Docking of Endothelial Nitric Oxide Synthase (eNOS) to the Mitochondrial Outer Membrane

Despite growing evidence for a mitochondrial localization of nitric oxide (NO) synthase and a broadening spectrum of NO actions on mitochondrial respiration and apoptosis, the basis for interaction between the enzyme and the organelle remain obscure. Here we investigated mitochondrial localization of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells and human embryonic kidney cells transfected or infected with eNOS expression vectors. Copurification of eNOS with mitochondria was observed in both human umbilical vein endothelial cells and eNOS-expressing human embryonic kidney cells. Immunodetectable eNOS was cleaved from mitochondria by proteinase K treatment, suggesting eNOS association with the outer mitochondrial membrane. Localization of eNOS to a proteinase K-cleavable site on the cytoplasmic face of the outer membrane was confirmed by immunogold labeling of non-permeabilized mitochondria. Markers for mitochondrial subfractions ruled out the possibility of eNOS association with an intramitochondrial site or inverted mitochondrial particles. Denaturation of eNOS did not attenuate association with mitochondria. Mutant eNOS lacking a pentabasic amino acid sequence within the autoinhibitory domain (residues 628–632 of the bovine eNOS) showed dramatically reduced binding to the mitochondrial but not to the plasma membrane, which was associated with increased oxygen consumption. Collectively, these findings argue in favor of eNOS localization to the outer mitochondrial membrane in endothelial cells and identify elements of a novel anchoring mechanism. Despite growing evidence for a mitochondrial localization of nitric oxide (NO) synthase and a broadening spectrum of NO actions on mitochondrial respiration and apoptosis, the basis for interaction between the enzyme and the organelle remain obscure. Here we investigated mitochondrial localization of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells and human embryonic kidney cells transfected or infected with eNOS expression vectors. Copurification of eNOS with mitochondria was observed in both human umbilical vein endothelial cells and eNOS-expressing human embryonic kidney cells. Immunodetectable eNOS was cleaved from mitochondria by proteinase K treatment, suggesting eNOS association with the outer mitochondrial membrane. Localization of eNOS to a proteinase K-cleavable site on the cytoplasmic face of the outer membrane was confirmed by immunogold labeling of non-permeabilized mitochondria. Markers for mitochondrial subfractions ruled out the possibility of eNOS association with an intramitochondrial site or inverted mitochondrial particles. Denaturation of eNOS did not attenuate association with mitochondria. Mutant eNOS lacking a pentabasic amino acid sequence within the autoinhibitory domain (residues 628–632 of the bovine eNOS) showed dramatically reduced binding to the mitochondrial but not to the plasma membrane, which was associated with increased oxygen consumption. Collectively, these findings argue in favor of eNOS localization to the outer mitochondrial membrane in endothelial cells and identify elements of a novel anchoring mechanism. An association of NOS-like proteins with mitochondria has previously been demonstrated immunohistochemically (1Bates T.E. Loesch A. Burnstock G. Clark J.B. Biochem. Biophys. Res. Commun. 1995; 213: 896-900Crossref PubMed Scopus (186) Google Scholar, 2Kobzik L. Stringer B. Balligand J.L. Reid M.B. Stamler J.S. Biochem. Biophys. Res. Commun. 1995; 211: 375-381Crossref PubMed Scopus (358) Google Scholar, 3Bates T.E. Loesch A. Burnstock G. Clark J.B. Biochem. Biophys. Res. Commun. 1996; 218: 40-44Crossref PubMed Scopus (223) Google Scholar, 4Frandsen U. Lopez-Figueroa M. Hellsten Y. Biochem. Biophys. Res. Commun. 1996; 227: 88-93Crossref PubMed Scopus (193) Google Scholar). Further supporting evidence was provided by the partial purification of mitochondrial nitric oxide synthase (mtNOS) 1The abbreviations used are: mtNOS, mitochondrial nitric oxide synthase; eNOS, endothelial NOS; rbeNOS, recombinant bovine eNOS; beNOS, bovine eNOS; AID, autoinhibitory domain; HEK, human embryonic kidney cells; HUVEC, human umbilical vein endothelial cell; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; BSA, bovine serum albumin; PBS, phosphate-buffered saline; VDAC, voltage-dependent anion-selective channel; COX, cytochrome c oxidase; L-NAME, nitro-l-arginine methyl ester; NO, nitric oxide; EBM-2, endothelial cell basal medium-2; Mn-SOD, manganese superoxide dismutase. activity (5Ghafourifar P. Richter C. FEBS Lett. 1997; 418: 291-296Crossref PubMed Scopus (537) Google Scholar, 6Tatoyan A. Giulivi C. J. Biol. Chem. 1998; 273: 11044-11048Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). There is an ongoing debate as to the identity of mtNOS; immunologic cross-reaction of mtNOS with antibodies against endothelial NOS (eNOS) (1Bates T.E. Loesch A. Burnstock G. Clark J.B. Biochem. Biophys. Res. Commun. 