Exploring the Regulation and Molecular Mechanism of Autophagy Using Saccharomyces cerevisiae as the Model System

Abstract

Macroautophagy/autophagy is a highly regulated cellular primarily catabolic process, conserved from yeast to more complex eukaryotes. During autophagy, cytoplasmic materials are delivered into the lysosome (or the analogous organelle, the vacuole, in yeast) for degradation by resident hydrolases; the resulting breakdown products are recycled back to the cytoplasm to maintain cellular homeostasis. Under normal nutrient conditions, autophagy occurs at a basal level, which can play a critical role in cell homeostasis. When cells face stimuli or stress, autophagy can be massively upregulated to handle the increased demand. The malfunction of autophagy is associated with various diseases, including cancer, aging, metabolic and neurodegenerative diseases. There has been substantial progress in understanding the regulation and molecular mechanisms of autophagy in different organisms; however, many questions about the most fundamental aspects of autophagy remain unresolved. For example, little is known about the complicated modulation of gene expression and specific transcriptional regulators of autophagy. Meanwhile, we currently have a limited understanding of most steps in the autophagic process. In Chapter II, I examine the question of whether yeast might employ a mechanism of promoter-proximal pausing, similar to that in higher eukaryotes. The promoter-proximal pausing is defined by a temporal halt of transcription within ~100 nucleotides downstream of the transcription start site (TSS). The DRB sensitivity-inducible factor (DSIF, composed of SUPT4H1 and SUPT5H) works with negative elongation factor (NELF) to impart the pausing. Upon specific stimulation, the temporal state of pausing will be reversed, and the status of DSIF can be changed to promote transcription elongation. The budding yeast expresses homologs of SUPT4H1 and SUPT5H, named Spt4 and Spt5, but there is no clear evidence of the presence of NELF. Therefore, most studies have focused on the positive role of the Spt4-Spt5 complex in transcription. Interestingly, our data show that the Spt4-Spt5 complex may act in a gene-specific manner in yeast, with Spt4 having an additional and clear negative role for certain ATG genes under growing conditions. Chapter III further investigates the mechanism of both nonselective and selective autophagy. In budding yeast, autophagosome formation occurs at the phagophore assembly site (PAS), a specific perivacuolar location that works as an organizing center for the recruitment of different autophagy-related (Atg) proteins. However, the PAS is a poorly defined structure, and one unanswered question is what determines the localization of the PAS. The first part in Chapter III reveals that the vacuolar membrane protein Vac8 is required for the correct vacuolar localization of the PAS. We also provide evidence that Vac8 anchors the PAS to the vacuolar membrane by recruiting the Atg1-Atg13 initiation complex. In the second part of Chapter III, we examine another aspect of the dynamic role of intracellular membranes and autophagy machinery, in this case focusing on a type of selective autophagy, pexophagy (specifically degrade surplus or damaged peroxisomes). Recently, studies have implicated the role of membrane contact sites in nonselective autophagy, but no papers had been published on the topic of pexophagy. We found that endoplasmic reticulum (ER)-mitochondria contact sites are necessary for efficient pexophagy, and disruption of the ER-mitochondria encounter structure (ERMES) results in a severe defect in pexophagy. Together the research presented in this dissertation expands our understanding of regulation and molecular mechanism of autophagy using Saccharomyces cerevisiae as the model system.