Novel Molecular Mechanisms of Membrane Traffic in Yeast

[摘要] Proper membrane traffic is important to target cargo proteins to their designated locations in the cell and maintain homeostasis. Cargo protein traffic through the endomembrane system is a highly regulated process that depends, in part, on clathrin-coated vesicles. Clathrin-coated vesicles are used by the cell in endocytosis and post-Golgi traffic. Clathrin coat formation depends on the action of many proteins that help in the formation of clathrin vesicles. Some of these proteins include clathrin adaptors, proteins that select cargo to be packaged, vesicle terminating proteins, and other proteins that regulate each step of vesicle formation. This thesis addresses two related topics. The first topic explores the role of membrane trafficking proteins in cell survival in stress conditions, such as nutrient starvation. To survive nutrient starvation, cells can use a process known as autophagy. Autophagy is a mechanism to recycle cellular components to their molecular components. Although it was thought to contribute to survival during glucose starvation, our investigation indicates that autophagy is not essential for survival in glucose starvation for the budding yeast, Saccharomyces cerevisiae. Instead, we found that during glucose starvation the clearance of cell surface proteins and their traffic to, and degradation at, the vacuole (the lysosome in yeast) promotes cell survival. Furthermore, we found that autophagy is inhibited in glucose starved cells. These results suggest that endocytosis and endosomal traffic, not autophagy, is the mechanism used by the cell to survive in glucose starvation.The second topic covered by this thesis is the characterization of Art1/Ldb19, a member of the α-arrestins family.This topic started with the investigation of the clearance of cell surface proteins in glucose starved cells. This response was similar to the clearance of cell surface proteins caused by heat shock and cycloheximide treatment. These cell surface clearance events depend on the function of α-arrestins, a family of proteins involved in the ubiquitination of cargo proteins. Based on this similarity, we initiated an investigation of α-arrestins in glucose starvation-induced endocytosis. We found that the α-arrestin Art1/Ldb19 was required for efficient clearance of cell surface proteins in glucose starvation. Interestingly, Art1 localizes to internal structures that resemble trans-Golgi network (TGN) localization of clathrin adaptors at steady-state conditions. This localization could suggest an additional role for Art1, a known endocytic protein, in TGN traffic. The second part of this thesis explores the role of Art1 at the TGN. Using fluorescence microscopy approaches, we found that Art1 localizes to the late TGN and could potentially interact with the clathrin adaptor, AP-1. Additionally, we found genetic interactions of ART1 with another clathrin adaptor, GGA2, and a hypomorphic allele of clathrin. Finally, we found that deletion of ART1 suppresses secretion defects observed in cells that express a hypomorphic allele of clathrin. In summary, the doctoral research presented here provides novel mechanisms of membrane trafficking, with particular relevance for α-arrestins-dependent TGN traffic. Specifically, we found a novel mechanism for cell survival during glucose starvation independent of autophagy. Additionally, our data supports emerging evidence that suggest a role for Art1 at the TGN, in addition to its established role in endocytosis.

[发布机构] University of Michigan

[效力级别] trans-Golgi Network (TGN)

[关键词] membrane traffic;trans-Golgi Network (TGN);arrestins;glucose starvation;Art1/Ldb19;clathrin-mediated traffic;Molecular;Cellular and Developmental Biology;Health Sciences;Science;Cell and Developmental Biology