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J. Biol. Chem., Vol. 276, Issue 19, 15776-15782, May 11, 2001
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From the Department of Cell Biology, Cleveland Clinic
Foundation, The Lerner Research Institute, Cleveland, Ohio 44195
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
[3H]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
[3H]palmitate was not significant even after 24 h.
These results show that [3H]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.
Palmitoylation of Caveolin-1 in Endothelial Cells Is
Post-translational but Irreversible*
*
This work was supported by National Institutes of Health
Grant HL/CA54519 and National Aeronautics and Space Administration Grant 96-HEDS-04 (to P. L. F.), and by a Fellowship of the American Heart Association, Ohio Valley Affiliate (to M. -O. P.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cell
Biology, Cleveland Clinic Foundation, The Lerner Research Institute, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-8053; Fax: 216-444-9404; E-mail: foxp@ccf.org.
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