Trafficking of endothelial nitric-oxide synthase in living cells - Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion

To examine endothelial nitric-oxide synthase (eNOS) trafficking in living e ndothelial cells, the eNOS deficient endothelial cell Line ECV304 was stabl y transfected with an eNOS-green fluorescent protein (GFP) fusion construct and characterized by functional, biochemical, and microscopic analysis. eN OS-GFP was co localized with Golgi and plasma membrane markers and produced NO in response to agonist challenge. Localization in the plasma membrane w as dependent on the palmitoylation state, since the palmitoylation mutant o f eNOS (C15S/C26S eNOS-GFP) was excluded from the plasma membrane and was c oncentrated in a diffuse perinuclear pattern. Fluorescence recovery after p hotobleaching (FRAP) revealed eNOS-GFP in the perinuclear region moving 3 t imes faster than the plasmalemmal pool, suggesting that protein-lipid or pr otein-protein interactions are different in these two cellular domains. FRA P of the palmitoylation mutant was two times faster than that of wild-type eNOS-GFP, indicating that palmitoylation was influencing the rate of traffi cking, Interestingly, FRAP of C15S/C26S eNOS-GFP but not wild-type eNOS-GFP fit a model of protein diffusion in a lipid bilayer, These data suggest th at the regulation of eNOS trafficking within the plasma membrane and Golgi are probably different mechanisms and not due to simple diffusion of the pr otein in a lipid bilayer.