Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible.
Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents subsequent repalmitoylation.
Department of Cell Biology, Cleveland Clinic Foundation, The Lerner Research Institute, Cleveland, Ohio 44195, USA.
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D000818:Animals; D001011:Aorta; D020126:Brefeldin A / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D021984:Cyclophilin A / Q000378:metabolism; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins; D004730:Endothelium, Vascular / Q000378:metabolism*; D019811:Hydroxylamine / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
D002352:Carrier Proteins; D051242:Caveolin 1; D022461:Caveolins; D000081406:Peptidyl-Prolyl Isomerase F; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D019811:Hydroxylamine; D019308:Palmitic Acid; D003513:Cycloheximide; D008715:Methionine; D021984:Cyclophilin A; D021983:Cyclophilins; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase