Andrew L. Paulsel

Traffic through late endolysosomal compartments is regulated by sequential signaling of small G proteins of the Rab5 and Rab7 families. The Saccharomyces cerevisiae Vps-C protein complexes CORVET (class C core vacuole/endosome tethering complex) and HOPS (homotypic fusion and protein transport) interact with endolysosomal Rabs to coordinate their signaling activities. To better understand these large and intricate complexes, we performed interaction surveys to assemble domain-level interaction topologies for the eight Vps-C subunits. We identified numerous intersubunit interactions and up to six Rab-binding sites. Functional modules coordinate the major Rab interactions within CORVET and HOPS. The CORVET-specific subunits, Vps3 and Vps8, form a subcomplex and physically and genetically interact with the Rab5 orthologue Vps21. The HOPS-specific subunits, Vps39 and Vps41, also form a subcomplex. Both subunits bind the Rab7 orthologue Ypt7, but with distinct nucleotide specificities. The in vivo functions of four RING-like domains within Vps-C subunits were analyzed and shown to have distinct functions in endolysosomal transport. Finally, we show that the CORVET- and HOPS-specific subunits Vps3 and Vps39 bind the Vps-C core through a common region within the Vps11 C-terminal domain (CTD). Biochemical and genetic experiments demonstrate the importance of these regions, revealing the Vps11 CTD as a key integrator of Vps-C complex assembly, Rab signaling, and endosomal and lysosomal traffic.

VPS9 domains can act as guanosine nucleotide exchange factors (GEFs) against small G proteins of the Rab5 family. Saccharomyces cerevisiae vps9Δ mutants have trafficking defects considerably less severe than multiple deletions of the three cognate Rab5 paralogs (Vps21, Ypt52, and Ypt53). Here, we show that Muk1, which also contains a VPS9 domain, acts as a second GEF against Vps21, Ypt52, and Ypt53. Muk1 is partially redundant with Vps9 in vivo, with vps9Δ muk1Δ double mutant cells displaying hypersensitivity to temperature and ionic stress, as well as profound impairments in endocytic and Golgi endosome trafficking, including defects in sorting through the multivesicular body. Cells lacking both Vps9 and Muk1 closely phenocopy double and triple knock-out strains lacking Rab5 paralogs. Microscopy and overexpression experiments demonstrate that Vps9 and Muk1 have distinct localization determinants. These experiments establish Muk1 as the second Rab5 GEF in budding yeast.

Small G proteins of the Rab5 family control traffic through early compartments of the endolysosomal system. Saccharomyces cerevisiae, like mammals, contains three Rab5 paralogs: Vps21, Ypt52, and Ypt53. We present experimental analyses of the roles of the overlapping and distinct functions of these Rabs, and we analyze the proteins that activate and terminate their signaling activities. We demonstrate that Rab5 signaling is essential for the biogenesis of morphologically normal endosomal multivesicular bodies (MVBs) and for the correct targeting of MVB cargos en route from the Golgi or plasma membrane. In addition to the known nucleotide exchange factor Vps9, we identify Muk1 as a second partially redundant activator of Rab5 family members, and we present biochemical and functional studies of the relative contributions of Vps9 and Muk1. As with the Rab5 paralogs themselves, Vps9 and Muk1 are essential for correct sorting of cargo into MVB intraluminal vesicles. Finally, we demonstrate that signaling by Vps21, the most biologically important of the three yeast Rab5 family members, is selectively terminated by the Gyp3 Rab GAP.

Thesis (Ph.D.)--University of Washington, 2013