Studies of the molecular mechanism and signaling regulation of autophagy in Saccharomyces cerevisiae.

摘要

Autophagy is a highly conserved cellular degradation process in which portions of cytosol and organelles are sequestered into a double-membrane vesicle, an autophagosome, and delivered into a degradative organelle, the vacuole/lysosome, for breakdown and eventual recycling of the resulting macromolecules. Malfunction of autophagy has been linked to a wide range of human pathologies, including cancer, neurodegeneration and pathogen infection. Identification of many autophagy-related, ATG, genes in yeast that are essential to drive this cellular process, and the finding of orthologs in other organisms, reveals the conservation of the autophagic machinery in all eukaryotes. In addition to this, complex signaling cascades controlling autophagy have also begun to emerge, with TOR as a central but far from exclusive player. In this thesis, (1) we summarize our current knowledge about the machinery and molecular mechanism of autophagy. (2) We highlight the recent advances in identifying and understanding the core molecular machinery and signaling pathways that are involved in mammalian autophagy. (3) We elucidate a molecular mechanism for linking the degradative and recycling roles of autophagy. We show that in contrast to published studies Atg22 is not directly required for the breakdown of autophagic bodies within the lysosome/vacuole. Instead, we demonstrate that Atg22, Avt3 and Avt4 are redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagic degradation. The release of autophagic amino acids allows the maintenance of protein synthesis and viability during nitrogen starvation. We propose a "recycling" model that includes the efflux of macromolecules from the lysosome/vacuole as the final step of autophagy. (4) We used genetic analyses to elucidate the mechanism by which the stress-responsive, cyclin-dependent kinase, Pho85 and its corresponding cyclins antagonistically modulate autophagy in Saccharomyces cerevisiae . When complexed with cyclins Pho80 and Pc15, Pho85 negatively regulates autophagy through downregulating the protein kinase Rim15, and the transcription factors Pho4 and Gcn4. The cyclins Clg1, Pcl1 and Pho80, in concert with Pho85, positively regulate autophagy through promoting the degradation of Sic1, a negative regulator of autophagy that targets Rim15. Our results suggest a model in which Pho85 has opposing roles in autophagy regulation.