Post-Translational Regulation of Autophagy in Saccharomyces Cerevisiae

Macroautophagy/autophagy is primarily a self-eating process that recycles cytosolic components such as misfolded or aggregated proteins and dysfunctional organelles for homeostasis and survival in unfavorable conditions. This highly conserved and constitutive pathway has to be tightly regulated; either too much or too little autophagy can be detrimental. Dysregulation of this pathway is related to various diseases that include neurodegeneration, cancer and infection, as well as aging-related disorders. Autophagy is stringently regulated at different levels including transcriptionally, post-transcriptionally, translationally and post-translationally. A thorough understanding of the mechanisms involved is crucial to allow the manipulation of autophagy for the treatment of diseases. Although 41 autophagy-related (ATG) genes have been identified, we have a limited understanding of the complex network of regulatory factors that control this process. Atg9 is the only transmembrane protein in the core autophagy machinery (and one of only two Atg membrane proteins in fungi), which is absolutely required for autophagosome biogenesis and autophagy activity. Unlike other Atg proteins, Atg9 has a distinctive feature with regard to its subcellular localization: This protein travels between peripheral sites and the phagophore assembly site (PAS) where the autophagosome is formed, presumably delivering membranes from different donors to the PAS for autophagosome biogenesis. Post-translational modifications (PTMs) including phosphorylation, ubiquitination, glycosylation, methylation, represent a subset of regulatory mechanisms that are critical for modulating autophagy in order to adapt to different types of environmental stress. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27. Besides, we identified a second novel mechanism of Atg9 regulation in autophagy through ubiquitination. In growing conditions, Atg9 is synthesized at a basal level and cells are primed for autophagy. We show that in this situation Atg9 is ubiquitinated and targeted for degradation in a proteasome-dependent manner, thereby limiting autophagy to a basal level. However, when cells are deprived of nutrients, autophagy is highly induced, necessitating an increase in the amount of Atg9; the proteasome-dependent reduction of Atg9 protein levels is subsequently reduced to facilitate the increase in autophagy. Thus, the post-translational ubiquitination of Atg9 provides an additional mechanism that allows cells to maintain appropriate levels of autophagy, and—by ending this modification—to rapidly respond and adapt to environmental stresses.PHDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/144192/1/yuchenf_1.pd