BENE, a novel raft-associated protein of the MAL proteolipid family, interacts with caveolin-1 in human endothelial-like ECV304 cells

The MAL proteolipid, an integral protein present in glycolipid- and cholest erol-enriched membrane (GEM) rafts, is an element of the machinery necessar y for apical sorting in polarized epithelial Madin-Darby canine kidney cell s. MAL was the first member identified of an extended family of proteins th at have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of t his family, named BENE, which was found to be expressed in endothelial-like ECV304 cells and normal human endothelium, Human BENE was characterized as a proteolipid protein predominantly present in GEM rafts in ECV304 cells, Coimmunoprecipitation experiments revealed that BENE interacted with caveol in-1, Confocal immunofluorescence and electron microscopic analyses indicat ed that BENE mainly accumulated into intracellular vesicular/tubular struct ures that partially colocalize with internal caveolin-1, In response to cel l surface cholesterol oxidation, BENE redistributed to the dilated vesicula r structures that concentrate most of the caveolin-1 originally on the cell surface, After cessation of cholesterol oxidation, a detectable fraction o f the BENE molecules migrated to the plasmalemma accompanying caveolin-1 an d then returned progressively to its steady state distribution. Together, t hese features highlight the BENE proteolipid as being an element of the mac hinery for raft-mediated trafficking in endothelial cells.