title: Sorting of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase): identification of a necessary and sufficient Golgi/endosomal retention signal in Stv1p.
abstract: Subunit a of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase) is responsible for both proton translocation and subcellular localization of this highly conserved molecular machine. Inclusion of the Vph1p isoform causes the V-ATPase complex to traffic to the vacuolar membrane, whereas incorporation of Stv1p causes continued cycling between the trans-Golgi and endosome. We previously demonstrated that this targeting information is contained within the cytosolic, N-terminal portion of V-ATPase subunit a (Stv1p). To identify residues responsible for sorting of the Golgi isoform of the V-ATPase, a random mutagenesis was performed on the N terminus of Stv1p. Subsequent characterization of mutant alleles led to the identification of a short peptide sequence, W(83)KY, that is necessary for proper Stv1p localization. Based on three-dimensional homology modeling to the Meiothermus ruber subunit I, we propose a structural model of the intact Stv1p-containing V-ATPase demonstrating the accessibility of the W(83)KY sequence to retrograde sorting machinery. Finally, we characterized the sorting signal within the context of a reconstructed Stv1p ancestor (Anc.Stv1). This evolutionary intermediate includes an endogenous W(83)KY sorting motif and is sufficient to compete with sorting of the native yeast Stv1p V-ATPase isoform. These data define a novel sorting signal that is both necessary and sufficient for trafficking of the V-ATPase within the Golgi/endosomal network.
authors.affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Finnigan
authors.forename: Glen E
authors.initials: GE
authors.lastname: Cronan
authors.forename: Hae J
authors.initials: HJ
authors.lastname: Park
authors.forename: Sankaranarayanan
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Florante A
authors.initials: FA
authors.lastname: Quiocho
authors.forename: Tom H
authors.initials: TH
authors.lastname: Stevens
mesh_terms: D020816:Amino Acid Motifs; D011992:Endosomes / Q000201:enzymology* / Q000235:genetics; D019143:Evolution, Molecular; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D021382:Protein Sorting Signals / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000201:enzymology* / Q000235:genetics
chemical_list: D007527:Isoenzymes; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases