{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T00:48:00Z","timestamp":1792716480116,"version":"4.5.0"},"reference-count":48,"publisher":"Rockefeller University Press","issue":"7","content-domain":{"domain":["rupress.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[1997,6,30]]},"abstract":"<jats:p>Catalytically active endothelial nitric oxide synthase (eNOS) is located on the Golgi complex and in the caveolae of endothelial cells (EC). Mislocalization of eNOS caused by mutation of the N-myristoylation or cysteine palmitoylation sites impairs production of stimulated nitric oxide (NO), suggesting that intracellular targeting is critical for optimal NO production. To investigate the molecular determinants of eNOS targeting in EC, we constructed eNOS\u2013green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The full-length eNOS\u2013GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, suggesting that the GFP tag does not interfere with eNOS localization or function. Experiments with different size amino-terminal fusion partners coupled to GFP demonstrated that the first 35 amino acids of eNOS are sufficient to target GFP into the Golgi region of NIH 3T3 cells. Additionally, the unique (Gly-Leu)5 repeat located between the palmitoylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoylation and thus localization, but not for N-myristoylation, membrane association, and NOS activity. The palmitoylation-deficient mutants displayed a more diffuse fluorescence pattern than did WT eNOS\u2013GFP, but still were associated with intracellular membranes. Biochemical studies also showed that the palmitoylation-deficient mutants are associated with membranes as tightly as WT eNOS. Mutation of the N-myristoylation site Gly-2 (abolishing both N-myristoylation and palmitoylation) caused the GFP fusion protein to distribute throughout the cell as GFP alone, consistent with its primarily cytosolic nature in biochemical studies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells via the first 35 amino acids, including N-myristoylation and palmitoylation sites, and its overall membrane association requires N-myristoylation but not cysteine palmitoylation. These results suggest a novel role for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex.<\/jats:p>","DOI":"10.1083\/jcb.137.7.1525","type":"journal-article","created":{"date-parts":[[2002,7,26]],"date-time":"2002-07-26T12:47:50Z","timestamp":1027687670000},"page":"1525-1535","update-policy":"https:\/\/doi.org\/10.1083\/jcb.crossmarkpolicy","source":"Crossref","is-referenced-by-count":160,"title":["The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study"],"prefix":"10.1083","volume":"137","author":[{"given":"Jianwei","family":"Liu","sequence":"first","affiliation":[{"name":"*Molecular Cardiobiology Program and the Department of Pharmacology, Boyer Center for Molecular Medicine, \u2021Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06536-0812"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas E.","family":"Hughes","sequence":"additional","affiliation":[{"name":"*Molecular Cardiobiology Program and the Department of Pharmacology, Boyer Center for Molecular Medicine, \u2021Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06536-0812"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William C.","family":"Sessa","sequence":"additional","affiliation":[{"name":"*Molecular Cardiobiology Program and the Department of Pharmacology, Boyer Center for Molecular Medicine, \u2021Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06536-0812"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[1997,6,30]]},"reference":[{"key":"2023072823512036700_B1","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1073\/pnas.87.2.682","article-title":"Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme","volume":"87","author":"Bredt","year":"1990","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B2","doi-asserted-by":"crossref","first-page":"802","DOI":"10.1126\/science.8303295","article-title":"Green fluorescent protein as a marker for gene expression","volume":"263","author":"Chalfie","year":"1994","journal-title":"Science (Wash DC)"},{"key":"2023072823512036700_B3","doi-asserted-by":"crossref","first-page":"11728","DOI":"10.1073\/pnas.91.24.11728","article-title":"Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin","volume":"91","author":"Chun","year":"1994","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B4","doi-asserted-by":"crossref","first-page":"1421","DOI":"10.1083\/jcb.131.6.1421","article-title":"Caveolin cycles between plasma membrane caveolae and the Golgi complex by microtubule-dependent and microtubule-independent