromatin’, ‘nuclear inclusion body’). Drosophila Stock Center and are listed with the corresponding stock number:
(4) proteins with non-mitochondrial annotation (Supplementary Table 4; ptc-Gal4 (2017), Act5c-Gal4/CyO (4414), nub-Gal4 (25754), w1118 (6326), tub-
previously curated false positive list of 2410 human proteins that are not anno- Gal80ts; tub-Gal4/TM6b (Perrimon Lab), UAS-Luciferase (35788), Desat-Gal4
tated to be mitochondrial2,55). (Oenocyte) (65405), repo-Gal4 (Glia) (7415), Mef2-Gal4 (Muscle) (27390),
(5) All other proteins. Lpp-Gal4 (Fat body) (Perrimon Lab, see transgene in 67043 for information),
We then normalized the TMT ratios in order to account for differences in elav-Gal4 (Neurons) (8760), Myo1a-Gal4 (Gut) (Perrimon Lab, see transgene
total protein quantity between samples within the TMT 6-plex experiments. To in 67057 for information), Hml-Gal4 (Hemocytes) (30140).
do this, the Log2(TMT ratios) corresponding to ligase experimentals/negative
control (Log2(126/127), Log2(128/129), Log2(130/131), Log2(129/127) for rep- Western blotting of Drosophila adults. For experiments Figure 3g and
licate 1, and (Log2(130/131), Log2(129/126), Log2(127/126) Log2(128/126) for Supplementary Figure 12, adult flies were aged 3 days after eclosion from
replicate 2) were normalized to the median for class (3) proteins for the nuclear pupal cases (13 days old after egg deposition). For each condition, five females
dataset or class (4) proteins for the mitochondrial matrix data set, which was and five males were lysed in RIPA buffer (Thermo Fisher, 89900) on ice using a
set to 0 (i.e. TMT ratios set to 1). To calculate optimal cut-offs, we then cal- blue pestle in a microcentrifuge tube. Samples were centrifuged at 14,000 xg for
culated the true positive rate (TPR) and false positive rate (FPR) we would 20 min at 4 °C. Supernatant was retained and transferred to a new centrifuge
obtain if we retained only proteins above that TMT ratio. We defined TPR as tube. Protein concentration was calculated using a BCA kit (Pierce 23225)
the fraction of class (1) proteins for the nuclear dataset or class (2) proteins for and RIPA buffer was added to samples to normalize to 4 µg/µL. Normalized
the mitochondrial matrix data set above the TMT ratio in question, and FPR protein samples were mixed with an equal volume of 4× SDS sample buffer
as the fraction of class (3) proteins for the nuclear dataset or class (4) proteins and boiled for 5 min at 95 °C. 10 µg/sample was loaded onto a 4-20% Mini-
above the TMT ratio in question. We selected TMT ratios that maximize the PROTEAN TGX PAGE gel (Bio-Rad 4561095), transferred to Immobilon-FL
difference between TPR and FPR as our cutoffs (Supplementary Fig. 10f,g). PVDF membrane (Millipore IPFL00010), incubated in PBS + 0.1% Tween
After applying cutoffs to each replicate, we then intersected both to pro- (PBST) for 15 min, and blocked overnight in 3% BSA in PBST (PBST-BSA)
duce the final proteomes (Supplementary Table 6 for nuclear proteomes, at 4 °C. To detect biotinylated proteins, blots were incubated with 0.3 µg/mL
Supplementary Table 7 for mitochondrial matrix proteomes). Overlap of streptavidin-HRP (Thermo Fisher S911) in PBST-BSA for 1 hour at room
proteins between proteomes obtained with BioID, TurboID, and miniTurbo temperature. Blots were washed extensively with PBST and exposed using Pico
for both the nucleus and mitochondrial matrix are shown in Supplementary Chemiluminescent Substrate (Thermo Fisher 34577). To detect expressed V5-
Figure 10k, Supplementary Table 6 for nucleus, and Supplementary tagged ligases, blots were incubated with 1:10,000 mouse anti-V5 (Invitrogen
Table 7 for mitochondrial matrix. To assess the specificity of our proteomes R960-25) with PBST-BSA overnight at 4 °C, washed with PBST, incubated with
(Figure 2h), we calculated the percentage of proteins present in class (1) for 1:5000 anti-mouse Alexa 800 (Thermo Fisher A32730), washed with PBST,
the nuclear proteomes and class (2) for the mitochondrial matrix proteomes and imaged on an Aerius Fluorescent imager (LI-COR 9250).
(Supplementary Table 3 for nuclear specificity, Supplementary Table 4 for
mitochondrial specificity). To assess the recall/sensitivity of our proteomes, Immunohistochemistry of Drosophila wing discs. For Figure 3d, wandering
we determined the fraction of true positive proteins present in each proteome. 3rd instar larvae were bisected and inverted to expose the imaginal discs. Inverted
230 True positive nuclear proteins are shown in Supplementary Table 3 and carcasses were fixed for 20 min in 4% paraformaldehyde in 1× PBS. Fixed car-
were assembled using Cell Atlas data (proteins detected by validated antibod- casses with attached wing discs were permeabilized with PBS + 0.1% Triton-X100
ies in nuclear bodies, nuclear membrane, nuclear speckles, nucleoli, fibrillary (PBST) for 20 min and blocked with PBST + 5% normal goat serum (PBST-NGS)
centers, nucleoplasm, or nucleus) and hyperLOPIT data51 (hyperLOPIT loca- for 1 hour. Blocked carcasses were incubated overnight at 4 °C in PBST-NGS with
tion annotated to nucleus, or nucleus-chromatin) and have been shown to be 1:500 mouse anti-V5 (Invitrogen R960-25) and 1:500 streptavidin-555 (Invitrogen
expressed in HEK cells56. 173 True positive mitochondrial matrix proteins are S32355). Carcasses were washed 3× with PBST and incubated for 1 hour at room
obtained from previous work2 and shown in Supplementary Table 4. temperature in PBST-NGS with 1:500 anti-mouse Alexa 647 (Thermo Fisher
A-21236) and 1:1000 DAPI (stock 1mg/ml). Samples were washed with three
Generation of UAS-ligase transgenic Drosophila lines. V5-BioID, V5- times with PBST, once with PBS, and equilibrated in 70% Glycerol/1× PBS. Wing
TurboID, and V5-miniTurboID coding sequence was PCR amplified from discs were dissected away from the carcass and mounted onto glass slides with
CMV-plasmids using the same F and R primers: Vectashield mounting media (Vector Labs H-1000) and glass coverslip. Mounted
V5-ligase_F: ccgcggccgcccccttcaccATGGGCAAGCCCATCCCC samples were imaged on a Zeiss 780 confocal microscope.
V5-ligase_R gggtcggcgcgcccacccttCTATTAGTCCAGGGTCAGGCG
DNA fragments were cloned into pEntr plasmids (Invitrogen) using Quantitation of fluorescence signal intensity from Drosophila wing discs in
Gibson assembly (NEB). pEntr_V5-ligase entry plasmids were recombined Figure 3e. Average signal intensity of fluorescence of streptavidin-555 in wing
into pWalium10-roe57 using Gateway LR Clonase II Enzyme (Invitrogen). discs was measured using raw images obtained under identical confocal set-
pWalium10-roe contains 10× UAS enhancer elements for Gal4-controlled tings and under non-saturating exposure settings. Using ImageJ software, the
expression, attB sequence, and a white+ transgene. Transgenic flies were polygon tool was used to select a rectangular region of the ptc-Gal4 expressing

domain in the wing pouch. The average signal intensity in this selected region media (2.5 mL total volume) for 3-4 h, after which time Lipofectamine-
was determined separately for the streptavidin-555 channel and the anti-V5 containing media was replaced with fresh serum-containing media. After
channel. The average signal intensity in control samples (very low background ~2 h, cells were trypsinized and seeded in triplicate into wells of each fibronec-
staining) was subtracted from signal intensity of experimental conditions tin-coated 96-well plate at 2000 cells/well in 50:50 serum-containing MEM:
(BioID, turboID, miniturboID). For each wing disc, the signal intensity of DMEM with or without 50 µM biotin. The stable cell lines were seeded into
streptavidin-555 was normalized to the signal intensity of anti-V5 (streptavi- wells in the same manner. An additional triplicate of coated wells without cells
din-555/anti-V5). Fold change was determined by normalizing streptavidin- served as background subtraction in each plate. One plate was immediately
555/anti-V5 values from TurboID and miniturboID to values from BioID. assayed after plating for cell viability by the CellTiter-Glo 2.0 Luminescent
Measurements were taken from at least three wing discs for each condition. Viability assay (Promega). Subsequent plates were assayed at the indicated
time points.
Quantification of adult Drosophila wing size and survival after ligase
expression during development in Supplementary Figure 13. UAS-V5-ligase C. elegans strains and culture conditions. Experiments on Caenorhabditis
transgenes were expressed during development by crossing with different elegans were performed with wild type (N2) or transgenic strains expressing
Gal4-expressing lines and their effects on the adult assessed. extrachromosomal arrays. The age and sex of animals involved in experi-
To determine if larval wing disc expression of ligases affects adult wing mor- ments are indicated in figure legends and methods below. The Stanford’s
phology, nub-Gal4 was crossed with UAS-V5-ligase transgenes and the resulting Administrative Panel on Laboratory Animal Care (APLAC) deems C. elegans
progeny analyzed. nub-Gal4 was crossed with wild-type flies (w1118) as a negative used in this study as invertebrates and not subject to formal review and
control. Adult flies were aged 3 days after eclosion from pupal cases. Wings were approval by the committee.
removed from adults, placed in a drop of 50% Permount/50% Xylenes on a glass Unless otherwise noted, C. elegans strains were cultured and maintained
slide, and a coverslip added. Mounted wings were imaged using a light micro- at 20 °C on E. coli OP50 bacteria as previously described59. To deplete the
scope with a 10× objective. Wing area was measured using the polygon selection animals of excess biotin, worms were grown for 2 generations on biotin aux-
tool in ImageJ. Wings quantified and imaged are from female flies. otrophic E. coli (MG1655bioB:kan)60 and washed twice with 1× M9 solu-
To determine if developmental expression of ligases reduces survival to tion. Biotin auxotrophic E. coli MG1655bioB:kan was kindly donated by
adulthood, we crossed UAS-ligase lines with different Gal4 lines that express Dr. John E. Cronan, University of Illinois. Embryos dissected from one day-
in major tissue types (Muscle, Fat, Neurons, Glia, Gut, Oenocytes, Hemocytes) old adults of the following genotypes were compared for this study: JLF289
or ubiquitously (Act5c-Gal4). To quantify toxicity, we counted the number of (wowEx66[ges1p::3×HA:BioID::unc-54, myo-2p:mCherry::unc-54]), JLF290
surviving adult animals after undergoing ~10 days of development (from ferti- (wowEx67[ges1p::3×HA:BioID::unc-54, myo-2p::mCherry::unc-54]), JLF291
lized egg through pupal stages) expressing UAS-ligase under Gal4 control, and (wowEx68[ges1p::3×HA::TurboID::unc-54, myo-2p::mCherry::unc-54]),
compared to the number of wild-type siblings. UAS-Luciferase was used as a JLF292 (wowEx69[ges1p::3×HA::TurboID::unc-54, myo-2p::mCherry::unc-
negative control transgene, which is widely considered as non-toxic to cells. 54]), JLF293 (wowEx70[ges1p::3×HA::miniTurbo::unc-54, myo-2p:mCherry::
As an example, the following crossing scheme was used for Act5c-Gal4: unc-54]), JLF294 (wowEx71[ges1p::3×HA::miniTurbo::unc-54, myo-2p:
P0 Act5c-Gal4/CyO × UAS-V5-ligase (homozygous) mCherry::unc-54]).
Segregation of the Act5c-Gal4 chromosome and CyO balancer chromosome
