The regulation of glucose starvation-induced cell surface protein clearance in yeast



 DESCRIPTION (provided by applicant): Correct intracellular membrane traffic ensures that transmembrane, secreted and organelle lumen resident "cargo" proteins are correctly positioned within the cell. Membrane traffic is essential for normal cell physiology. It regulates cell proliferation and behavior by controlling the signaling capacity of cells, their adhesive propertie, and their capacity to internalize nutrients. Defects in traffic are associated with cancer and with cell death during stress. Membrane traffic and its regulation are ancient processes that are highly conserved. Due to its ease of use, the yeast Saccharomyces cerevisiae provides an ideal model system for the study of such conserved pathways. Recently, the Duncan lab described that upon glucose starvation, yeast cells internalize many cell surface proteins and degrade them in the vacuole. This process is essential for cell survival; however, the mechanism that regulates this change in membrane traffic is unknown. Using a candidate approach, I discovered one protein that is important for the internalization and degradation of proteins during glucose starvation. In this proposal, I will test the model that the candidate protein is regulated in a glucose-dependent manner in yeast to drive cell surface protein degradation and ultimately cell survival. My preliminary data shows that the candidate protein reduces the delivery of cargo proteins to the vacuole during glucose starvation but not when glucose is present. The data are consistent with a role for the candidate in endocytosis or in sorting at intracellular compartments. Thus, I will first determine at which membrane the candidate protein acts during glucose starvation using epistasis analysis coupled with biochemistry and fluorescence microscopy. Next, I will determine the molecular mechanism for how this protein is regulated during glucose starvation. I will test if the candidate protein is subject to post-translational modification or if its interactions with binding partners change during glucose starvation. To do this, I will apply approaches such as immuno-blot analysis or GST-pull-down and immuno-precipitation coupled with mass spectrometry to detect and identify changes in post-translational modification and binding proteins during glucose starvation. I will then test th functional significance of the post-translational modification or binding partner by making mutations that affect the modification or interaction. These studies will provide the necessary data to understand the mechanism regulating glucose starvation induced changes in membrane traffic. Understanding these regulatory mechanisms will allow us to understand how yeast cells survive glucose starvation and provide a frame-work to test if this same mechanism is used by cancer cells or other human cells under stress conditions related to disease.