1995; 213: 896-900Crossref PubMed Scopus (186) Google Scholar, 3Bates T.E. Loesch A. Burnstock G. Clark J.B. Biochem. Biophys. Res. Commun. 1996; 218: 40-44Crossref PubMed Scopus (223) Google Scholar, 7Lacza Z. Puskar M. Figueroa J.P. Zhang J. Rajapakse N. Busija D.W. Free Radic. Biol. Med. 2001; 31: 1609-1615Crossref PubMed Scopus (126) Google Scholar), neuronal NOS (4Frandsen U. Lopez-Figueroa M. Hellsten Y. Biochem. Biophys. Res. Commun. 1996; 227: 88-93Crossref PubMed Scopus (193) Google Scholar), and inducible NOS (6Tatoyan A. Giulivi C. J. Biol. Chem. 1998; 273: 11044-11048Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) have been reported. A recent investigation (8Kanai A.J. Pearce L.L. Clemens P.R. Birder L.A. VanBibber M.M. Choi S.Y. de Groat W.C. Peterson J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14126-14131Crossref PubMed Scopus (322) Google Scholar) has identified a neuronal NOS in cardiomyocyte mitochondria. It has been suggested that mtNOS plays important roles in oxidative stress and apoptosis (9Ghafourifar P. Bringold U. Klein S.D. Richter C. Biol. Signals Recept. 2001; 10: 57-65Crossref PubMed Scopus (87) Google Scholar, 10Ghafourifar P. Bringold U. Klein S.D. Richter C. J. Biol. Chem. 1999; 274: 31185-31188Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), regulation of mitochondrial respiration (11Brown G.C. Biochim. Biophys. Acta. 2001; 1504: 46-57Crossref PubMed Scopus (517) Google Scholar, 12Ghafourifar P. Richter C. Biol. Chem. 1999; 380: 1025-1028Crossref PubMed Scopus (66) Google Scholar), and modulation of intracellular Ca2+ homeostasis (13Bringold U. Ghafourifar P. Richter C. Free Radic. Biol. Med. 2000; 29: 343-348Crossref PubMed Scopus (76) Google Scholar). The potential distribution of mtNOS in subfractions of mitochondria has been explored but remains enigmatic. Some indirect evidence suggests that mtNOS is localized to the inner mitochondrial membrane. Indeed, immunohistochemical findings suggest that mtNOS co-localizes with succinate dehydrogenase, a mitochondrial marker for the inner membrane (2Kobzik L. Stringer B. Balligand J.L. Reid M.B. Stamler J.S. Biochem. Biophys. Res. Commun. 1995; 211: 375-381Crossref PubMed Scopus (358) Google Scholar). Further support for an inner membrane localization of NOS in mitochondria came from NOS activity assays, which indicated that specific activity in submitochondrial particles and crude fractions was higher than that of mitochondrial homogenates or permeabilized mitochondria (14Giulivi C. Poderoso J.J. Boveris A. J. Biol. Chem. 1998; 273: 11038-11043Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). In contrast, a recent report (15Reiner M. Bloch W. Addicks K. J. Histochem. Cytochem. 2001; 49: 1605-1610Crossref PubMed Scopus (27) Google Scholar) suggested that eNOS localizes to the outer membrane of mitochondria, as judged from immunoelectron microscopy of intact endothelial cells. In the present study, we sought to examine the molecular targeting of eNOS to mitochondria. Using three independent experimental approaches, we have verified that eNOS is present on mitochondria of HUVEC and specifically localized to the cytoplasmic face of the outer mitochondrial membrane. In vitro experiments with isolated mitochondria show that native and denatured eNOS interact with a proteinase K-hydrolyzable protein that participates in eNOS recruitment to mitochondria. Deletion analysis revealed a stretch of five basic amino acids in the autoinhibitory domain (AID) of eNOS that is required for anchoring the enzyme to the mitochondrial outer membrane. Tethering of eNOS by its AID would predictably disinhibit enzymatic activity, allowing for NO synthesis by the mitochondrial membrane-bound eNOS. One of the consequences of the reduced mitochondrial association of AID-deleted eNOS is the increase in oxygen consumption. Reagents and Antibodies—ATP, mannitol, phenylmethylsulphonyl fluoride, proteinase K, and nicotinamide adenine dinucleotide were obtained from Sigma. Precast gradient 4–20% Tris-glycine gels (1.0 mm) were obtained from Invitrogen. Mouse anti-eNOS monoclonal antibody was purchased from Transduction Laboratories (Los Angeles, CA). Mouse anti-human cytochrome oxidase subunit II monoclonal antibody, Alexa-Fluor goat anti-mouse IgG, and Alexa-Fluor goat anti-rabbit IgG were obtained from Molecular Probes (Eugene, OR). Rabbit