Palmitoylation of Inducible Nitric-oxide Synthase at Cys-3 Is Required for Proper Intracellular Traffic and Nitric Oxide Synthesis

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and ·NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker β-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the ·NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization. A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and ·NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker β-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the ·NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization. The gaseous radical nitric oxide (·NO) 1The abbreviations used are: ·NO, radical nitric oxide; ER, endoplasmic reticulum; GFP, green fluorescent protein; IFN, interferon; LPS, lipopolysaccharide; NOS, nitric-oxide synthase; TGN, trans-Golgi network; WT, wild-type; Br, bromo; β-cop, β-coatomer protein. modulates biological function in a wide range of tissue types, acting either as a signaling molecule or as a toxin. Three human NOS isoforms have been cloned and characterized. Among them, NOS2 (sometimes referred to as inducible NOS or iNOS) is mostly involved in the synthesis of the large amounts of ·NO that appear in inflammatory and immunologic processes (1MacMicking J. Xie Q.W. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3500) Google Scholar, 2Stuehr D.J. Biochim. Biophys. Acta. 1999; 1411: 217-230Crossref PubMed Scopus (816) Google Scholar). Both crystallographic and enzymatic studies performed with recombinant proteins expressed in Escherichia coli have shown that the N terminus end of the three mammalian NOSs is not involved in ·NO synthesis but rather in subcellular targeting of the mature polypeptide chain (2Stuehr D.J. Biochim. Biophys. Acta. 1999; 1411: 217-230Crossref PubMed Scopus (816) Google Scholar, 3Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2516) Google Scholar). For instance, the PDZ domain of NOS1 (residues 1-90) interacts with dystrophin and becomes localized to the sarcolemma of fast twitch fibers (4Bredt D.S. J. Cell Sci. 2003; 116: 9-15Crossref PubMed Scopus (129) Google Scholar). In fact, deletion of the first 226 amino acids of NOS1 results in a catalytic protein that synthesizes ·NO at a similar rate than the full-length protein (5Brenman J.E. Chao D.S. Xia H. Aldape K. Bredt D.S. Cell. 1995; 82: 743-752Abstract Full Text PDF PubMed Scopus (850) Google Scholar). Likewise, the N-terminal end of NOS3 is covalently and irreversibly myristoylated at Gly-2 and reversibly palmitoylated at Cys-15 and Cys-26 in a well described process responsible for its targeting to caveolae (6Gratton J.P. Fontana J. O'Connor D.S. García-Cardeña G. McCabe T.J. Sessa W.C. J. Biol. Chem. 2000; 275: 22268-22272Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 7Feron O. Saldana F. Michel J.B. Michel T. J. Biol. Chem. 1998; 273: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). In addition, deletion of the first 52 amino acids of NOS3 does not affect catalytic activity, reflecting that this sequence stretch is not part of the enzymatic machinery but is involved in intracellular traffic (8Rodriguez-Crespo I. Moenne-Loccoz P. Loher T.M. Ortiz de Montellano P.R. Biochemistry. 1997; 36: 8530-8538Crossref PubMed Scopus (55) Google Scholar). As in the case of NOS1 and NOS3, the N-terminal end of NOS2 is very likely involved in subcellular targeting. In fact, deletion of the first 65 amino acids of a recombinant NOS2 expressed in E. coli results in a mutant protein that behaves like the wild-type counterpart (9Ghosh D.K. Wu C. Pitters E. Moloney M. Werner E.R. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10609-10619Crossref PubMed Scopus (154) Google Scholar). However, much less is known about the post-translational modifications of NOS2 in vivo within eukaryotic cells, such as acylation, phosphorylation, or its binding to other cellular proteins. Interestingly, when a peptide corresponding to the first 17 amino acids of NOS2 is used to elicit a rabbit antiserum the resulting antibodies recognize a soluble form of the enzyme, but not its membrane-bound counterpart (10Ringheim G.E. Pan J. Biochem. Biophys. Res. Commun. 