Analysis of the Molecular Mechanism of Autophagosome Formation in the Yeast Saccharomyces Cerevisiae.

Abstract

As a lysosomal/vacuolar degradative pathway conserved in eukaryotic organisms, autophagy mediates the turnover of long-lived proteins and excess or aberrant organelles. During autophagy, a double-membrane vesicle, the autophagosome, envelops part of the cytoplasm and delivers it to the lysosome/vacuole for breakdown and eventual recycling of the degradation products. Genetic screening in yeast has led to the identification of over 30 autophagy-related (ATG) genes and most of them are involved in the autophagosome formation process. In Saccharomyces cerevisiae, most Atg proteins reside at the phagophore assembly site (PAS), which is involved in autophagosome biogenesis. One reason of our limited understanding of the PAS function is the lack of stoichiometric information regarding the Atg proteins at this site. In this dissertation, we describe a fluorescence microscopy-based method to study the quantitative properties of Atg proteins at the PAS. We found that in response to autophagy induction, the amount of most Atg proteins remains unchanged at the PAS whereas we see an enhanced recruitment of Atg8 and Atg9 at this site. This method is applicable to many related studies and will provide a new way to study the property and functions of Atg proteins. Another essential issue in autophagy that has not been resolved is the origin of the lipids that form the autophagosome. We showed that two post-Golgi proteins, Sec2 and Sec4, are required for autophagy. The known function of Sec2 and Sec4 is to direct secretory vesicles from the Golgi to the secretion site on the plasma membrane. In sec2 and sec4 conditional mutant yeast, the anterograde movement of Atg9, a proposed membrane carrier, is impaired during starvation conditions. Similarly, in the sec2 mutant, Atg8 is inefficiently recruited to the PAS, resulting in the generation of fewer autophagosomes. We propose that following autophagy induction the function of Sec2 and Sec4 are diverted to direct membrane flow to autophagosome formation. In summary, most of the work presented in the dissertation is related to the autophagosome formation process and its regulation mechanism. It can inspire new questions and will be the cornerstone for future analysis.