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 pr otein 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 o rthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human protei ns 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. D eletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. Ln con trast, 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 compartme nts. Gel filtration chromatography of COS7 cell cytosol showed that both re combinant and endogenous hVps35, hVps29, and hVps26 coelute as a large comp lex (similar to 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 retro mer complex.