1995; 210: 711-716Crossref PubMed Scopus (16) Google Scholar). In contrast, both the particulate and the soluble NOS2 were recognized by a serum elicited against the C terminus end of the protein (10Ringheim G.E. Pan J. Biochem. Biophys. Res. Commun. 1995; 210: 711-716Crossref PubMed Scopus (16) Google Scholar). Likewise, the presence of a post-translational modification in NOS2 resulting in membrane attachment with an increased molecular mass has also been reported (11Vodovotz Y. Russell D. Xie Q.W. Bogdan C. Nathan C. J. Immunol. 1995; 154: 2914-2925PubMed Google Scholar, 12Navarro-Lérida I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar). Although this membrane association of NOS2 has been well characterized in a number of tissues (11Vodovotz Y. Russell D. Xie Q.W. Bogdan C. Nathan C. J. Immunol. 1995; 154: 2914-2925PubMed Google Scholar, 12Navarro-Lérida I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar, 13Gath I. Closs E.I. Godtel-Armbrust U. Schmitt S. Nakane M. Wessler I. Förstermann U. FASEB J. 1996; 10: 1614-1620Crossref PubMed Scopus (122) Google Scholar, 14Schmidt H.H. Warner T.D. Nakane M. Forstermann U. Murad F. Mol. Pharmacol. 1992; 41: 615-624PubMed Google Scholar), the mechanisms underlying the translocation of NOS2 to the particulate fraction and its subsequent dissociation is poorly characterized. We demonstrate in this article that the palmitoylation of NOS2 at Cys-3 is a process necessary for its intracellular transit toward subcellular domains where ·NO synthesis is required. In fact, the site-directed mutant where this Cys-3 has been converted into a Ser residue did not incorporate the radioactive fatty acid and irreversibly aggregated in the Golgi compartment, becoming completely devoid of activity. Cell Culture and Materials—Glutamine, antibiotics, cell culture media (Dulbecco's modified Eagle's medium), sulfanilic acid (4-aminobenzenesulfonic acid), N-(1-naphthyl)ethylenediamine dihydrochloride, LPS (from Salmonella enteriditis), brefeldin A, monensin, 2-Br-palmitate, 8-Br-palmitate, nickel-nitrilotriacetic acid resin, transfection reagents Escort-III and Escort-IV, and Hoescht were purchased from Sigma. Trypsin-EDTA and fetal bovine serum were from BioWhittaker Europe. The source of the various antibodies used in this work is as follows: anti-caveolin-1 and were from and were purchased from and purchased from Sigma. The rabbit serum in as described I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar, I. Álvarez-Barrientos A. Gavilanes F. Rodriguez-Crespo I. J. Cell Sci. PubMed Google Scholar). from human from resin, and and were from The were in modified Eagle's with fetal bovine and in a at of into myotubes performed in modified Eagle's with and in the absence of serum for I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar). of and of the ·NO performed as reported I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar, I. Álvarez-Barrientos A. Gavilanes F. Rodriguez-Crespo I. J. Cell Sci. PubMed Google Scholar). and of the 16 NOS2 have described the and of the full-length wild-type NOS2 I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar), the sequence We have created the mutant NOS2-GFP the single and the and as a full-length NOS2-GFP and as a We the NOS2 the with a at the end and a at the The of the NOS chimeras of NOS2 to the a in the the of the as described I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar, I. Álvarez-Barrientos A. Gavilanes F. Rodriguez-Crespo I. J. Cell Sci. PubMed Google Scholar). The were into the and We the NOS2 in with and the into the corresponding of a I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar, I. Álvarez-Barrientos A. Gavilanes F. Rodriguez-Crespo I. J. Cell Sci. PubMed Google Scholar). In this we a at the end of the as well as a at the end by a with a NOS2-GFP with for and for to a within the NOS2 the from this in the case of the large chimeras and in the case of the into that been with NOS2-GFP used to and in culture Escort-III and the of transfected with the NOS2 were with L. 1995; PubMed Scopus Google in the case of the chimeras described in and with palmitic acid in the other as described I. Álvarez-Barrientos A. Gavilanes F. Rodriguez-Crespo I. J. Cell Sci. PubMed Google Scholar). Both of radioactive and similar a with the media the were and were with culture The were in and the proteins were with antibodies