Acylation targets endothelial nitric oxide synthase to plasmalemmal caveolae

The endothelial isoform of nitric oxide synthase (ecNOS) is a key determinant of vascular tone and platelet aggregation. We showed previously that ecNOS is targeted to the endothelial paniculate subcellular fraction by two distinct acylations, N-terminal myristoylation and reversible thiopalmitoylation. We now explore the specific paniculate fraction to which ecNOS is targeted. Some palmitoylated proteins are localized to plasmalemmal caveolae. Caveolae are specialized plasma membrane invaginations that may serve to localize cell surface signaling domains, and may contain Gproteins, receptors and calcium channels. Using a newly-developed detergent-free method, we isolated caveolae from endothelial cells and found that ecNOS activity (assayed by 3H-citrulline formation) and protein expression (analyzed by immunoblot) is >9-fold enriched in caveolae relative to plasma membrane fractions. Immunoelectron microscopy also specifically localized ecNOS to endothelial caveolae. In COS-7 cells transfected with cDNA encoding wild-type ecNOS, the enzyme is targeted to caveolae. By contrast, neither palmitoylation- nor myristoylation-deficient ecNOS mutants are localized to caveolae in transfected COS-7 cells. Acylation of ecNOS is therefore required for its caveolar targeting. The localization of ecNOS to plasmalemmal caveolae may play a key role both in enzyme activation and in extracellular NO generation; agonist-induced depalmitoylation of caveolar ecNOS may serve as an important feedback mechanism for NO signaling.