LAUSR

Endosomes undergo a maturation process highlighted by a reduction in lumenal pH, a conversion of surface markers that prime endosome-lysosome fusion, and the sequestration of ubiquitinated transmembrane protein cargoes within intralumenal vesicles (ILVs). We investigated ILV cargo sorting in mutant strains of the budding yeast Saccharomyces cerevisiae that are deficient for either the lysosomal/vacuolar signaling lipid PI(3,5)P2 or the Doa4 ubiquitin hydrolase that deubiquitinates ILV cargoes. Disruption of PI(3,5)P2 synthesis or Doa4 function causes a defect in the sorting of a subset of ILV cargoes. We show that these cargo-sorting defects are suppressed by mutations that disrupt Vph1, which is a subunit of Vacuolar H+-ATPase (V-ATPase) complexes that acidify late endosomes and vacuoles. We further show that Vph1 dysfunction increases endosome abundance and disrupts vacuolar localization of Ypt7 and Vps41, two critical mediators of endosome-vacuole fusion. Because V-ATPase inhibition attenuates endosome-vacuole fusion and rescues the ILV cargo-sorting defects in yeast lacking PI(3,5)P2 and Doa4 activity, our results suggest that the V-ATPase performs a role in the coordination of ILV cargo sorting with the membrane fusion machinery.