Role of Soi3p in localization of the processing protease Kex2p to the <italic>trans</italic>-Golgi network through assembly of vacuolar ATPase.

Abstract

The yeast (<italic>Saccharomyces cerevisiae</italic>) 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 <italic>trans </italic>-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 <italic>SOI3/RAV1</italic> 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 <italic>soi3</italic> 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 <italic>vph1</italic>Delta 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 <italic>skp1</italic> 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.