steps","volume":"131","author":"Conrad","year":"1995","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B5","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1083\/jcb.133.5.1027","article-title":"Differential distribution of \u03b1 subunits and \u03b2\u03b3 subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas","volume":"133","author":"Denker","year":"1996","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B6","doi-asserted-by":"crossref","first-page":"7154","DOI":"10.1074\/jbc.271.12.7154","article-title":"G-protein palmitoyltransferase activity is enriched in plasma membranes","volume":"271","author":"Dunphy","year":"1996","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B7","doi-asserted-by":"crossref","first-page":"22810","DOI":"10.1074\/jbc.271.37.22810","article-title":"Endothelial nitric oxide synthase targeting to caveolae","volume":"271","author":"Feron","year":"1996","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B8","doi-asserted-by":"crossref","first-page":"1788","DOI":"10.1073\/pnas.88.5.1788","article-title":"Calmodulin-dependent endothelium-derived relaxing factor\/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells","volume":"88","author":"Forstermann","year":"1991","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B9","doi-asserted-by":"crossref","first-page":"6448","DOI":"10.1073\/pnas.93.13.6448","article-title":"Targeting of nitric oxide synthase to endothelial caveolae via palmitoylation","volume":"93","author":"Garc\u00eda-Carde\u00f1a","year":"1996","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B10","doi-asserted-by":"crossref","first-page":"27237","DOI":"10.1074\/jbc.271.44.27237","article-title":"Endothelial nitric oxide synthase is regulated by tyrosine phosphorylation and interacts with caveolin-1","volume":"271","author":"Garc\u00eda-Carde\u00f1a","year":"1996","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B11","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1083\/jcb.133.5.1007","article-title":"Multiple features of the p59fynsrc homology 4 domain define a motif for immune receptor\u2013based activation motif (ITAM) binding and for plasma membrane localization","volume":"133","author":"Gauen","year":"1996","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B12","first-page":"147","article-title":"Characterization of an acyltransferase acting on p21N-ras protein in a cell-free system","volume":"1078","author":"Gutierrez","year":"1991","journal-title":"Biochem Biophys Acta"},{"key":"2023072823512036700_B13","doi-asserted-by":"crossref","first-page":"4845","DOI":"10.1073\/pnas.93.10.4845","article-title":"Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera","volume":"93","author":"Htun","year":"1996","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B14","doi-asserted-by":"crossref","first-page":"1744","DOI":"10.1126\/science.274.5293.1744","article-title":"A role for endothelial NO synthase in LTP revealed by adenovirus-mediated inhibition and rescue","volume":"274","author":"Kantor","year":"1996","journal-title":"Science (Wash DC)"},{"key":"2023072823512036700_B15","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1083\/jcb.125.3.639","article-title":"Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes","volume":"125","author":"Ley","year":"1994","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B16","doi-asserted-by":"crossref","first-page":"11691","DOI":"10.1016\/S0021-9258(17)32626-1","article-title":"Identification of covalently bound amino-terminal myristic acid in endothelial nitric oxide synthase","volume":"269","author":"Liu","year":"1994","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B17","doi-asserted-by":"crossref","first-page":"12333","DOI":"10.1021\/bi00038a029","article-title":"Biosynthesis and palmitoylation of endothelial nitric oxide synthase: mutagenesis of palmitoylation sites, cysteine 15 and\/or 26, argue against depalmitoylation induced translocation of the enzyme","volume":"34","author":"Liu","year":"1995","journal-title":"Biochemistry"},{"key":"2023072823512036700_B18","doi-asserted-by":"crossref","first-page":"13277","DOI":"10.1021\/bi961720e","article-title":"Palmitoylation of endothelial nitric oxide synthase is necessary for optimal stimulated release of nitric oxide: implications for caveolae localization","volume":"35","author":"Liu","year":"1996","journal-title":"Biochemistry"},{"key":"2023072823512036700_B19","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/0896-6273(95)90279-1","article-title":"The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function","volume":"14","author":"Marshall","year":"1995","journal-title":"Neuron"},{"key":"2023072823512036700_B20","doi-asserted-by":"crossref","first-page":"6252","DOI":"10.1073\/pnas.90.13.6252","article-title":"Phosphorylation and subcellular translocation of endothelial nitric oxide synthase","volume":"90","author":"Michel","year":"1993","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B21","doi-asserted-by":"crossref","first-page":"10945","DOI":"10.1016\/S0021-9258(18)67479-4","article-title":"Topology of mannosidase II in rat liver Golgi membranes and release of the catalytic domain by selective proteolysis","volume":"261","author":"Moreman","year":"1986","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B22","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1016\/S0896-6273(00)80044-6","article-title":"Labeling neural cells using adenoviral gene transfer of membrane targeted GFP","volume":"16","author":"Moriyoshi","year":"1996","journal-title":"Neuron"},{"key":"2023072823512036700_B23","doi-asserted-by":"crossref","first-page":"2800","DOI":"10.1073\/pnas.91.7.2800","article-title":"Receptor regulation of G-protein palmitoylation","volume":"91","author":"Mumby","year":"1994","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B24","doi-asserted-by":"crossref","first-page":"10339","DOI":"10.1073\/pnas.92.22.10339","article-title":"VIP21\/caveolin is a cholesterol-binding protein","volume":"92","author":"Murata","year":"1995","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B25","doi-asserted-by":"crossref","first-page":"10436","DOI":"10.1021\/bi00090a020","article-title":"Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins","volume":"32","author":"Peitzsch","year":"1993","journal-title":"Biochemistry"},{"key":"2023072823512036700_B26","doi-asserted-by":"crossref","first-page":"10480","DOI":"10.1073\/pnas.88.23.10480","article-title":"Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells","volume":"88","author":"Pollock","year":"1991","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B27","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/0378-1119(92)90691-H","article-title":"Primary structure of the Aequorea victoria green-fluorescent protein","volume":"111","author":"Prasher","year":"1992","journal-title":"Gene (Amst)"},{"key":"2023072823512036700_B28","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1016\/0092-8674(94)90104-X","article-title":"Myristoylation and palmitoylation of Src family members: the fats of the matter","volume":"76","author":"Resh","year":"1994","journal-title":"Cell"},{"key":"2023072823512036700_B29","doi-asserted-by":"crossref","first-page":"3507","DOI":"10.1128\/MCB.15.7.3507","article-title":"Myristoylation and differential palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and association with caveolae","volume":"15","author":"Robbins","year":"1995","journal-title":"Mol Cell Biol"},{"key":"2023072823512036700_B30","doi-asserted-by":"crossref","first-page":"11776","DOI":"10.1073\/pnas.92.25.11776","article-title":"Mutagenesis of palmitoylation sites in endothelial nitric oxide synthase identifies a novel motif for dual acylation and subcellular targeting","volume":"92","author":"Robinson","year":"1995","journal-title":"Proc Natl Acad Sci USA"},{"key":"2023072823512036700_B31","doi-asserted-by":"crossref","first-page":"13713","DOI":"10.1016\/S0021-9258(19)76485-0","article-title":"Palmitoylation, sulfation, and glycosylation of the alpha subunit of the sodium channel. Role of post-translational modifications in channel assembly","volume":"262","author":"Schmidt","year":"1987","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B32","doi-asserted-by":"crossref","first-page":"1217","DOI":"10.1083\/jcb.127.5.1217","article-title":"Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules","volume":"127","author":"Schnitzer","year":"1994","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B33","doi-asserted-by":"crossref","first-page":"14399","DOI":"10.1074\/jbc.270.24.14399","article-title":"Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexin, and GTPases","volume":"270","author":"Schnitzer","year":"1995","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B34","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1126\/science.274.5285.239","article-title":"Role of GTP hydrolysis in fission of caveolae directly from plasma membranes","volume":"274","author":"Schnitzer","year":"1996","journal-title":"Science (Wash DC)"},{"key":"2023072823512036700_B35","doi-asserted-by":"crossref","first-page":"921","DOI":"10.1161\/01.RES.72.4.921","article-title":"Mutation of N-myristoylation site converts endothelial cell nitric oxide synthase from a membrane to a cytosolic protein","volume":"72","author":"Sessa","year":"1993","journal-title":"Circ Res"},{"key":"2023072823512036700_B36","doi-asserted-by":"crossref","first-page":"17641","DOI":"10.1074\/jbc.270.30.17641","article-title":"The Golgi association of endothelial nitric oxide synthase is necessary for the efficient synthesis of nitric oxide","volume":"270","author":"Sessa","year":"1995","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B37","doi-asserted-by":"crossref","first-page":"6518","DOI":"10.1074\/jbc.271.11.6518","article-title":"Acylation targets endothelia nitric-oxide synthase to plasmalemmal caveolae","volume":"271","author":"Shaul","year":"1996","journal-title":"J Biol Chem"},{"key":"2023072823512036700_B38","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1083\/jcb.126.2.353","article-title":"Cysteine 3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae","volume":"126","author":"Shenoy-Scaria","year":"1994","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B39","doi-asserted-by":"crossref","first-page":"927","DOI":"10.1083\/jcb.124.6.927","article-title":"Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65","volume":"124","author":"Shi","year":"1994","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B40","doi-asserted-by":"crossref","first-page":"1185","DOI":"10.1083\/jcb.127.5.1185","article-title":"Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation","volume":"127","author":"Smart","year":"1994","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B41","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1083\/jcb.124.3.307","article-title":"Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae","volume":"124","author":"Smart","year":"1994","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B42","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1083\/jcb.131.4.929","article-title":"Hormonal regulation of caveolae internalization","volume":"131","author":"Smart","year":"1995","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B43","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1083\/jcb.126.2.331","article-title":"A signal located within amino acids 1\u201327 of GAD65 is required for its targeting to the Golgi complex region","volume":"126","author":"Solimena","year":"1994","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B44","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1006\/viro.1995.0016","article-title":"Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm","volume":"213","author":"Stauber","year":"1995","journal-title":"Virology"},{"key":"2023072823512036700_B45","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1083\/jcb.114.6.1113","article-title":"A heterotrimeric G protein, G\u03b1i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC\u2013PK1 epithelial cells","volume":"114","author":"Stow","year":"1991","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B46","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1083\/jcb.136.5.1023","article-title":"Rapid plasma membrane anchoring of newly synthesized p59fyn: selective requirement for NH2-terminal myristoylation and palmitoylation at cysteine-3","volume":"136","author":"van't Hof","year":"1997","journal-title":"J Cell Biol"},{"key":"2023072823512036700_B47","doi-asserted-by":"crossref","first-page":"703","DOI":"10.1006\/bbrc.1996.1417","article-title":"Bradykinin-stimulated protein tyrosine phosphorylation promotes endothelial nitric oxide synthase translocation to the cytoskeleton","volume":"226","author":"Venema","year":"1996","journal-title":"Biochem Biophys Res Commun"},{"key":"2023072823512036700_B48","doi-asserted-by":"crossref","first-page":"503","DOI":"10.1074\/jbc.270.2.503","article-title":"Lipid modifications of trimeric G proteins","volume":"270","author":"Wedegaertner","year":"1995","journal-title":"J Biol Chem"}],"container-title":["The Journal of Cell Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/rupress.org\/jcb\/article-pdf\/137\/7\/1525\/1859641\/14518.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/rupress.org\/jcb\/article-pdf\/137\/7\/1525\/1859641\/14518.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,7,28]],"date-time":"2023-07-28T19:52:09Z","timestamp":1690573929000},"score":1,"resource":{"primary":{"URL":"https:\/\/rupress.org\/jcb\/article\/137\/7\/1525\/15521\/The-First-35-Amino-Acids-and-Fatty-Acylation-Sites"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1997,6,30]]},"references-count":48,"journal-issue":{"issue":"7","published-print":{"date-parts":[[1997,6,30]]}},"URL":"https:\/\/doi.org\/10.1083\/jcb.137.7.1525","relation":{},"ISSN":["0021-9525","1540-8140"],"issn-type":[{"value":"0021-9525","type":"print"},{"value":"1540-8140","type":"electronic"}],"subject":[],"published":{"date-parts":[[1997,6,30]]}}}