Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes

• Published in: Molecular biology of the cell Vol. 11; no. 12; pp. 4105 - 4116
• Main Authors: Haft, C R, de la Luz Sierra, M, Bafford, R, Lesniak, M A, Barr, V A, Taylor, S I
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01-12-2000
• Subjects: Amino Acid Sequence; Animals; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cloning, Molecular; COS Cells; Humans; Macromolecular Substances; Membrane Proteins - genetics; Models, Biological; Molecular Sequence Data; Saccharomyces - genetics; Saccharomyces cerevisiae Proteins; Sequence Homology; Two-Hybrid System Techniques; Vesicular Transport Proteins
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.11.12.4105

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.