results in two possible F1 progeny genotypes: Transgenic ligase strain construction for C. elegans. C. elegans codon-
F1 (genotype 1) Act5c-Gal4/UAS-V5-ligase optimized ligase genes BioID and TurboID (containing the 3 worm introns
F1 (genotype 2) CyO/UAS-V5-ligase present in GFP) and miniTurbo (containing 2 worm introns present in GFP)
The CyO chromosome has a dominant Cy mutation that causes adult flies were synthesized (IDT) and inserted into pJF241 to produce plasmids pAS28,
to have curly wings. Therefore genotype 1 flies have straight wings and express pAS31, and pAS32, respectively. Transgenic worms were generated by inject-
the ligase transgene, and genotype 2 have curly wings and do not express the ing 50ng/µL ligase gene and 2.5ng/µL of the co-injection marker myo-2p::
transgene. The fraction of surviving flies expressing a given UAS-transgene mCherry into day 1 N2 hermaphrodites.
is calculated as: # genotype 1/(# genotype 1 + # genotype 2)
For example, a survival fraction of 0.5 indicates that equal numbers of Western blotting of C. elegans adults. Ligase expression and biotinylation
genotype 1 and genotype 2 were observed in the adult population, and that (Fig. 3i, Supplementary Fig. 15g) were assessed by Western blotting one day-
no reduction in survival from expressing a UAS-transgene during develop- old adult worm lysates. For each condition, 50 N2 hermaphrodites (wild-
ment occurred. type) or worms expressing a ligase transgene were transferred to Eppendorf
Similar crossing schemes were used for tissue-specific Gal4 lines. Gal4 lines tubes containing 1mL of M9 and washed once. Excess M9 was removed until
that are normally maintained as a homozygous stock were first outcrossed to ~50 µL of M9 remained and an additional 50 µL of 4× sample buffer was
an appropriate balancer line to obtain Gal4/Balancer flies, which were then added. Worms were boiled at 95 °C for 10 min, vortexed 10 seconds, and cen-
crossed with UAS-Luciferase or UAS-TurboID. Gal4 lines on chromosome II trifuged at 13,000×g for 5 min at 4 °C. Equal volume of lysate was loaded onto a
were used with a CyO balancer, and Gal4 lines on chromosome III were used 4-20% Mini-PROTEAN TGX PAGE gel (Bio-Rad), transferred to a nitrocel-
with a TM3, Sb balancer. lulose membrane (0.4 µm, Bio-Rad), and stained with Ponceau S solution.
Adult flies were aged >3 days after eclosion from pupal cases before being Blots were blocked in 5% milk PBST solution, probed with anti-HA (1:5000,
counted. Females and males of the same genotype were counted together. rat monoclonal, Roche) and anti-tubulin (1:5000, rat monoclonal, Abcam)
For imaging whole adults, flies were frozen at -20 °C overnight and images primary antibodies, and detected with secondary antibody (1:5000, goat anti-
of adult flies were obtained using a dissection microscope connected to a rat IRDye 680RD, Licor) and streptavidin-IRDye (1:5000, 800CW, Licor). Blots
digital camera. were imaged on LI-COR Odyssey CLx.
To determine if changes in the fraction of surviving flies were statistically
significant, a two-sided Chi-square test was applied to the number of adult Immunohistochemistry and microscopy of C. elegans. To visualize ligases
flies for genotype 1 and genotype 2, comparing UAS-Luciferase to a UAS-ligase and biotinylation (Fig. 3j), embryos were isolated from one day-old adults,
transgene. fixed, and stained as previously described61. Briefly, embryos were attached to
poly-lysine coated microscope slides with Teflon spacers. Slides were frozen
Mammalian cell viability assays. For each experiment presented in on dry ice and embryos were permeabilized by freeze-crack and fixed in 100%
Supplementary Figure 14, five sterile, white, clear bottom 96-well plates were MeOH for 5 min at −20 °C. Embryos were washed in PBS then PBST, and sub-
pre-coated with 100 µL 50 µg/mL human fibronectin in MEM for at least sequently incubated in anti-HA primary antibody (Abcam, 1:200) overnight
20 min at 37 °C under 5% CO2. For transiently transfected samples, HEK 293T at 4 °C to visualize ligase expression. Embryos were washed in PBST and then
cells were plated in 6-wells and transfected at ~90% confluency using 0.8 µL incubated in CY3-anti-mouse secondary antibody (Jackson Immunoresearch
Lipofectamine2000 (Life Technologies) and 200 ng plasmid in serum-free Laboratories, 1:200) Streptavidin Alexa Fluor 488 (Invitrogen, 1:200), and