and as described I. Álvarez-Barrientos A. Gavilanes F. Rodriguez-Crespo I. J. Cell Sci. PubMed Google Scholar, J.B. Mol. Biol. Cell. 1999; 10: PubMed Scopus Google Scholar). A transfected but in the absence of also to the of NOS2-GFP and that in were by and membranes of various chimeras performed with the of the serum by of or palmitic acid into the proteins by and of the A performed a and its mutant to that the first amino acids of the N-terminal end of the sequence J.B. Mol. Biol. Cell. 1999; 10: PubMed Scopus Google Scholar). of NOS2 nitric oxide in and we performed the to I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar). A of of with of a sulfanilic acid and of a The to for and the at in of were as In addition, in transfected with the various NOS2 we also performed the to as described I. Ortiz de Montellano P.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of NOS in E. coli and cloned in the used to where the for I. Ortiz de Montellano P.R. Biochem. Biophys. 1996; PubMed Scopus Google Scholar). of media were used for protein at The protein as described Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). of with transfected with the various chimeras were with and for subcellular of the the The were at a wild-type NOS2-GFP antibodies and into that were in of the with for the other with a of of Both and muscular myotubes with a of LPS and a NOS2 be both by and I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar). of with low concentrations of the palmitoylation and 8-Br-palmitate to a in ·NO synthesis Likewise, when NOS2 transfected in cells, the of 8-Br-palmitate a in NOS2 activity in both the of NOS2 remained In with I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar), when we used the wild-type NOS2-GFP to a particulate the be into a perinuclear of the with 8-Br-palmitate In of NOS2 palmitoylation both and 8-Br-palmitate in both in ·NO synthesis and the subcellular of NOS2. NOS2 on we performed site-directed mutagenesis of the N-terminal end of the residue at into a created a recombinant when transfected in cells, devoid of activity and a large to the In addition, the C3S NOS2 mutant to perinuclear in with the shown by the wild-type protein when with the wild-type NOS2 transfected in both in the soluble and particulate an of the C3S mutant completely in the particulate fraction that either the inactive C3S mutant with membrane or the results in an aggregated protein. results also a for the to Cys-3 in the wild-type protein that be by a Ser the proper subcellular sorting of the C3S NOS2 mutant becomes Cys-3 of NOS2 palmitoylated within mammalian cells, we transfected with the of wild-type and C3S mutant with the acid of palmitic acid as described L. 1995; PubMed Scopus Google Scholar). As in the full-length wild-type the radioactive unlike the C3S that to show similar amounts of protein in both myotubes were with palmitic acid and NOS2 induced a of LPS and I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar), a at be in the of to the proinflammatory almost in the of both and myotubes that not been with the of In addition, we recombinant NOS2 expressed in E. coli as described Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). As the as a single of in a Using this recombinant NOS2 Cys-3 is not we concentrations of 8-Br-palmitate its enzymatic activity. In with the results with palmitoylation of NOS2 on mammalian cells, NOS2 does not its ·NO activity in the range of 8-Br-palmitate the in activity in both transfected and NOS2 when with and 8-Br-palmitate be of in the sorting of NOS2 when the of palmitic acid is is to that is a palmitoylation of NOS2 and its proper ·NO synthesis. The in the activity of NOS2 transfected in were also the to As of wild-type NOS2 with and 8-Br-palmitate to of as when the of were the Likewise, the C3S mutant of NOS2 less than of the activity of its wild-type a of the of a palmitic acid to NOS2 a the transfected with the wild-type NOS2-GFP and with acid were by with or of the be in the presence of the results that the NOS2 and the a of NOS2 with the Golgi and we the intracellular traffic of both the NOS2 and C3S chimeras when transfected in We performed a of both with a Golgi marker in or and the cell in is used as a marker of the Golgi and trans-Golgi with