Differential Control of ErBB2 Surface Expression in Breast Cancer Cells by an Alternatively Spliced Form of ERBIN
Abstract
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.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2005
- Accession Number
- ADA439144
Entities
People
- Kathryn H. Ching
Organizations
- Georgetown University