DAPI (Sigma, 1:10000). Embryos were mounted in Vectashield (Vector to right: 512, 586, 466, 563, 286, 524, 513, 459. 95% confidence intervals (CI)
Laboratories) and stored at 4°C. Samples were imaged using 405 nm, 488 for the difference in proportions of BioID, TurboID, and miniTurboID com-
nm, and 561 nm lasers, a Yokogawa X1 confocal spinning disk head, and a pared to Luciferase were -0.02945 to 0.09046, 0.2757 to 0.384, and -0.02958
60× PLAN APO oil objective (NA=1.4) on a Nikon Ti-E inverted microscope to 0.09206 for normal food, and -0.04278 to 0.104, -0.05999 to 0.08728, and
(Nikon Instruments) equipped with a 1.5× magnifying lens. Images were -0.07191 to 0.07838 for biotin food. p-values for all datapoints - Columns 1/2:
captured using NIS Elements software (Nikon) and an Andor Ixon Ultra 0.3113, Columns 1/3: <0.0001, Columns 1/4: 0.3027, Columns 5/6: 0.4109,
back thinned EM-CCD camera, at a sampling rate of 0.5µm. All samples Columns 5/7: 0.718, Columns 5/8: 0.9369. This experiment was performed
were imaged with the same camera and laser settings, with the exception of twice with similar results. Supplementary Figure 13f, Data was analyzed as
embryos expressing miniTurbo. To avoid pixel saturation, a 25% reduction in Supplementary Figure 13d. Sample size values (n) from left column to
in camera exposure was used to capture the streptavidin-AF488 signal in right: 350, 367, 196, 339, 203, 284, 194, 287, 214, 232, 215, 305, 240, 346. 95%
miniTurbo expressing embryos. Thus, the miniTurbo images and quanti- confidence intervals (CI) for each Gal4 line from left to right were -0.06874
fications in Figure 3 for streptavidin-AF488 are an underrepresentation of to 0.0807, 0.04977 to 0.2228, -0.07546 to 0.1081, -0.03849 to 0.148, - 0.07737
the signal resulting from biotinylation. Images were processed and assem- to 0.1115, 0.1398 to 0.3063, -0.07858 to 0.08951. p-values for all datapoints
bled in NIS Elements and Adobe InDesign. In Figure 3j, images shown are - Oenocytes: 0.8719, Glia: 0.0017, Muscle: 0.7295, Fat body: 0.2333, Neurons:
maximum intensity projections of two Z-slices with brightness adjusted for 0.7143, Gut: <0.0001, Hemocytes: 0.8917. This experiment was performed
visual clarity. twice with similar results.
Figure 3i, experiment was repeated 5 times with similar results, with the
Quantitation of fluorescence signal intensity in C. elegans intestine. Bean exception that miniTurbo expression was detectable in only 2 of those 5
and comma stage embryos were chosen for analyses. For each embryo, one replicates. Embryo imaging results shown in Figure 3j and Supplementary
slice of the Z-stack was used for analysis. A custom Python script including the Figure 15a are representative images of complete quantitative data shown in
modules scikit-image, matplotlib, and NumPy combined with ImageJ was used Figure 3k. Figure 3k samples sizes (n) from left column to right column are
to analyze C. elegans imaging data. A threshold for the HA:ligase signal was 26, 18, 11, 16, 25, 8, 19, 23, 14, 14, 23, 9. Statistical significance was assessed
calculated by the Otsu method to create a mask to isolate the intestine region via Mann-Whitney U test (two-sided). Error bars were calculated using
for each embryo. Background streptavidin-AF488 signal for each embryo s.e.m. Supplementary Figure 15c–f, statistical significance was assessed via
was determined by drawing a square in the anterior portion of the embryo Mann-Whitney U test (two-sided). Error bars were calculated using s.e.m.
outside of the intestine and calculating the average pixel intensity within that Supplementary Figure 16a–c, sample sizes (n) are indicated within an in col-
square. The average background pixel intensity of streptavidin-AF488 was umn titled ‘Replicates’. Statistical significance was assessed via Mann-Whitney
then subtracted from the average streptavidin-AF488 pixel intensity within U test (two-sided). Error bars were calculated using s.e.m.
the isolated intestine region, and the resulting corrected average was plotted
for each embryo (Fig. 3k). To measure the ratio of streptavidin-AF488 to HA: For further detail on the experimental design, reagents, statistics, reproduc-
ligase pixel intensities, each pixel value for streptavidin-AF488 within the ibility, software, and data collection methods used in this study, please refer
isolated intestine region was corrected for background and then divided by its to the Life Sciences Reporting Summary.
corresponding HA:ligase pixel value. Then the average of the ratio values for
each embryo was plotted (Supplementary Fig. 15). Statistical significance was Code availability. The Python script used for C. elegans imaging analysis is
determined using the Mann-Whitney U test (Fig. 3k). Samples were blinded available from the corresponding author upon reasonable request.
for statistical analysis.
Data availability. Source data for Figure 2e and Supplementary Figures 8c–g
Quantitation of C. elegans viability in Supplementary Fig. 16. C. elegans are provided in the paper in Supplementary Table 5. Source data for Figure 2h
strains expressing ligase variants were maintained at 20 °C on either biotin+ and Supplementary Figure 9f–k are provided in the paper in Supplementary
(OP50) or biotin- (MG1655(bioB:kan)) E. coli. For BioID and TurboID in Tables 6 and 7. The original mass spectra may be downloaded from MassIVE
each bacteria condition, a one-day old adult worm expressing the ligase trans- (http://massive.ucsd.edu) using the identifier: MSV000082304. The data is
gene from an extrachromosomal array and a sibling one-day old adult worm directly accessible via ftp://massive.ucsd.edu/MSV000082304. Any additional
lacking the transgene (control) were placed on separate plates containing the data that support the findings of this study are available from the corresponding
appropriate bacteria. For each plate, the adult was removed after laying eggs author upon reasonable request.
for 4 hours and the remaining embryos on the plate were counted. Three days
later the number of living worms were counted and viability was calculated
38. Weaver, L.H., Kwon, K., Beckett, D. & Matthews, B.W. Corepressor-induced
by dividing the number of hatched worms by the number of eggs that were organization and assembly of the biotin repressor: a model for allosteric activation
initially laid. Worms were kept on biotin+ or biotin- bacteria for two genera- of a transcriptional regulator. Proc. Natl. Acad. Sci. USA 98, 6045–6050 (2001).
39. Chao, G. et al. Isolating and engineering human antibodies using yeast surface
tions. Developmentally delayed worms were defined as worms that were larval display. Nat. Protoc. 1, 755–768 (2006).
stage or non-gravid at the time of counting. 40. Jan, C.H., Williams, C.C. & Weissman, J.S. LOCAL TRANSLATION. response to
comment on “Principles of ER cotranslational translocation revealed by proximity-
specific ribosome profiling”. Science 348, 1217 (2015).
Statistics. Figure 3d,e, wing disc imaging results are representative of at least 41. Colby, D.W. et al. Engineering antibody affinity by yeast surface display. Methods
10 discs present on the microscope slide, and at least 3 of which were imaged. Enzymol. 388, 348–358 (2004).
42. Ausubel, F.M. et al. Current protocols in molecular biology. Mol. Biol. 1, 13.2.1 (2003).
Sample sizes (n) in e from left column to right are 5, 6, 3. Error bars were cal-
43. Wood, Z.A., Weaver, L.H., Brown, P.H., Beckett, D. & Matthews, B.W. Co-repressor
culated using s.e.m. This experiment was performed twice with similar results. induced order and biotin repressor dimerization: a case for divergent followed by
Supplementary Figure 13c, sample sizes (n) from left column to right are 17, convergent evolution. J. Mol. Biol. 357, 509–523 (2006).
44. Xu, Y. & Beckett, D. Evidence for interdomain interaction in the Escherichia coli
14, 17, 15, 19, 18, 19, 18. Error bars were calculated using s.e.m. This experi- repressor of biotin biosynthesis from studies of an N-terminal domain deletion
ment was performed twice with similar results. Supplementary Figure 13d, mutant. Biochemistry 35, 1783–1792 (1996).
for each food type, a two-sided Chi-square test was used to determine if the 45. Eginton, C., Cressman, W.J., Bachas, S., Wade, H. & Beckett, D. Allosteric coupling
via distant disorder-to-order transitions. J. Mol. Biol. 427, 1695–1704 (2015).
difference in proportions (measure of effect) of UAS-ligase transgenes to UAS- 46. Hung, V. et al. Spatially resolved proteomic mapping in living cells with the
Luciferase was statistically significant. Sample size values (n) from left column engineered peroxidase APEX2. Nat. Protoc. 11, 456–475 (2016).

47. Apweiler, R. et al. UniProt: the universal protein knowledgebase. Nucleic Acids 55. Pagliarini, D.J. et al. A mitochondrial protein compendium elucidates complex I
Res. 45, D158–D169 (2017). disease biology. Cell 134, 112–123 (2008).
48. Käll, L., Krogh, A. & Sonnhammer, E.L.L. A combined transmembrane topology 56. Sultan, M. et al. A global view of gene activity and alternative splicing by deep
and signal peptide prediction method. J. Mol. Biol. 338, 1027–1036 (2004). sequencing of the human transcriptome. Science 321, 956–960 (2008).
49. Gene Ontology Consortium. Gene Ontology Consortium: going forward. Nucleic Acids 57. Perkins, L.A. et al. The transgenic RNAi project at Harvard medical school: resources
Res. 43, D1049–D1056 (2015). and validation. Genetics 201, 843–852 (2015).
50. Ashburner, M. et al.; The Gene Ontology Consortium. Gene ontology: tool for the 58. Markstein, M., Pitsouli, C., Villalta, C., Celniker, S.E. & Perrimon, N. Exploiting
unification of biology. Nat. Genet. 25, 25–29 (2000). position effects and the gypsy retrovirus insulator to engineer precisely expressed
51. Thul, P.J. et al. A subcellular map of the human proteome. Science 356, eaal3321 transgenes. Nat. Genet. 40, 476–483 (2008).
(2017). 59. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77,
52. Muthusamy, B. et al. Plasma Proteome Database as a resource for proteomics 71–94 (1974).
research. Proteomics 5, 3531–3536 (2005). 60. Ortega-Cuellar, D. et al. Biotin starvation with adequate glucose provision causes
53. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E.L.L. Predicting paradoxical changes in fuel metabolism gene expression similar in rat (Rattus
transmembrane protein topology with a hidden Markov model: application to norvegicus), nematode (Caenorhabditis elegans) and yeast (Saccharomycces
complete genomes. J. Mol. Biol. 305, 567–580 (2001). cerevisiae). J. Nutrigenet. Nutrigenomics 3, 18–30 (2010).
54. Calvo, S.E., Clauser, K.R. & Mootha, V.K. MitoCarta2.0: an updated inventory of 61. Leung, B., Hermann, G.J. & Priess, J.R. Organogenesis of the Caenorhabditis
mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2016). elegans intestine. Dev. Biol. 216, 114–134 (1999).