I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar, I. Álvarez-Barrientos A. Gavilanes F. Rodriguez-Crespo I. J. Cell Sci. PubMed Google Scholar, J.B. Mol. Biol. Cell. 1999; 10: PubMed Scopus Google Scholar, D. A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the other proteins involved in the the endoplasmic reticulum and the Golgi as well as in antibodies against used as to Y. T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the subcellular of the full-length and C3S mutant of NOS2 the sorting were be the mutant in perinuclear that with both and β-cop, the wild-type a the we the for the and we the shown in Although the wild-type NOS2 with the Golgi in perinuclear the C3S mutant an almost with and that the sorting of the protein has been in the Golgi of the with of the C3S with reflecting that the wild-type has the Golgi in transit to to the the other of with in with the C3S where of its with the is an almost with both the and and the of the C3S of the and of the results not that the full-length NOS2-GFP the traffic machinery of the in transit the but also that the palmitoylation of the residue at of its sequence is required for this and of NOS2-GFP in the that the N-terminal end of NOS2 hydrophobic amino acids and with three basic residues at and the for NOS2 we in amino acids at the N-terminal end of full-length NOS2-GFP in that an residue at as well as and that the full-length chimeras were from the cell and with perinuclear to a Interestingly, of an residue a mutant protein that with the membrane Although the of hydrophobic residues for basic residues seem to in when with the wild-type the of residues at the N-terminal end of NOS2 a in perinuclear in the NOS2 NOS2 activity has been shown to be by G. P. J. S. Sessa W.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and by within muscular I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google we that the full-length NOS2 various targeting the of the N-terminal end in the absence of from the binding that is residues we created chimeras of NOS2-GFP the first amino acids of NOS2 to to subcellular by the N-terminal acylation, a targeting to caveolae. chimeras were of wild-type NOS2-GFP and also of and unlike the full-length of a both the wild-type and NOS2-GFP chimeras a membrane in we the subcellular with the radioactive fatty acid we that palmitoylation be with a of the recombinant in the of the subcellular transit leading to the the other the that to such as and the and in a perinuclear C and the presence of both the basic residues Lys-6 and with the hydrophobic residue Pro-4 seem to be in the proper palmitoylation of the chimeras as well as subcellular In addition, at in the case of and chimeras the targeting by first amino acids of NOS2 a transit the trans-Golgi and in the membrane by in as shown in of the full-length has the and is in transit to the membrane in a the other to the in subcellular by the and chimeras we that of the reported for NOS2 be responsible for this targeting with or of NOS2 in in the case of endothelial NOS, palmitoylation of Cys-15 and Cys-26 is necessary for its translocation to caveolae (6Gratton J.P. Fontana J. O'Connor D.S. García-Cardeña G. McCabe T.J. Sessa W.C. J. Biol. Chem. 2000; 275: 22268-22272Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 7Feron O. Saldana F. Michel J.B. Michel T. J. Biol. Chem. 1998; 273: 3125-3128Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), we domains in where proteins Interestingly, in transfected the wild-type NOS2 the C3S mutant with A in the of in with large We the targeting of and wild-type NOS2-GFP The of the wild-type NOS2-GFP in the the of the and a very of protein with caveolae where a single mutant C3S to with caveolae. The chimeras acids of both wild-type and C3S NOS2 were and a very fraction of the wild-type NOS2 not the C3S be in caveolae of with the membrane of a of the wild-type to from a caveolae transfected with the full-length NOS2 were at and the reflecting that NOS2 to NOS2 with intracellular membranes the of membranes not in in and be in with the in the In fact, to full-length and NOS2-GFP chimeras with in the membrane and in intracellular of the Golgi in the to Golgi with the of NOS2 and with that the proper sorting of NOS2 be indispensable to activity. we the effect of