Analysis of the Molecular Mechanism of Autophagosome Formation in Yeast and Zebrafish Models.

Abstract

Autophagy is a conserved intracellular degradative pathway induced by various stress or developmental signals in eukaryotes, and its malfunction contributes to a variety of diseases. During autophagy, cargos such as cytosolic proteins, damaged organelles and invasive pathogens are engulfed into double-membrane autophagosomes, transported to, and degraded in the lysosome/vacuole. Over 30 ATG (autophagy-related) genes have been identified in the budding yeast S. cerevisiae. To understand the molecular mechanism controlling membrane delivery during autophagy, I studied protein interactions involving Atg9, the only known transmembrane protein required for autophagosome formation. In yeast, Atg9 cycles between peripheral sites and the phagophore assembly site (PAS), suggesting its role in supplying membrane for autophagosome nucleation and expansion. Through a yeast two-hybrid screen aimed to find interaction partners of Atg9, I identified Atg11, a component involved in autophagic cargo recognition. Subsequently, I demonstrated that Atg11 mediates Atg9 movement to the PAS along the actin cytoskeleton. Thus, my model suggests that the anterograde transport of Atg9 to the PAS mediated by Atg11 may serve as a membrane shuttle for autophagosome biogenesis. Furthermore, I characterized the self-interaction of Atg9 and generated an Atg9 mutant defective in this interaction. This mutation results in abnormal autophagy, due to altered phagophore formation as well as inefficient membrane delivery to the PAS. Based on my analyses, I propose a model suggesting dual functions for the Atg9 complex: by reversibly binding to another Atg9 molecule, Atg9 can both promote lipid transport from the membrane origins, and help assemble an intact phagophore membrane. I also extended my analysis on autophagy in the zebrafish model, which represents a unique system to study autophagy due to its rapid embryonic development and technical advantages in high-throughput drug screens. I identified two zebrafish Atg8 (an autophagosome marker protein) homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing GFP-tagged versions of the corresponding proteins. I observed a high level of autophagy activity in zebrafish embryos, which can be further upregulated by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. Thus, I established a convenient zebrafish tool to assay autophagic activity during embryogenesis in vivo.