A chimeric transmembrane domain directs endothelial nitric-oxide synthase palmitoylation and targeting to plasmalemmal caveolae

Abstract

The endothelial nitric-oxide synthase (eNOS), a key signaling protein, undergoes a series of covalent modifications, including co-translational N- myristoylation at Gly2, as well as post-translational thiopalmitoylation at Cys15 and Cys26. Myristoylation of eNOS is required for the subsequent palmitoylation of the enzyme, and both acylations are required for the efficient subcellular targeting of eNOS to plasmalemmal caveolae. We constructed chimeric cDNAs encoding proteins comprised of various acylation- deficient eNOS mutants fused at their N termini to the hydrophobic transmembrane domain of the glycoprotein CD8 and characterized these constructs in transient transfection experiments in COS-7 cells. One construct (termed CD8-myr-eNOS) encodes a fusion protein comprised of the eNOS myristoylation-deficient mutant coupled to the CD8 transmembrane domain. In biosynthetic labeling experiments using [3H]palmitic acid, we found that the CD8-myr-eNOS chimera undergoes palmitoylation. Subcellular fractionation showed that the CD8-myr-eNOS chimera is targeted to caveolae. We also constructed and characterized a cDNA encoding the CD8 transmembrane domain fused to the palmitoylation-deficient mutant eNOS (in which Cys15 and Cys26 are changed to serine). This chimera (termed CDS-myr-·palm-eNOS) did not undergo palmitoylation, indicating that the palmitoylation seen with the CD8-myr-eNOS fusion protein occurs on the same residues as in the wild- type enzyme. Importantly, the CD8-myr-·palm-eNOS fusion protein remained efficiently targeted to caveolae, in contrast to the palm-eNOS mutant lacking the CD8 transmembrane domain, which has nominal caveolar localization. A construct encoding the CD8 transmembrane domain alone was insufficient for selective targeting to caveolae. These results indicate that membrane targeting per se, but not necessarily myristoylation, is sufficient for eNOS palmitoylation and localization to plasmalemmal caveolae, and suggest further that sequences within eNOS itself, in addition to its palmitoylation sites, facilitate the selective localization of the enzyme within caveolae.