Mapping of Vps21 and HOPS binding sites in Vps8 and effect of binding site mutants on endocytic trafficking.
Vps8 is a subunit of the CORVET tethering complex, which is involved in early-to-late endosome fusion. Here, we examine the role of Vps8 in membrane fusion at late endosomes in Saccharomyces cerevisiae. We demonstrate that Vps8 associates with membranes and that this association is independent of the class C/HOPS core complex and, contrary to a previous report, also independent of the Rab GTPase Vps21. Our data indicate that Vps8 makes multiple contacts with membranes. One of these membrane binding regions could be mapped to the N-terminal part of the protein. By two-hybrid analysis, we obtained evidence for a physical interaction between Vps8 and the Rab5 homologue Vps21. In addition, the interaction with the HOPS core complex was confirmed by immunoprecipitation experiments. By deletion analysis, the Vps21 and HOPS binding sites were mapped in Vps8. Deletions that abrogated HOPS core complex binding had a strong effect on the turnover of the endocytic cargo protein Ste6 and on vacuolar sorting of carboxypeptidase Y. In contrast, deletions that abolished Vps21 binding showed only a modest effect. This suggests that the Vps21 interaction is not essential for endosomal trafficking but may be important for some other aspect of Vps8 function.
Pawelec
Agnes
A
Institut für Lebensmittelwissenschaft und Biotechnologie, Fg. Gärungstechnologie (150f), Universität Hohenheim, Stuttgart, Germany.
Arsić
Janja
J
Kölling
Ralf
R
D001665:Binding Sites; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; C103153:VPS8 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C086866:VPS21 protein, S cerevisiae; D020691:rab GTP-Binding Proteins