Biosynthesis and palmitoylation of endothelial nitric oxide synthase: mutagenesis of palmitoylation sites, cysteines-15 and/or -26, argues against depalmitoylation-induced translocation of the enzyme.
The presence of myristoylated endothelial nitric oxide synthase (eNOS) in cytosolic fractions of bovine aortic endothelial cells (BAEC) suggests that N-myristoylation of eNOS is not sufficient for membrane localization. Therefore, we examined if posttranslational palmitoylation was another molecular signal for the membrane attachment of eNOS. Metabolic labeling showed incorporation of [3H]palmitic acid into the membrane-bound, but not the cytosolic, form of eNOS. Fatty acid analysis demonstrated the labeled fatty acid incorporated into eNOS was palmitate, linked via a hydroxylamine-labile thioester bond. Biosynthesis and turnover studies show that the turnover of palmitate is much faster than the protein itself. However, the rates of palmitoylation and depalmitoylation were not affected by bradykinin or ionomycin treatment of BAEC. To examine the contribution of palmitoylation to the membrane association of eNOS, we mutated cysteine-15 and -26. Both mutations markedly diminished palmitoylation of eNOS, but did not significantly alter its membrane association. Additionally, [3H]palmitic acid was not incorporated into nonmyristoylated mutant eNOS (G2A eNOS), suggesting that myristoylation is necessary for subsequent palmitoylation of the enzyme. Taken together, our data imply that palmitoylation does not play a major role in membrane association of eNOS and other amino acid sequences, in conjunction with N-myristoylation, are necessary and sufficient for membrane association of the enzyme.
Liu
J
J
Molecular Cardiobiology Program, Yale University School of Medicine, New Haven, Connecticut 06536-0812, USA.
García-Cardeña
G
G
Sessa
W C
WC
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation; D003545:Cysteine / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D004730:Endothelium, Vascular / Q000201:enzymology*; D015759:Ionomycin / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D014162:Transfection
D009227:Myristic Acids; D010169:Palmitic Acids; D011994:Recombinant Proteins; D019814:Myristic Acid; D019308:Palmitic Acid; D015759:Ionomycin; D019001:Nitric Oxide Synthase; D003545:Cysteine; D001920:Bradykinin; D002118:Calcium