Differential Control of ErBB2 Surface Expression in Breast Cancer Cells by an Alternatively Spliced Form of ERBIN
Annual summary rept. 1 May 2002-30 Apr 2005
GEORGETOWN UNIV WASHINGTON DC
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Snf7p and Vps20p are involved in yeast MVB structure, formation and function. We cloned and characterized 3 human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, 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 structures induced by overexpressing an AlPase-defective Vps4-A mutant. Overexpressed hVps20 showed an 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 GST-capture with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with AIP1, a mammalian Brolp-containing protein involved in cellular vacuolization and apoptosis. Mapping experiments showed that the N-terminus of Alp1 containing both a Brol and an alpha-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Brol-containing molecule, rhophilin-2. Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AlP1. Immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures. These results Suggest that mammalian Vps20, AIP1 and Snf7 proteins and a new component UEV3 may play roles in MVB function.
- Medicine and Medical Research