Bro1 binds the Vps20 subunit of ESCRT-III and promotes ESCRT-III regulation by Doa4.

The budding of intralumenal vesicles (ILVs) at endosomes requires membrane scission by the ESCRT-III complex. This step is negatively regulated in yeast by Doa4, the ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted as cargoes into ILVs. Doa4 acts non-enzymatically to inhibit ESCRT-III membrane scission activity by directly binding the Snf7 subunit of ESCRT-III. This interaction inhibits the remodeling/disassembly of Snf7 polymers required for the ILV membrane scission reaction. Thus, Doa4 is thought to have a structural role that delays ILV budding while it also functions enzymatically to deubiquitinate ILV cargoes. In this study, we show that Doa4 binding to Snf7 in vivo is antagonized by another ESCRT-III subunit, Vps20. Doa4 is restricted from interacting with Snf7 in yeast expressing a mutant Vps20 allele that constitutively binds Doa4. This inhibitory effect of Vps20 is suppressed by overexpression of another ESCRT-III-associated protein, Bro1. We show that Bro1 binds directly to Vps20, suggesting that Bro1 has a central role in relieving the antagonistic relationship that Vps20 has toward Doa4.

Vacuolar H+-ATPase dysfunction rescues intralumenal vesicle cargo sorting in yeast lacking PI(3,5)P2 or Doa4.

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 ubiquitylated transmembrane protein cargos 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 deubiquitylates ILV cargos. Disruption of PI(3,5)P2 synthesis or Doa4 function causes a defect in sorting of a subset of ILV cargos. We show that these cargo-sorting defects are suppressed by mutations that disrupt Vph1, 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 crucial mediators of endosome-vacuole fusion. Because V-ATPase inhibition attenuates this fusion and rescues the ILV cargo-sorting defects in yeast that lack PI(3,5)P2 or Doa4 activity, our results suggest that the V-ATPase has a role in coordinating ILV cargo sorting with the membrane fusion machinery. This article has an associated First Person interview with the first author of the paper.

The ubiquitin hydrolase Doa4 directly binds Snf7 to inhibit recruitment of ESCRT-III remodeling factors in S. cerevisiae.

The ESCRT-III protein complex executes reverse-topology membrane scission. The scission mechanism is unclear but is linked to remodeling of ESCRT-III complexes at the membrane surface. At endosomes, ESCRT-III mediates the budding of intralumenal vesicles (ILVs). In Saccharomyces cerevisiae, ESCRT-III activity at endosomes is regulated through an unknown mechanism by Doa4, an ubiquitin hydrolase that deubiquitylates transmembrane proteins sorted into ILVs. We report that the non-catalytic N-terminus of Doa4 binds Snf7, the predominant ESCRT-III subunit. Through this interaction, Doa4 overexpression alters Snf7 assembly status and inhibits ILV membrane scission. In vitro, the Doa4 N-terminus inhibits association of Snf7 with Vps2, which functions with Vps24 to arrest Snf7 polymerization and remodel Snf7 polymer structure. In vivo, Doa4 overexpression inhibits Snf7 interaction with Vps2 and also with the ATPase Vps4, which is recruited by Vps2 and Vps24 to remodel ESCRT-III complexes by catalyzing subunit turnover. Our data suggest a mechanism by which the deubiquitylation machinery regulates ILV biogenesis by interfering with ESCRT-III remodeling.