Acylation targets endothelial nitric oxide synthase (enos) to plasmalemmal caveolae

eNOS generates the key signalling molecule nitric oxide (NO) in response to intralumenal hormonal and mechanical stimuli. We designed studies to determine whether eNOS is localized to caveolae, which are invaginated plasma membrane (PM) microdomains implicated in signal transduction. Using immunoblot analysis, eNOS protein was detected in caveolar membrane (CM) fractions isolated from endothelial cell PM by a newly-developed detergent-free method; eNOS protein was not found in noncaveolar plasma membrane (NCPM). Similarly, NOS enzymatic activity was 9.4-fold enriched in CM versus PM, and it was undetectable in NCPM. 51-86% of total NOS activity in postnuclear supernatant was recovered in PM, and 57-100% of activity in PM was recovered in CM. Immunoelectron microscopy for eNOS localized the enzyme to endothelial caveolae, whereas coated pits and smooth PM were devoid of immunosignal. Furthermore, eNOS was targeted to caveolae in COS-7 cells transfected with wild-type eNOS cDNA. Studies with eNOS mutants revealed that both myristoylation and palmitoylation are required to target eNOS to caveolae, and that each acylation process enhancing targeting by 10-fold. Thus, acylation targets eNOS to caveolae. Localization to this microdomain is likely to optimize eNOS activation and extracellular NO release.