anti-fumarase polyclonal antibody was obtained from Accurate Chemical and Scientific Corp. (Westbury, NY). Mouse anti-human VDAC monoclonal antibody (31HL) was purchased from Calbiochem (San Diego, CA). Mouse anti-cytochrome c monoclonal antibody was obtained from BD Biosciences (Los Angeles, CA). Mouse anti-Na+/K+ á-1 subunit monoclonal antibody was purchased from Research Diagnostics, Inc. (Flanders, NJ). Mouse anti-Golgi marker 58K protein monoclonal antibody was obtained from Abcam (UK). Rabbit anti-caveolin-1 polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse IgG horseradish peroxidase-conjugated whole antibody from sheep and anti-rabbit IgG horseradish peroxidase-conjugated whole antibody from donkey were obtained from Amersham Biosciences (UK), Percoll was from Amersham Biosciences (Piscataway, NJ), DMEM was from Biofluids (Rockville, MD), and EBM-2 was a product of Clonetics (San Diego, CA). Cell Culture—Human umbilical vein endothelial cells (HUVEC) were grown in EBM-2 medium containing 2% fetal bovine serum, and used in experiments at the 6th passage. Human embryonic kidney (HEK) cells and HEK cells stably transfected with a pcDNA3 vector that engenders stable expression of wild-type bovine eNOS (HEK-eNOS; a gift of Dr. William Sessa, Yale University School of Medicine) or a truncated enzyme lacking the autoinhibitory domain (HEK-ΔeNOS, 628–632 deletion) were cultured in DMEM containing 10% fetal bovine serum. Construction, Propagation, and Purification of Adenoviral Vectors for Bovine eNOS (beNOS) Protein Expression—Construction of recombinant Ad-GFP-beNOS viruses was carried out following the manufacturer's protocol (Adeno-Quest, Quantum Biotechnologies Inc., Montreal, Canada). Briefly, cDNA-encoding bovine eNOS was subcloned into pQBIAd-GFP-IRES (ligating the flanking BamHI sites eNOS to BglII cloning site on the vector). The vector carries an adenovirus origin of replication and packaging sequence, cytomegalovirus promoter, and an internal ribosome entry site element that provides expression of green fluorescent protein (GFP) and the inserted gene (wild-type eNOS or an eNOS deletion mutant lacking the oligonucleotide sequence that encodes amino acid residues 628–632, RRKRK) in a one-to-one ratio. The vector cloning site (Bg1II) is followed by a DNA sequence homologous to Ad5 9.4–15.5 map units. Recombinant adenoviruses were produced by homologous recombination during cotransfection of the linearized plasmid vector with the QBI viral DNA into HEK-293A cells. Transfected 293A monolayers were overlaid with an agar-DMEM mixture and incubated at 37 °C with 5% CO2. Expression of GFP and the appearance of plaques were tracked by fluorescence microscopy, and plaques were isolated as soon as they were identifiable. Plaque lysates were subjected to three rounds of plaque purification, expanded on HEK 293A cell monolayers, and finally purified on CsCl step and continuous gradients, respectively. Viral titers ranged from 5 × 1012 viral particles per ml and were stored at –20 °C in a glycerol/BSA storage solution. Adenoviral Infection of HEK Cells—HEK cells were plated in 10-cm plates to reach about 60–70% confluence at the time of transfection. The culture medium was removed, and 200 virus particles/cell were added to each plate in 2-ml serum-free DMEM. After incubation for 1 h at 37 °C with shaking, 8 ml of fresh complete DMEM were added. Cells were harvested for the isolation of mitochondria 6–24 h after infection. Isolation of Mitochondria—All procedures were carried out at 4 °C unless were isolated by as K. de B. Biochim. Biophys. Acta. PubMed Scopus (311) Google Scholar). Briefly, cells were with PBS, into mannitol, and with a Cell homogenates were at × for in a the was and the was at × for The mitochondria was with mitochondria were obtained by Percoll gradient K. de B. Biochim. Biophys. Acta. PubMed Scopus (311) Google Scholar). Briefly, crude mitochondrial were in mannitol, 1 5 BSA, and was ml of Percoll in mannitol, 1 BSA, and at × for in a The mitochondrial was from the of the with to and in a of to a of of mitochondria was to the protocol by A. U. B. J. N. Biol. 2000; PubMed Scopus Google Scholar). in were with five of to the outer membrane by After of incubation on an of was added to the were isolated by and in of Recombinant Bovine eNOS to mitochondria and of BSA, 5 nicotinamide adenine 1 were for 5 at °C the of 1 of purified recombinant bovine eNOS After at °C for 1 of were added to the were by and with protein and analysis by K were with proteinase K at a of a for at was added to a of and were incubated for a at were by and was by procedures on 4–20% Tris-glycine gels were to and incubated with The were incubated