Autophagy is a highly regulated cellular degradation system that engulfs cytosol, damaged organelles, protein aggregates and invading microorganisms into a double-membrane vesicle termed autophagosome that delivers cargoes to endolysosomes for degradation. Dysfunction of autophagy has been implicated in a broad spectrum of human diseases including cancers. It is still largely unknown how this process is regulated biochemically. One unsolved question in autophagy is how autophagosome fuses with lysosome. Genetic analysis suggests important roles of multiple SNARE proteins in the autophagic membrane fusion, however, whether these SNAREs function as fusogens and how their fusogenic activities are regulated in an autophagy specific manner is unknown. We found that autophagic SNAREs Syntaxin17 (STX17)-SNAP29-Vamp8 assemble into a fusion competent four-helices bundle and functions as a basal fusion machinery, their assembly on autophagosomes, as well as the fusogenic activity, is promoted by autophagosome membrane binding protein ATG14. ATG14 colocalizes with STX17 on the complete autophagosome where the oligomerized ATG14 physically interacts with STX17-SNAP29 SNARE binary complex to promote autophagosome fusion with lysosome. This regulated membrane fusion in vitro recapitulates stress induced autophagosome fusion with lysosome in vivo. Our preliminary data also suggest that the retrieval of autophagic SNAREs from autolysosomes is likely mediated by TECPR1 through its interaction with STX17. We therefore hypothesize that the autophagic membrane fusion is tightly controlled by the core STX17-SNAP29-VAMP8 SNARE complex and two SNARE-binding proteins ATG14 and TECPR1. In this study, we aim to determine how ATG14 regulates STX17-SNAP29-VAMP8 mediated membrane fusion in our state-of-the-art biochemical and genetic assays (Aim 1). The recruitment and retrieval of autophagic proteins on mature autophagosomes (complete autophagosome and autolysosome) is scarcely studied. We will utilize the autophagic fusogenic proteins as readouts to study the mechanism of the trafficking to and the retrieving away from the mature autophagosomes by comprehensive cell biology and biochemical approaches (Aim 2). At last, our study suggests that autophagic membrane fusion is tightly regulated. ATG14 homo-oligomerization plays a crucial role in mediating in its binding to autophagic SNAREs. We will utilize biochemical and structural biology approaches to investigate the regulatory mechanism in autophagic membrane tethering/fusion (Aim 3). These studies will provide new insights into the molecular mechanism of membrane fusion in autophagy and help us to design new classes of drugs for the treatment of human diseases caused by autophagy dysfunction.
Dysfunction of autophagy has been implicated in a broad spectrum of human diseases including cancers, neurodegeneration diseases, infectious diseases and metabolic diseases. This project investigates the molecular and biochemical mechanisms underlying membrane fusion in autophagy, which will provide novel insights into the core autophagy fusion machinery and this knowledge will help us to design new chemicals to regulate autophagy specifically and set up foundation for drug discovery for autophagy involved human diseases.
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