Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport inSaccharomyces cerevisiae

Abstract

Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLR181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP - conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Δ) or VTA1 (vta1Δ) leads to similar class E Vps- phenotypes resembling those of vps4Δ, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.

Exploring the ESCRTing machinery in eukaryotes

“…The stoichiometry and size of the ESCRT-III complex has not been determined and additional components might be present, for example, the highly charged, small coiled coil SNF7 domain proteins VPS60 or CHMP5 and VPS46 or CHMP1 and the larger coiled coil protein VTA1 or SBP1 [25,27,56,57] Table S5) and their gene products interact with VPS4 (SNF7) and VPS60, respectively [25,56,57,59]. The overall shared amino acid identity between yeast and the putative human and plant homologs is low (15% and 14%, respectively).…”

“…The stoichiometry and size of the ESCRT-III complex has not been determined and additional components might be present, for example, the highly charged, small coiled coil SNF7 domain proteins VPS60 or CHMP5 and VPS46 or CHMP1 and the larger coiled coil protein VTA1 or SBP1 [25,27,56,57] (see Supplementary material Table S5). Direct and indirect associations with ESCRT-III proteins have been suggested for these 'nonclassical' ESCRT-III components [25] (N. Schlager and M.T.…”

“…If VTA1 is deleted, the resulting phenotypes are not typical of class E VPS phenotypes and, thus, VTA1 might function in a subset of sorting events. VTA1 and SBP1 homologs are single-copy genes in all analyzed genomes (see Supplementary material Table S5) and their gene products interact with VPS4 (SNF7) and VPS60, respectively [25,56,57,59]. The overall shared amino acid identity between yeast and the putative human and plant homologs is low (15% and 14%, respectively).…”

“…The protein can be subdivided into three parts: at the N-terminal, 150 amino acids form an as yet uncharacterized DUF605 and a coiled coil domain; at the Cterminus, a second coiled coil domain shows a high level of sequence identity between yeast and the human and plant homologs (52.8% and 40.0%, respectively). Together with the Nterminal coiled coil domain, the short C-terminal domain is responsible for the interaction with VPS4 [57]. The two terminal domains are separated by a highly diverse stretch of between 100 (human) and 300 (rice) amino acids.…”

“…[62]. VPS4 proteins act by binding, catalyzing and energizing the dissociation of the ESCRT-III complex and the release of the membrane-associated SNF7 domain proteins into the cytoplasm for further rounds of sorting [57,63]. In diverse protein-protein interaction studies, it has been shown that VPS4 and its two mammalian homologs VPS4B or SKD1 and VPS4A or SKD2 interact with 'classical' ESCRT-III complex components (SNF7 or CHMP4, VPS2 or CHMP2, VPS20 or CHMP6) and the associated proteins VPS46 or CHMP1, and VTA1 or SBP1 [20][21][22]24,25,[63][64][65][66].…”