Identification of a major protein on the cytosolic face of caveolae.
Cav-p60, a specific and ubiquitous caveolar protein, was immunoprecipitated from solubilized rat adipocyte plasma membranes and identified as similar to a GeneBank entry annotated mouse polymerase transcript release factor (PTRF) by MALDI-TOF and MS-MS of major fragments. Cloning and virtual translation of the corresponding rat adipocyte cDNA sequence revealed 98.7% identity with mouse PTRF. In vitro translation of this sequence produced a protein, which was recognized by antibodies to both cav-p60 and PTRF. EM gold labeling studies showed that a rabbit antiserum against murine PTRF immunolabeled caveolae specifically in adipocytes from both mouse and rat. In view of the reported function of the protein, which is exerted in the cell nucleus, its subcellular localization was investigated. We found that the protein could be purified by differential solubilization of a plasma membrane fraction followed by SDS-PAGE, and that the protein was as abundant as caveolin in this fraction. We were unable to detect the protein in cell nuclei by subcellular fractionation or fluorescence microscopy. The results show that in a large number of cell types, PTRF is essentially located to caveolae, and that each caveola harbors many copies of the protein. Consequently, we suggest the name Cavin for this protein.
Vinten
J
J
Department of Medical Physiology, Panum Institute, University of Copenhagen, Denmark. J.Vinten@mfi.ku.dk
Johnsen
A H
AH
Roepstorff
P
P
Harpøth
J
J
Tranum-Jensen
J
J
D017667:Adipocytes / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D021941:Caveolae / Q000737:chemistry*; D022461:Caveolins / Q000737:chemistry*; D002462:Cell Membrane / Q000648:ultrastructure; D003600:Cytosol / Q000737:chemistry*; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment
D022461:Caveolins; C112745:Cavin1 protein, mouse; C506791:Cavin1 protein, rat; D008565:Membrane Proteins; D016601:RNA-Binding Proteins