Parat M O, Fox P L
Department of Cell Biology, Cleveland Clinic Foundation, The Lerner Research Institute, Cleveland, Ohio 44195, USA.
J Biol Chem. 2001 May 11;276(19):15776-82. doi: 10.1074/jbc.M006722200. Epub 2001 Feb 13.
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.
小窝蛋白-1是一种棕榈酰化蛋白，参与称为小窝的质膜亚结构域中信号分子的组装以及细胞内胆固醇转运。小窝蛋白-1 C末端的三个半胱氨酸残基会发生棕榈酰化，这对于小窝蛋白-1靶向小窝并非必需。蛋白质棕榈酰化是一种翻译后可逆修饰，可能受到调控，进而可能调节靶蛋白的构象、膜结合、蛋白质-蛋白质相互作用以及细胞内定位。我们对主动脉内皮细胞中[³H]棕榈酸掺入小窝蛋白-1进行了详细分析。棕榈酸与小窝蛋白-1的连接对羟胺敏感，因此推测是硫酯键。然而，与预期相反，蛋白质合成抑制剂环己酰亚胺和嘌呤霉素完全阻断了棕榈酸的掺入。在显示特异性的平行实验中，主动脉内皮细胞特异性一氧化氮合酶的棕榈酰化不受这些试剂的影响。蛋白质转运抑制剂布雷菲德菌素A和莫能菌素阻断了小窝蛋白-1的棕榈酰化，表明这种修饰不是共翻译的，而是需要小窝蛋白-1从内质网和高尔基体转运到质膜。此外，与小窝蛋白-1形成复合物的亲免素伴侣，即FK506结合蛋白52、亲环素A和亲环素40，对于小窝蛋白-1的棕榈酰化并非必需，因为结合亲免素的试剂不会抑制棕榈酰化。脉冲追踪实验表明，小窝蛋白-1的棕榈酰化基本上是不可逆的，因为即使在24小时后，[³H]棕榈酸的释放也不显著。这些结果表明，[³H]棕榈酸掺入仅限于新合成的小窝蛋白-1，不是因为掺入仅发生在合成过程中，而是因为小窝蛋白-1上棕榈酸的持续存在阻止了后续的再棕榈酰化。