with horseradish peroxidase-conjugated IgG and with an antibodies were used at the following anti-eNOS antibody antibody anti-fumarase antibody anti-cytochrome c oxidase antibody anti-cytochrome c antibody and antibody antibodies were used at a of were incubated with 10% fetal bovine serum in for h and with anti-eNOS monoclonal antibody at for 1 h at were three with PBS, incubated for 1 h with anti-mouse antibody at at and three with were in PBS, and fluorescence was by a fluorescence at of and of fractions were with 10% fetal bovine serum in for h at incubated with anti-eNOS monoclonal antibody at for 1 or were and in 200 of goat anti-mouse IgG with 5 in containing and for 1 h at were to a and in A and in were for in 2% acid in and in at 4 were with with and with an and in were at a on an were with and in a at of cells were transfected with and adenovirus at cells were with and containing and in the at a of 1 cell was in a with a was an to a the of each succinate and were added to that in from mitochondrial was as the of in an of and was as per per cells. The of eNOS and expression on were as from the of adenovirus HEK cells was as the of of from each of cells were the and the for each and were of HUVEC fractions that were for eNOS. from analysis of eNOS protein by suggest that mitochondrial eNOS is of lysates The of eNOS after the mitochondria from HUVEC were with proteinase K and but was to mitochondrial by proteinase K has been used to proteins from the of mitochondrial outer membrane K. G. B. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, N. W. J. Biol. Chem. Full Text PDF PubMed Google Scholar, W. A. N. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (66) Google Scholar). the submitochondrial localization of NOS in HUVEC, subfractions of mitochondria were identified by VDAC for outer membrane M. J. Biol. 1997; PubMed Scopus Google Scholar), cytochrome c for J. N. Cell Biol. PubMed Scopus Google Scholar), for inner membrane K. de B. Biochim. Biophys. Acta. PubMed Scopus (311) Google Scholar), and for K. de B. Biochim. Biophys. Acta. PubMed Scopus (311) Google Scholar). The on of the outer mitochondrial membrane marker VDAC was to after with proteinase K, with VDAC in the outer mitochondrial membrane and from complete by proteinase K S. M. M. PubMed Scopus Google Scholar, C. M. W. N. J. Cell Biol. 2001; PubMed Scopus Google Scholar). The of cytochrome and were not by of mitochondria with proteinase K, the complete of eNOS, that eNOS was not in the inner mitochondrial membrane, or of mitochondria. suggest that eNOS is on the of the outer mitochondrial membrane. In the of we sought independent of eNOS localization to the outer mitochondrial membrane by immunoelectron microscopy In antibody, but not particles were not in association with purified mitochondria eNOS was observed on the outer of the outer membrane of mitochondria to antibody The of eNOS distribution was as in the In with and with proteinase K in the of immunoelectron eNOS we of a wild-type HEK cells lacking eNOS, and infected the cells with vector eNOS of eNOS protein in lysates and mitochondrial fractions was by of a in eNOS was in both lysates and isolated mitochondria. after of eNOS and at eNOS expression was in both HEK cell lysates and in purified mitochondria. The of the isolated mitochondria was on the of a mitochondrial marker and the of protein for or plasma suggest that the isolated mitochondria were in COX, but of and plasma Collectively, these suggest that eNOS with the mitochondrial and that to with or plasma examine the possibility of an of eNOS from the mitochondrial fractions obtained from wild-type HEK cells were incubated with the recombinant bovine eNOS in the of in that with mitochondria in vitro and association was not by of eNOS with 8 eNOS, both native and was by of with proteinase K suggest that eNOS to the outer membrane of mitochondria. the of mitochondria with proteinase K was to the incubation with eNOS, eNOS binding was suggesting the of an eNOS binding on the of mitochondrial outer membrane. were to of eNOS by mitochondria. purified mitochondria from cells were in and subfractions were that inner membrane, and in that the of isolated mitochondria with proteinase K the of eNOS in the mitochondria which the outer membrane was was reduced with that present in an of intact mitochondria. on the would that anti-eNOS antibodies to the protein on non-permeabilized mitochondria. examine isolated mitochondria were incubated with anti-eNOS antibodies as and followed by the of the The of eNOS is in obtained from cells and HUVEC showed fluorescence that of mitochondria obtained from wild-type HEK cells. 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