CONTROL OF RAB SMALL G-PROTEINS IN YEAST ENDOSOMAL TRAFFIC

Abstract

The protein trafficking pathway in eukaryotic cells is essential for maintenance of organelle identity, nutrient uptake, and adaptation to extracellular stimuli. The endocytic pathway is tasked with uptake of plasma membrane receptors and bulk extra-cellular material as well as interaction with the secretory pathway. The Rab5 family of small GTPases are essential in maintaining the endocytic pathway. Rab5 proteins are active only when bound to GTP. Due to the slow intrinsic rate of GTP loading, Rab5 relies on the VPS9 domain for activation. VPS9 domains are highly conserved and function as activators of Rab5 by promoting GDP release and GTP loading. The diversity of members of the Rab5 subfamily and their activators indicates that this family may multiple roles in endocytosis. VPS9-domain proteins are themselves highly regulated. An emerging theory in this field suggests that modulating their localization may influence Rab5 localization and therefore function. To address this I investigate possible regulatory roles of Saccharomyces cerevisiae Vps9 as influenced by ubiquitin. Yeast Vps9 can both bind ubiquitin and become ubiquitylated, though it has remained unclear what, if any, influence these functions have on Vps9 activity. These studies lead to the discovery of Muk1, an alternate yeast Rab5 activator with a VPS9-domain. The yeast vacuole or mammalian lysosome is considered the terminal compartment of endocytosis and the HOPS protein complex is essential for regulating membrane fusion at this organelle. This complex contains several carboxy-terminal RING domains. While most RING domains have been shown to possess E3 ubiquitin ligase activity, the HOPS RING domains remain largely uncharacterized. Finally, I examined HOPS RING domain mutants to address their function for vacuole morphology and their relation to ubiquitin.