title: Structural features of vps35p involved in interaction with other subunits of the retromer complex.
abstract: The penta-subunit retromer complex of yeast mediates selective retrieval of membrane proteins from the prevacuolar endosome to the trans Golgi network. In this study, we set out to generate a panel of vps35 dominant-negative mutants that disrupt retromer-mediated cargo sorting. Mapping of the mutations revealed two types of alterations leading to dominant-negative behavior of the 944-amino acid protein: (i) mutations at or near the R(98) residue or (ii) C-terminal truncations exemplified by a nonsense mutation at codon 733. Both could be suppressed by overexpression of wild-type Vps35p, suggesting that these dominant-negative mutants compete for interactions with other retromer subunits. Interestingly, Vps35-R(98)W expression destabilized Vps26p while having no effect on Vps29p stability, while Vps35-Q(733)* expression affected Vps29p stability but had no effect on Vps26p. Measurement of Vps35/Vps26 and Vps35/Vps29 pairwise associations by coimmunoprecipitation in the presence or absence of other retromer subunits indicated that the R(98) residue, which is part of a conserved PRLYL motif, is critical for Vps35p binding to Vps26p, while both R(98) and residues 733-944 are needed for efficient binding to Vps29p.
authors: Division of Biological Sciences, 401 Tucker Hall, University of Missouri, Columbia, MO 65211, USA.
authors: Ricardo
authors: R
authors: Restrepo
authors: Division of Metabolism, Endocrinology & Diabetes, University of Michigan, 5560 MSRB2, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.
authors: Xiang
authors: X
authors: Zhao
authors: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
authors: Harald
authors: H
authors: Peter
authors: Division of Metabolism, Endocrinology & Diabetes, University of Michigan, 5560 MSRB2, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.
authors: Bao-Yan
authors: BY
authors: Zhang
authors: Division of Metabolism, Endocrinology & Diabetes, University of Michigan, 5560 MSRB2, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA.
authors: Peter
authors: P
authors: Arvan
authors: Division of Biological Sciences, 401 Tucker Hall, University of Missouri, Columbia, MO 65211, USA.
authors: Steven F
authors: SF
authors: Nothwehr
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry; D003062:Codon; D011992:Endosomes / Q000378:metabolism; D005799:Genes, Dominant; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D003062:Codon; D008565:Membrane Proteins; C089990:PEP8 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C416672:VPS29 protein, S cerevisiae; C076106:VPS35 protein, S cerevisiae; D033921:Vesicular Transport Proteins