A Novel RING Finger Protein Complex Essential for a Late Step in Protein Transport to the Yeast Vacuole

Abstract

Protein transport to the lysosome-like vacuole in yeast is mediated by multiple pathways, including the biosynthetic routes for vacuolar hydrolases, the endocytic pathway, and autophagy. Among the more than 40 genes required for vacuolar protein sorting (VPS) inSaccharomyces cerevisiae, mutations in the four class CVPS genes result in the most severe vacuolar protein sorting and morphology defects. Herein, we provide complementary genetic and biochemical evidence that the class C VPSgene products (Vps18p, Vps11p, Vps16p, and Vps33p) physically and functionally interact to mediate a late step in protein transport to the vacuole. Chemical cross-linking experiments demonstrated that Vps11p and Vps18p, which both contain RING finger zinc-binding domains, are components of a hetero-oligomeric protein complex that includes Vps16p and the Sec1p homologue Vps33p. The class C Vps protein complex colocalized with vacuolar membranes and a distinct dense membrane fraction. Analysis of cells harboring a temperature-conditionalvps18 allele (vps18 tsf ) indicated that Vps18p function is required for the biosynthetic, endocytic, and autophagic protein transport pathways to the vacuole. In addition,vps18 tsf cells accumulated multivesicular bodies, autophagosomes, and other membrane compartments that appear to represent blocked transport intermediates. Overproduction of either Vps16p or the vacuolar syntaxin homologue Vam3p suppressed defects associated with vps18 tsf mutant cells, indicating that the class C Vps proteins and Vam3p may functionally interact. Thus we propose that the class C Vps proteins are components of a hetero-oligomeric protein complex that mediates the delivery of multiple transport intermediates to the vacuole.

The late stages of autophagy: how does the end begin?

“…Class C Vps/HOPS proteins consist of a tethering protein complex including an SM (Sec1/Munc18-like) protein and a Rab GEF (GDP/GTP exchanger) for Ypt7, and are involved in fusion. 36,46 Recently, it was found that the mammalian HOPS complex binds to UVRAG, which is a Beclin-1 binding protein. 47 Beclin-1, the mammalian homologue of yeast Atg6, also interacts with class III phosphatidylinositol 3-kinase/ hVps34 and is essential for autophagosome formation.…”

Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

“…However, the promiscuity of SNARE-SNARE interactions may allow other post-Golgi t-SNAREs to substitute at least in part for the loss of the relevant late Golgi t-SNARE. In fact, loss of the vacuolar t-SNAREs Vam3p and Vam7p results in less severe defects in vacuolar morphology and function than loss of the class C proteins Vps11p, Vps18p, Vps16p, and Vps33p, which act together to regulate the fusion of multiple transport intermediates with the vacuole, and high levels of the prevacuolar t-SNARE Pep12p suppress mutations in VAM3 (Darsow et al, 1997;Rieder and Emr, 1997). It is also difficult to extrapolate from the phenotypes resulting from defects in t-SNAREs, because these proteins are likely to regulate a number of different transport events.…”

“…These include Vac1p, the yeast homologue of EEA1, which binds the Rab5 homologue Vps21p as well as the Sec1-like Vps45p and the endosomal t-SNARE Pep12p (Burd et al, 1997;Peterson et al, 1999;Tall et al, 1999). Class C Vps proteins include Vps11p, Vps18p, Vps16p, and the Sec1-like Vps33p, which form a complex that, together with the vacuolar t-SNARE Vam3p, the SNAP-25-like Vam7p, and the Rab protein Ypt7p, is required for the fusion of multiple transport intermediates with the vacuole (Haas et al, 1995;Darsow et al, 1997;Rieder and Emr, 1997;Sato et al, 1998;Ungermann and Wickner, 1998). Class E VPS gene products regulate transit through the PVC, whereas components of the retromer complex are thought to act at the PVC to sort proteins into retrograde vesicles that are targeted to fuse with the TGN (Seaman et al, 1998;Nothwehr et al, 1999).…”

TheSaccharomyces cerevisiaev-SNARE Vti1p Is Required for Multiple Membrane Transport Pathways to the Vacuole

“…Deletion of vps genes necessary for fusion of transport vesicles with the PVC or required for fusion of membranes derived from the PVC and from the ALP pathway with the vacuole results in a growth defect at 37°C (Piper et al, 1994;Becherer et al, 1996;Rieder and Emr, 1997). vti1-1 cells do not have a growth defect at the nonpermissive temperature.…”

“…The adaptor complex AP3 is required for ALP but not for CPY transport to the vacuole Stepp et al, 1997). Several proteins have been identified as being involved in both ALP and CPY transport to the vacuole, for example, Vam3p, Vam7p and a vacuolar protein complex consisting of Vps18p, Vps11p, Vps16p, and Vps33p (Darsow et al, 1997;Piper et al, 1997;Wada et al, 1997;Rieder and Emr, 1997;Sato et al, 1998;Srivastava and Jones, 1998).…”