title: Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p.
abstract: Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 null mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.
authors.affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, USA. nothwehrs@missouri.edu
authors.forename: S F
authors.initials: SF
authors.lastname: Nothwehr
authors.forename: S A
authors.initials: SA
authors.lastname: Ha
authors.forename: P
authors.initials: P
authors.lastname: Bruinsma
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins; D016764:Cell Polarity; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle / Q000378:metabolism; D013489:Suppression, Genetic; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; C087335:PEP1 protein, S cerevisiae; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle; C076106:VPS35 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D000469:Alkaline Phosphatase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C061833:STE13 protein, S cerevisiae