UC Berkeley

Autophagy is a conserved pathway critical for homeostasis in all eukaryotic cells. Autophagy provides a mechanism for cells to respond to a large variety of cellular stresses through a vesicle-based degradation process. Most of the gene products and other factors required for the execution of autophagy have been elucidated, but the biochemical reactions by which these components work in concert to engulf cargo in a double membrane vesicle, called an autophagosome, is still unclear. In mammalian cells, the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is essential for early steps in autophagy because it generates a membrane lipid, phosphatidylinositol 3-phosphate (PtdIns3P), that is necessary to recruit downstream autophagy factors to cellular membranes. However, it was not known whether the PI3KC3-C1 also contributes to phagophore formation in other ways.

The research described in this dissertation focused on one of the subunits of PI3KC3-C1, Atg14L, which is essential for the complex to initiate autophagy. I used an in vitro reconstitution assay where I added back purified PI3KC3-C1 or various mutant derivatives of the complex to extracts derived from a Cas9-generated Atg14L-deficient cell line. As a biochemical readout for examining the functions of Atg14L, I used a cell-free reaction that reproduces a key early step in autophagosome membrane generation, lipidation of a cytoplasmic protein, LC3. In this assay, I found that Atg14L (and the other subunits of PI3KC3-C1) are required for LC3 lipidation, whereas mutants of the complex that abrogate its catalytic activity and membrane curvature-sensing capability did not support LC3 lipidation. In addition, PI3KC3-C1 activity, through Atg14L and its membrane curvature- sensing motif, is required for efficient membrane recruitment of the downstream PtdIns3P-binding effector, WIPI2, but not for recruitment of Atg16L, a key factor required for LC3 lipidation.

In an effort to find other cofactors that may also regulate PI3KC3-C1, I found that NRBF2 binds Atg14L and attenuates its autophagy-stimulating function and, hence, serves as a negative regulator of autophagy. Under fed conditions, however, NRBF2 is phosphorylated; phosphorylation null mutants show elevated autophagy under fed conditions. In another study, I uncovered that Atg14L acts as a vesicle tether that supports vesicle fusion when soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are incorporated into the vesicle. The fusogenic activity of Atg14L requires oligomerization through conserved cysteine repeats near the N-terminus of Atg14L.