both brefeldin A and monensin on the NOS2 activity and subcellular both in myotubes with and in transfected with a full-length NOS2-GFP A is known to to the dissociation of and other proteins from Golgi resulting in Golgi to the as the Golgi J. J. Cell Biol. 1992; 116: PubMed Scopus Google Scholar). the other the monensin is used to the of the Golgi and to in eukaryotic G. P. G. J. Cell Biol. PubMed Scopus Google Scholar). of NOS2 in muscular myotubes by the of both brefeldin A and monensin drastically the ·NO synthesis to and of the in the in a of the Golgi by a in the NOS2-GFP transfected in also severely affected by with brefeldin A and monensin the of ·NO to and of the with the Likewise, affected the intracellular of NOS2-GFP with brefeldin A resulting in large perinuclear and monensin the of of NOS2 the The of a the we created both a full-length and a that a residue at an residue is in the the Ser residue at the C3S mutant is completely devoid of activity and becomes in the of the we to a at the N-terminal end of NOS2 at a wild-type to some Both the and chimeras in perinuclear is the and C3S mutant In fact, the full-length NOS2 mutant activity when with the polypeptide we palmitic acid and the transfected in cells, a of the be a residue at the N-terminal end of NOS2 a polypeptide that becomes palmitoylated and the wild-type in of subcellular and activity. The described in this work is the that inducible nitric-oxide synthase is with palmitic acid in Cys-3 of its both in NOS2 induced in muscular myotubes with proinflammatory as well as in transfected NOS2 and a However, when recombinant NOS2 expressed in a bacterial the not by of 8-Br-palmitate that a effect when to transfected with NOS2. NOS3, where palmitoylation is a for with and protein NOS2 the for its proper sorting the its synthesis and to the the proper of the NOS2 within the be a process to the of NOS2 be to subcellular with a large of its or its of Cys-3 for Ser in the of fatty acid protein and in subcellular membranes of the Golgi with a of NOS activity. Both basic amino acids and the hydrophobic residue at in of the palmitoylated seem to be for the the subcellular of NOS2 and its as studies performed by and E. F. Biol. Res. PubMed Google Scholar, E. F. C. Sci. U. S. A. 2000; PubMed Scopus Google have that in cells, about of the NOS2 protein with in where it for In fact, of NOS2 at a single on its N terminus E. F. C. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). studies have shown in NOS2 also with becoming the that NOS2 to the I. Portolés M.T. Álvarez-Barrientos A. Gavilanes F. Bosca L. Rodriguez-Crespo I. J. Cell Sci. 2004; 117: 1687-1697Crossref PubMed Scopus (18) Google Scholar). The results described herein with a of NOS2 that with in be when the wild-type and the C3S NOS2 were However, be when the wild-type protein and the C3S mutant were in of sorting the to the and trans-Golgi In fact, of the of the C3S with the marker β-cop, and with the marker The of this C3S with its and perinuclear that its proper subcellular sorting is for the palmitoylation and proper intracellular targeting of other N-terminal palmitoylated proteins such as or M. Cell Biol. 2003; Full Text Full Text PDF PubMed Scopus Google is well For instance, brefeldin A palmitoylation of that palmitoylation in the Golgi or trans-Golgi M. Cell Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). be that the N-terminal amino acids of NOS2 known to with the membrane protein protein of as well as with A. C. C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). it is not NOS2 interacts with proteins its sorting as well as the of it to be palmitoylation affect binding to proteins. we created the we a wild-type is in this to that of the N-terminal palmitoylated proteins the residue at rather than is the for instance, of or it is to in proteins that palmitoylated within the mammalian the at Cys-3 be the at when we characterized the of of the NOS2 in the wild-type and the the palmitoylation by a membrane localization. this not with caveolae when by and that the N-terminal end of NOS2 in a palmitoylation and membrane targeting sequence that the proper of the mature In the of the involved in NOS2 be a in the of inflammatory with increased NOS2

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