nature research | reporting summary
Corresponding author(s): Alice Ting NBT-TR43271B
Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency
in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.
Statistical parameters
When statistical analyses are reported, confirm that the following items are present in the relevant location (e.g. figure legend, table legend, main
text, or Methods section).
n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement
An indication of whether measurements were taken from distinct samples or whether the same sample was measured repeatedly
The statistical test(s) used AND whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested

A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons
A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND
variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted
Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes
Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated
Clearly defined error bars

State explicitly what error bars represent (e.g. SD, SE, CI)
Our web collection on statistics for biologists may be useful.
Software and code

Policy information about availability of computer code
Data collection ImageStudioLite was used to process Western blots imaged with Licor. UVP BioSpectrum Imaging System was used to acquire Western
blots imaged with Clarity Western ECL Blotting Substrates (BioRad). Slidebook 6.0 was used to collect mammalian cell imaging data. BD
CSampler software was used to acquire the FACS data.
Data analysis Slidebook 6.0 was used to analyze mammalian cell imaging data. ImageJ 1.50i was used to quantify Western blot data, Drosophila
imaging data, and to measure Drosophila wing area. Prism 7.0 was used to analyze numerical data. BD FACSDIVA v8.0 was used to
analyze the FACS data. A custom Python script including the modules scikit-image, matplotlib, and NumPy combined with ImageJ was
used to analyze C. elegans imaging data. The script can be made available upon request. R statistical programming language and ggplot2
were used to analyze and plot numerical data for C. elegans data.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers
April 2018
upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.
1
Nature Biotechnology: doi:10.1038/nbt.4201
Data

Policy information about availability of data
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:
- Accession codes, unique identifiers, or web links for publicly available datasets
- A list of figures that have associated raw data
- A description of any restrictions on data availability
Source data for Figure 2e and Supplementary Figures 8c-g are provided in the paper in Supplementary Table 25. Source data for Figure 2h and Supplementary
Figures 9f-k are provided in the paper in Supplementary Tables 64 and 76. The original mass spectra may be downloaded from MassIVE
(http://massive.ucsd.edu) using the identifier: MSV000082304. The data is directly accessible via ftp://massive.ucsd.edu/MSV000082304. Any additional data that
support the findings of this study are available from the corresponding author upon reasonable request.
Field-specific reporting
Please select the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/authors/policies/ReportingSummary-flat.pdf
Life sciences study design

All studies must disclose on these points even when the disclosure is negative.
Sample size All mammalian cell imaging results presented were representative of at least 10 independent fields of view. Sample size of mammalian cell
image fields of view was chosen in consistency with many other publications (e.g. PMID: 28650461). Drosophila fly wing disc imaging results
are representative of at least 10 discs present on the microscope slide, at least 3 of which were imaged. Full information on sample size in
figure legend and methods. Sample size of imaged wing discs was chosen as the maximum number of samples processed at the same time
without sacrificing consistency of tissue fixation, antibody staining, and slide mounting. The number of adult flies counted for survival assays
was performed to maximize accuracy, at least 286 adult flies/sample were counted. Full information on sample size in figure legend and
methods. Sample size of counted surviving adults was chosen as the maximum possible number of progeny flies collected from a single cross
to control for variables such as food quality. The number of flies counted is consistent with published protocols on quantifying survival in
Drosophila (PMID: 24751824). Measurements of wing size from adult flies were performed on at least 14 wings. Full information on sample
size in figure legend and methods. Images and quantification of Drosophila flies and tissues were representiverepresentative of results.
Sample size is indicated in Figure 3 and Supplementary Figure 12 legends. Sample size of wings were chosen as the maximum number of
wings processed at the same time. The number of wings quantified is consistent with many other publications (e.g. PMID: 25107277).
For all C. elegans experiments, full information on sample size and replicates are within figures, figure legends, and/or and methods. Images
and quantifications shown in Figure 3 and Supplementary figures and qualifications are representative of results. For consistency, two specific
stages of worm embryonic development were chosen for IF staining. Sample size of embryos imaged were chosen as the maximum number of
samples processed at the same time.
Data exclusions For consistency, a specific stage of worm development was chosen (embryos).
Embryos within that stage were chosen for quantitative analysis.
Replication The number of times an experimental finding was reproduced is indicated in the
associated figure legend for each experiment shown. For Figure 3i, the experiment was performed five times (n = 5). In biotin+ conditions,
BioID biotinylation activity was undetectable and TurboID gave robust biotinylation signal in all replicates (n = 5/5). Despite high activity
detected by immunofluorescence in embryos, we only detected some low level of biotinylation by miniTurbo in adults (n = 2/5), likely due to
its low expression levels. For all other experiments, attempts at replication were successful.
Randomization No randomization methods were used because this was not applicable for our experiments.Randomization of adult flies or larvae was not
used because freely-moving animals are too small to be assigned numbers for subsequent random selection. Fly wing discs and adult wings
were selected for imaging randomly, and adult flies were selected for lysis and western blotting randomly. Randomization does not apply to
counting survival of adult flies since all flies are counted. Worm embryos were selected for imaging randomly, and adult worms were selected
randomly for lysis and subsequent Western blotting. Randomization does not apply to counting survival and developmental delay of worms
since all worms in these experiments were counted.
Blinding Investigator was blinded to group allocation during statistical analyses for C.
elegans experiments.
April 2018
Reporting for specific materials, systems and methods
2
Materials & experimental systems Methods

