Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo.
HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.
Zhou
Minglong
M
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, Texas 77843-4466, USA.
Parr
Rebecca D
RD
Petrescu
Anca D
AD
Payne
H Ross
HR
Atshaves
Barbara P
BP
Kier
Ann B
AB
Ball
Judith M
JM
Schroeder
Friedhelm
F
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D049294:Phospholipid Transfer Proteins; D011233:Precipitin Tests; D011485:Protein Binding; D013347:Subcellular Fractions / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D002352:Carrier Proteins; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; C072087:sterol carrier proteins