Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes.
Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex ( approximately 220-440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.
Haft
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Diabetes Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1770, USA. carol_haft@nih.gov
de la Luz Sierra
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Bafford
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Lesniak
M A
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Barr
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Taylor
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D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D012440:Saccharomyces / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017385:Sequence Homology; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; C498296:VPS26A protein, human; C419572:VPS29 protein, human; C076106:VPS35 protein, S cerevisiae; C419573:VPS35 protein, human; D033921:Vesicular Transport Proteins