n/a Involved in the study n/a Involved in the study
Unique biological materials ChIP-seq
Antibodies Flow cytometry
Eukaryotic cell lines MRI-based neuroimaging
Palaeontology
Animals and other organisms
Human research participants
Antibodies
Antibodies used antibody, vendor, catalog number/lot number: mouse anti-myc, Calbiochem, OP10/D00158557; goat anti-mouse-HRP, BioRad,
170-6516; chicken anti-myc, Invitrogen, A-21281/1819879; goat anti-chicken-AlexaFluor647, Invitrogen, A21449/1081817;
rabbit anti-biotin, ImmuneChem, ICP0611; goat anti-rabbit-R-phycoerythrin, Life Technologies, P2771MP; mouse anti-V5,
Invitrogen, 46-0705/1847319/1865511; mouse anti-calreticulin, Calbiochem, 208912/D00154827; mouse anti-BCAP31,
Proteintech, 11200-1-AP/00018537; rabbit anti-Tom70, Proteintech, 14528-1-AP/00005668; mouse anti-Tom20, Santa Cruz
Biotechnology, sc-17764/B2117; mouse anti-HXK I, Santa Cruz Biotechnology, sc-46695/F1611; rabbit anti-NDUFS6, Abcam,
ab195808; rabbit anti-calnexin, Santa Cruz Biotechnology, sc-11397/I1914; rabbit anti-Tom20, Santa Cruz Biotechnology,
sc-11415/12711; goat anti-rabbit-AlexaFluor568, Invitrogen, A-11011/1704462; mouse anti-His6, Calbiochem, OB05; goat anti-
mouse-AlexaFluor647, Invitrogen, A21236; goat anti-mouse-AlexaFluor800, Invitrogen, A32730/RJ243418; mouse anti-HA,
Abcam, ab130275/GR250145-5; goat anti-mouse-CY3, Jackson Immunoresearch Laboratories, 115-165-166/117091; rat anti-
alpha tubulin, Abcam, ab6161; rat anti-HA, Roche, 11867423001; goat anti-rat IRDye 680RD, LI-COR, 92568076/C61115-06.
Validation mouse anti-myc (Calbiochem, OP10): Epitope Tagging - Munro, S., et al. 1986. Cell 46, 291. Immunoblotting - Evan, G.I., et al.
1985. Mol. Cell. Biol. 5, 3610; chicken anti-myc (Invitrogen, A-21281): several published flow cytometry applications cited on
website - https://www.thermofisher.com/antibody/product/Myc-Tag-Antibody-Polyclonal/A-21281; rabbit anti-biotin
(ImmuneChem, ICP0611): Udeshi et al in Nature Methods Vol.14 pages: 1167-1170 (2017); mouse anti-V5 (Invitrogen, 46-0705):
several published immunofluorescence and Western blotting applications cited on website - https://www.thermofisher.com/
antibody/product/V5-Tag-Antibody-Monoclonal/R960-25; mouse anti-calreticulin (Calbiochem, 208912): Nauseef, W.M., et al.
1995. J. Biol. Chem. 270, 4741. Jethmalani, S.M., et al. 1994. J. Biol. Chem. 269, 23603. Smith, M.J., and Koch, G.L.E. 1989. EMBO
J. 8, 3581; mouse anti-BCAP31 (Proteintech, 11200-1-AP): validation shown on website - https://www.ptglab.com/products/
BCAP31-Antibody-11200-1-AP.htm#validation; rabbit anti-Tom70 (Proteintech, 14528-1-AP): validation shown on website -
https://www.ptglab.com/products/TOM70-Antibody-14528-1-AP.htm#validation; mouse anti-Tom20 (Santa Cruz Biotechnology,
sc-17764): several published applications cited on website - https://www.scbt.com/scbt/product/tom20-antibody-f-10; mouse
anti-HXK I (Santa Cruz Biotechnology, sc-46695): several published applications cited on website - https://www.scbt.com/scbt/
product/hxk-i-antibody-g-1?requestFrom=search; rabbit anti-NDUFS6 (Abcam, ab195808): validation shown on website - http://
www.abcam.com/ndufs6-antibody-epr15957-37-ab195808.html; rabbit anti-calnexin (Santa Cruz Biotechnology, sc-11397):
several published applications cited on website - https://www.scbt.com/scbt/product/calnexin-antibody-h-70?
requestFrom=search; rabbit anti-Tom20 (Santa Cruz Biotechnology, sc-11415): several published applications cited on website -
https://www.scbt.com/scbt/product/tom20-antibody-fl-145; mouse anti-His6 (Calbiochem, OB05): Zentgraf, H., et al. 1995. Nucl.
Acids. Res. 23, 3347. Gu, J., et al. 1994. BioTechniques 17, 257. Sporeno, E., et al. 1994. J. Biol. Chem., 10991. Sisk, W.P., et al.
1994. J. Virol. 68, 766. Lu, T., et al. 1993. Anal. Biochem. 213, 318. Garner, J., et al. 1992. Cell 69, 833. Hochuli, E., et al. 1987. J.
Chromatogr. 411, 177; mouse anti-HA (Abcam, ab130275): several published applications cited on website - http://
www.abcam.com/ha-tag-antibody-16b12-ab130275-references.html; rat anti-alpha tubulin (Abcam, ab6161): several published
applications cited on website - http://www.abcam.com/tubulin-antibody-yol134-microtubule-marker-ab6161-references.html;
rat anti-HA (Roche, 11867423001): several published applications cited on website - https://www.sigmaaldrich.com/catalog/
product/roche/roahaha?lang=en&region=US&gclid=CjwKCAjwma3ZBRBwEiwA-
CsblLDIeASZObnaR5a0aQ7M7VoYIt3zRfhFWajwsOc-lAzvFLlHOW_VzRoCPycQAvD_BwE.
Eukaryotic cell lines

Policy information about cell lines
Cell line source(s) The source of the HEK 293T cell line was ATCC.
Authentication The cell line (HEK293T) was not authenticated.
Mycoplasma contamination The cells were not tested for mycoplasma contamination.
Commonly misidentified lines None. The only cell line used in this paper is HEK293T, which is not listed in the ICLAC
April 2018
(See ICLAC register) database.
Animals and other organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research
Laboratory animals Experiments on flies were performed with wild type or transgenic strains of Drosophila melanogaster. The age and sex of animals
3
Laboratory animals involved in experiments are indicated in figure legends and methods below. Unless otherwise noted, fly stocks were obtained
from the Bloomington Drosophila Stock Center and are listed with the corresponding stock number: ptc-Gal4 (2017), Act5c-Gal4/

CyO (4414), nub-Gal4 (25754), w1118 (6326), tub-Gal80ts; tub-Gal4/TM6b (Perrimon Lab), UAS-Luciferase (35788), Desat-Gal4
(Oenocyte) (65405), repo-Gal4 (Glia) (7415), Mef2-Gal4 (Muscle) (27390), Lpp-Gal4 (Fat body) (Perrimon Lab, see transgene in
67043 for information), elav-Gal4 (Neurons) (8760), Myo1a-Gal4 (Gut) (Perrimon Lab, see transgene in 67057 for information),
Hml-Gal4 (Hemocytes) (30140). All Details on the flies used are provided in the methods section and figure legends.
Caenorhabditis elegans experiments involved embryos or, hermaphrodite adultss. The age of animals involved in experiments
are indicated in figure legends or methods. N2 wild-type strain was obtained from the Caenorhabditis Genetics Center (CGC).
Wild animals None.
Field-collected samples None.
Flow Cytometry
Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
Methodology
Sample preparation All FACS experiments in this study used yeast. Sample preparation is described in detail in the following methods sections: Yeast
cell culture, Generation of BirA libraries for yeast display, Yeast display selections, Directed evolution of TurboID and miniTurbo.
Instrument Yeast display FACS sorting was performed on BD FACS Aria II cell sorter, and all FACS analysis was performed on BD Accuri flow
cytometer.
Software BD FACSDIVA software was used to perform FACS sorts and analyze all data. FACS analysis data (performed on BD Accuri flow
cytometer) was collected using BD CSampler software.
Cell population abundance Percentages of pre- and post-sort cell populations analyzed and sorted are described in detail in the following methods sections:
Yeast display selections, Directed evolution of TurboID and miniTurbo.
Gating strategy Descriptions of the gating strategies used are included in the methods sections "Yeast display selections" and "Directed evolution
of TurboID and miniTurbo" and are exemplified in Supplementary Figure 2j.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.
April 2018
4

10 1038@nbt 4201

Uploaded by

Copyright:

Available Formats

10 1038@nbt 4201

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

10 1038@nbt 4201

Uploaded by

Copyright:

Available Formats

letters

Efficient proximity labeling in living cells and organisms

nature biotechnology advance online publication 

 advance online publication nature biotechnology

c d TurboID (35 kD) N-terminal domain miniTurbo (28 kD)

e R118S G1 G2 G3 G3∆ miniTurbo TurboID

30 min biotin 3 h biotin

nature biotechnology advance online publication 

 advance online publication nature biotechnology

Relative biotinylation activity, normalized

to ligase expression level

c Nucleus Mitochondrial matrix ER membrane ER lumen

d e Secretory pathway specificity

f Subsecretory specificity g ER specificity h Nuclear specificity Mitochondrial specificity

proteome proteome size

80 80 88% 87% 84% 80

60 membrane 60 (lumen) 60 67%

nature biotechnology advance online publication 

 advance online publication nature biotechnology

Fold change signal

j Biotin DAPI Anti-HA Streptavidin-AF488

nature biotechnology advance online publication 

 advance online publication nature biotechnology

nature biotechnology doi:10.1038/nbt.4201

doi:10.1038/nbt.4201 nature biotechnology

nature biotechnology doi:10.1038/nbt.4201

doi:10.1038/nbt.4201 nature biotechnology

nature biotechnology doi:10.1038/nbt.4201

doi:10.1038/nbt.4201 nature biotechnology

nature biotechnology doi:10.1038/nbt.4201

doi:10.1038/nbt.4201 nature biotechnology

nature biotechnology doi:10.1038/nbt.4201

doi:10.1038/nbt.4201 nature biotechnology

doi:10.1038/nbt.4201 nature biotechnology

A description of all covariates tested

Clearly defined error bars

Our web collection on statistics for biologists may be useful.

Software and code

nature research | reporting summary

Life sciences study design

Reporting for specific materials, systems and methods

nature research | reporting summary

Eukaryotic cell lines

Authentication The cell line (HEK293T) was not authenticated.

(See ICLAC register) database.

Animals and other organisms

nature research | reporting summary

Wild animals None.

Field-collected samples None.

You might also like

nature biotechnology advance online publication

advance online publication nature biotechnology

nature biotechnology advance online publication

advance online publication nature biotechnology

nature biotechnology advance online publication

advance online publication nature biotechnology

nature biotechnology advance online publication

advance online publication nature biotechnology