The budding yeast, Saccharomyces cerevisiae, is an excellentmodel organism for the study of eukaryotic cellular processes such as endocytosis 1. Like other eukaryotic cells, yeast take up ...extracellular material by invagination of the plasma membraneto form a vesicle. The internalised material is transported to a membrane-bound compartment, the early endosome. As the early endosome matures, internal vesicles form within the lumen giving it the appearance of a multivesicular body (vesicles enclosed by a membrane). The machinery required for endosome maturation is highly conserved between yeast and mammalian cells. In mammalian cells this machinery is also required for the budding of enveloped viruses. Here we discuss how studies of endosome maturation in S. cerevisiae have given valuable insights into the mechanism by which clinically important enveloped viruses, including human immunodeficiency virus (HIV) and hepatitis B virus, are released from mammalian cells.
Sorting of membrane proteins into intralumenal endosomal vesicles, multivesicular body (MVB) sorting, is critical for receptor down regulation, antigen presentation and enveloped virus budding. Vps4 ...is an AAA ATPase that functions in MVB sorting. Although AAAATPases are oligomeric, mechanisms that govern Vps4 oligomerization and activity remain elusive. Vps4 has an N-terminal microtubule interacting and trafficking domain required for endosome recruitment, an AAA domain containing the ATPase catalytic site and a beta domain, and a C-terminal alpha helix positioned close to the catalytic site in the 3Dstructure. Previous attempts to identify the role of the C-terminal helix have been unsuccessful. Here, we show that the C-terminal helix is important for Vps4 assembly and ATPase activity invitro and function invivo, but not endosome recruitment or interactions with Vta1 or ESCRT-III. Unlike the beta domain, which is also important for Vps4 assembly, the C-terminal helix is not required invivo for Vps4 homotypic interaction or dominant-negative effects of Vps4-E233Q, carrying a mutation in the ATP hydrolysis site. Vta1 promotes assembly of hybrid complexes comprising Vps4-E233Q and Vps4 lacking an intact C-terminal helix invitro. Formation of catalytically active hybrid complexes demonstrates an intersubunit catalytic mechanism for Vps4. One end of the C-terminal helix lies in close proximity to the second region of homology (SRH), which is important for assembly and intersubunit catalysis in AAAATPases. We propose that Vps4 SRH function requires an intact C-terminal helix. Co-evolution of a distinct Vps4 SRH and C-terminal helix in meiotic clade AAAATPases supports this possibility.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Endocytic and biosynthetic trafficking pathways to the lysosome/vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that ...are destined for delivery to the lysosome/vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endosomal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a beta domain, which protrudes from the AAA domain, and a final C-terminal beta-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly conserved between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro. We show that the beta domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic functions in vivo. We also show that the beta domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function.PUBLICATION ABSTRACT
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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