A Family of Small Coiled-Coil–forming Proteins Functioning at the Late Endosome in Yeast

Abstract

The multispanning membrane protein Ste6, a member of the ABC-transporter family, is transported to the yeast vacuole for degradation. To identify functions involved in the intracellular trafficking of polytopic membrane proteins, we looked for functions that block Ste6 transport to the vacuole upon overproduction. In our screen, we identified several known vacuolar protein sorting (VPS) genes (SNF7/VPS32, VPS4, and VPS35) and a previously uncharacterized open reading frame, which we named MOS10 (more of Ste6). Sequence analysis showed that Mos10 is a member of a small family of coiled-coil-forming proteins, which includes Snf7 and Vps20. Deletion mutants of all three genes stabilize Ste6 and show a "class E vps phenotype." Maturation of the vacuolar hydrolase carboxypeptidase Y was affected in the mutants and the endocytic tracer FM4-64 and Ste6 accumulated in a dot or ring-like structure next to the vacuole. Differential centrifugation experiments demonstrated that about half of the hydrophilic proteins Mos10 and Vps20 was membrane associated. The intracellular distribution was further analyzed for Mos10. On sucrose gradients, membrane-associated Mos10 cofractionated with the endosomal t-SNARE Pep12, pointing to an endosomal localization of Mos10. The growth phenotypes of the mutants suggest that the "Snf7-family" members are involved in a cargo-specific event.

Control of Bro1-Domain Protein Rim20 Localization by External pH, ESCRT Machinery, and theSaccharomyces cerevisiaeRim101 Pathway

“…The recent analysis of Bro1-domain structure makes it clear that all Bro1-domain proteins may interact with Snf7 or its orthologues (Kim et al, 2005). Like Snf7 itself (Kranz et al, 2001;Babst et al, 2002), the characterized mammalian Snf7 orthologues have been localized to the endosomal surface and implicated in endosome trafficking (Katoh et al, 2003;Peck et al, 2004;Yorikawa et al, 2005). With that as a backdrop, our finding that Rim20 is localized to Snf7-containing endosomes is entirely expected.…”

“…Snf7 is an endosome-associated protein (Kranz et al, 2001;Babst et al, 2002). To determine whether Rim20 and Snf7 foci coincide, we examined cells expressing functional Rim20-GFP and Snf7-RFP fusions under alkaline conditions.…”

“…One additional feature of ESCRT proteins is that they hyperaccumulate on endosomes in the absence of the recycling factor Vps4 (Babst et al, 1998;Kranz et al, 2001). Defects in Vps4 ultimately result in ESCRT accumulation on a large, endosome-derived "class E" compartment found at the periphery of the vacuole.…”

“…Snf7 is one component of the ESCRT machinery (Kranz et al, 2001;Babst et al, 2002), a group of three protein complexes that sort endocytic cargo into MVBs (Babst, 2005). Once the cargo is sorted into presumed subdomains of the vesicle surface, it is de-ubiquinated through the action of Bro1-Doa4, and MVBs are produced by invagination of the surface subdomain.…”

“…Recent studies reveal that Bro1 functions as an adaptor, binding Snf7 to bring the ubiquitin hydrolase Doa4 (Amerik et al, 2000) into proximity with endocytic cargo proteins (Luhtala and Odorizzi, 2004), whose cytoplasmic domains are ubiquitinated (Raiborg et al, 2003;Babst, 2005). Rim20 is also thought to function as an adaptor, binding Snf7 to bring the protease Rim13 into proximity with Rim101, thus promoting Rim101 cleavage and activation Vincent et al, 2003).Snf7 is one component of the ESCRT machinery (Kranz et al, 2001;Babst et al, 2002), a group of three protein complexes that sort endocytic cargo into MVBs (Babst, 2005). Once the cargo is sorted into presumed subdomains of the vesicle surface, it is de-ubiquinated through the action of Bro1-Doa4, and MVBs are produced by invagination of the surface subdomain.…”