Structure and function of human Vps20 and Snf7 proteins.
Snf7p (sucrose non-fermenting) and Vps20p (vacuolar protein-sorting) are small coil-coiled proteins involved in yeast MVB (multivesicular body) structure, formation and function. In the present study, we report the identification of three human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, designated hVps20, that may have similar roles in humans. Immunofluorescence studies showed that hSnf7-1 and hSnf7-3 localized in large vesicular structures that also co-localized with late endosomal/lysosomal structures induced by overexpressing an ATPase-defective Vps4-A mutant. In contrast, overexpressed hVps20 showed a typical endosomal membrane-staining pattern, and co-expression of hVps20 with Snf7-1 dispersed the large Snf7-staining vesicles. Interestingly, overexpression of both hSnf7 and hVps20 proteins induced a post-endosomal defect in cholesterol sorting. To explore possible protein-protein interactions involving hSnf7 proteins, we used information from yeast genomic studies showing that yeast Snf7p can interact with proteins involved in MVB function. Using a glutathione S-transferase-capture approach with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with mouse AIP1 [ALG-2 (apoptosis-linked gene 2) interacting protein 1], a mammalian Bro1p [BCK1 (bypass of C kinase)-like resistance to osmotic shock]-containing protein involved in cellular vacuolization and apoptosis. Whereas mapping experiments showed that the N-terminus of AIP1 containing both a Bro1 and an alpha-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Bro1-containing molecule, rhophilin-2. Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AIP1. Additional immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures. Together these results suggest that mammalian Vps20, AIP1 and Snf7 proteins, like their yeast counterparts, play roles in MVB function.
Peck
Jeremy W
JW
Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, U.S.A.
Bowden
Emma T
ET
Burbelo
Peter D
PD
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism; D003001:Cloning, Molecular; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000737:chemistry; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D008247:Lysosomes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D025941:Protein Interaction Mapping; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D022161:Transport Vesicles / Q000737:chemistry; D033921:Vesicular Transport Proteins
C481529:CHMP4A protein, human; C481528:CHMP6 protein, human; D002352:Carrier Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D009687:Nuclear Proteins; C083890:SNF7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D002784:Cholesterol