Role of Soi3p in localization of the processing protease Kex2p to the <i>trans</i>-Golgi network through assembly of vacuolar ATPase
The yeast (Saccharomyces cerevisiae) protease Kex2p is the prototype of a family of eukaryotic proteases, members of which process pro-proteins in late compartments of the secretory pathway, the trans -Golgi network (TGN) and secretory granules. Genetic and biochemical approaches have been taken to understand how Kex2p is localized to the TGN. This dissertation has focused on the role of SOI3/RAV1 in Kex2p localization and assembly of vacuolar ATPase (V-ATPase).
Soi3p was recently found to play a role both in early-to-late endosome transport and in glucose-regulated assembly of V1 and V0 subassemblies of V-ATPase. V-ATPase is a multi-subunit complex responsible for the acidification of intracellular organelles. This work first tested the hypothesis that Soi3p function in V-ATPase assembly accounts for the role of Soi3p in trafficking at the early endosome. Indeed, V-ATPase mutants had growth phenotypes, genetic interactions, and protein trafficking defects similar to soi3 mutants. Soi3p was shown to function specifically through the assembly of Vph1-containing V-ATPase; moreover, loss of Vph1p caused accumulation of Soi3p on early endosomes. Soi3p was recruited to membranes under conditions that promote V-ATPase assembly and that recruitment was dependent on the V1 sector. Localization of Soi3p and V1 to non-vacuolar membranes in a vph1Δ mutant suggested that V-ATPase assembly occurs on early endosomes. Supporting this hypothesis, evidence was obtained for retrograde transport of V0 from the vacuole.
Skp1p, a component of SCF-type E3 ubiquitin ligases, was identified as a Soi3p-interacting protein. Analysis of a skp1 allele that abrogated the Soi3p-Skp1p interaction showed that although important for Kex2p trafficking, Skp1p was not required for V-ATPase assembly. However, Skp1p was shown to be important for proper Soi3p localization, demonstrating a role for Skp1p in Soi3p function.
Key findings of this work are that early endosome acidification and therefore maturation are regulated by assembly of V-ATPase and that V-ATPase assembly appears to go through an intermediate on early endosomes. From this work a model for the pathway of Soi3p-dependent V1-V0 assembly now emerges.
0379: Cellular biology