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 cholesterol-enriched membrane (GEM) rafts, is an element of the machinery necessary for apical sorting in polarized epithelial Madin-Darby canine kidney cells. MAL was the first member identified of an extended family of proteins that have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of this 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 caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the cell surface. After cessation of cholesterol oxidation, a detectable fraction of the BENE molecules migrated to the plasmalemma accompanying caveolin-1 and then returned progressively to its steady state distribution. Together, these features highlight the BENE proteolipid as being an element of the machinery for raft-mediated trafficking in endothelial cells.
de Marco
M C
MC
Centro de Biologia Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Spain.
Kremer
L
L
Albar
J P
JP
Martinez-Menarguez
J A
JA
Ballesta
J
J
Garcia-Lopez
M A
MA
Marazuela
M
M
Puertollano
R
R
Alonso
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D004727:Endothelium / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D010084:Oxidation-Reduction; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; C495540:CAV1 protein, human; D002352:Carrier Proteins; D051242:Caveolin 1; D022461:Caveolins; C110862:MALL protein, human; D008565:Membrane Proteins; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D011510:Proteolipids; D002784:Cholesterol