Nitric Oxide Synthase Generates Nitric Oxide Locally to Regulate Compartmentalized Protein S-Nitrosylation and Protein Trafficking

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Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking

Yasuko Iwakiri*†‡, Ayano Satoh§, Suvro Chatterjee¶, Derek K. Toomre§ʈ, Cecile M. Chalouni§, David Fulton**, Roberto J. Groszmann*‡, Vijay H. Shah¶, and William C. Sessa†,††

*Section of Digestive Diseases, Departments of †Pharmacology and §Cell Biology, and ʈInstitute for Cancer Research, Yale University School of Medicine, New Haven, CT 06510; ‡Hepatic Hemodynamic Laboratory, VA Connecticut Healthcare System, West Haven, CT 06516; ¶Gastroenterology Research Unit, Department of Physiology and Tumor Biology Program, Mayo Clinic, Rochester, MN 55905; and **Vascular Biology Center and Department of Pharmacology, Medical College of Georgia, Augusta, GA 30912

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved October 23, 2006 (received for review July 13, 2006) Nitric oxide (NO) is a highly diffusible and short-lived physiological ating NO, suggesting that the compartmentalization of NO equiv- messenger. Despite its diffusible nature, NO modifies thiol groups alents is physiologically important to regulate diverse cellular of specific cysteine residues in target proteins and alters protein function. (ii) In red blood cells, S-nitrosohemoglobin binds to and function via S-nitrosylation. Although intracellular S-nitrosylation transnitrosylates the plasma membrane anion exchange protein is a specific posttranslational modification, the defined localization AE1, whereas NO itself does not S-nitrosylate AE1 in the absence of an NO source (nitric oxide synthase, NOS) with protein S- of hemoglobin (13, 14). (iii) Inducible NOS (iNOS) can only nitrosylation has never been directly demonstrated. Endothelial S-nitrosylate cyclooxygenase after a direct protein–protein inter- NOS (eNOS) is localized mainly on the Golgi apparatus and in action; if the interaction between iNOS and cyclooxygenase is plasma membrane caveolae. Here, we show by using eNOS tar- prevented (with an inhibitory peptide), iNOS generates NO, but geted to either the Golgi or the nucleus that S-nitrosylation is does not S-nitrosylate cyclooxygenase (15). (iv) NOSs are them- concentrated at the primary site of eNOS localization. Furthermore, selves S-nitrosylated, thus NO would appear to be acting ‘‘very localization of eNOS on the Golgi enhances overall Golgi protein locally’’ (16, 17). (v) Privileged sites of S-nitrosylation have been CELL BIOLOGY S-nitrosylation, the specific S-nitrosylation of N-ethylmaleimide- identified in particular, mitochondrial procaspase-3 is constitu- sensitive factor and reduces the speed of protein transport from tively S-nitrosylated whereas the cytosolic form is not (5). (vi)OxyR the endoplasmic reticulum to the plasma membrane in a reversible is S-nitrosylated more readily by S-nitrosoglutathione (GSNO) than manner. These data indicate that local NOS action generates S-nitrosocysteine (CysNO) (18), whereas the opposite is true for organelle-specific protein S-nitrosylation reactions that can regu- hemoglobin (19). GSNO (but not CysNO) binds directly to OxyR, late intracellular transport processes. whereas it is too large to access the buried Cys in hemoglobin. In comparison to the actions of NO-regulating metal-centered endothelial nitric oxide synthase ͉ Golgi ͉ targeting processes (activation of soluble guanylyl cyclase and inhibition of cytochrome oxidase) or its interaction with other radicals, thiol itric oxide (NO), produced by the nitric oxide synthase (NOS) modification via S-nitrosylation is thought to require higher con- Nfamily of proteins, regulates a variety of important physiolog- centrations of NO among the primary biological reactions with NO ical responses, including vasodilation, respiration, cell migration, (20) to sustain protein S-nitrosylation in vitro (21). Therefore, the and apoptosis (1–5). NO has been considered to mediate these flux of NO generated by NOS may regulate specific cellular responses by activating the primary NO effector soluble guanylyl functions despite the diffusible and short-lived properties of NO; cyclase to produce cGMP (6) or by NO-based chemical modifica- however, this has never been directly demonstrated. tions of proteins or perhaps lipids. One clear example of cGMP- eNOS is unique among the NOS family members as it is localized independent actions of NO is protein thiol group modification by mainly to specific intracellular membrane domains, including the NO known as S-nitrosylation (7). This posttranslational control cytoplasmic aspect of the Golgi apparatus and plasma membrane mechanism regulates important physiological activities of proteins caveolae (22–25). Particularly, on the Golgi, it is shown that eNOS in response to endogenously or exogenously generated NO (8). is colocalized with well known Golgi proteins, such as Golgi matrix Thus, S-nitrosylation of proteins is an emerging area of investigation protein 130 (GM130), 53K Golgi protein, and mannosidase II for NO-mediated physiological responses (8). (22–24, 26). Previously, we hypothesized that a NO pool is formed NO is a lipophillic, highly diffusible, and short-lived physiological around and within the Golgi and may create a favorable environ- messenger (9). On the one hand, NO is thought to diffuse over a ␮ wide area (100 m), moving freely through membranes of neigh- Author contributions: Y.I., C.M.C., and R.J.G. designed research; Y.I., A.S., S.C., and C.M.C. boring cells (9). On the other hand, given the apparent promiscuity performed research; D.K.T., D.F., and V.H.S. contributed new reagents/analytic tools; Y.I., of NO, the question arises as to how S-nitrosylation might occur in S.C., D.K.T., C.M.C., V.H.S., and W.C.S. analyzed data; and Y.I., R.J.G., V.H.S., and W.C.S. a precisely regulated manner, i.e., protein S-nitrosylation occurs on wrote the paper. specific thiol residues in proteins that are targeted to specific The authors declare no conflict of interest. organelles in cells (10), and low concentrations of NO activate the This article is a PNAS direct submission. ryanodine receptor via thiol modification, whereas higher concen- Abbreviations: NOS, nitric oxide synthase; eNOS, endothelial NOS; GSNO, S-nitrosogluta- trations inhibit the receptor (11). There are several compelling thione; GSNOR, GSNO reductase; NLS, nuclear localization signal; RFP, red fluorescent arguments in favor of the concept that all sources of NO are not protein; DAF, diaminofluorescein; DAF-2T, DAF-2 triazole; DAF-2DA, 4-amino-5-methylamino-2Ј,7Јdifluorescein; SNAP, S-nitroso-N-acetyl-D-L-penicillamine; L-NAME, L-nitroargi- bioequivalent. (i) In cardiac myocytes, which express two forms of nine methylester; VSVG, vesicular stomatitis virus glycoprotein; ER, endoplasmic reticulum; NOS, gene deletion of either neuronal NOS or endothelial NOS NSF, N-ethylmaleimide-sensitive factor. (eNOS) exerts isoform-specific phenotypes, arguing that the source ††To whom correspondence should be addressed. E-mail: [email protected]. of NO favors local control of different cellular functions (12). This article contains supporting information online at www.pnas.org/cgi/content/full/ Moreover, in many circumstances, the actions of endogenously 0605907103/DC1. generated NO from NOS cannot be recapitulated by drugs liber- © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605907103 PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19777–19782 Downloaded by guest on September 30, 2021 Fig. 2. Kinetics of DAF-2DA (an intracellular NO indicator) in response to Fig. 1. The functional analysis of eNOS constructs. (A) Subcellular localiza- exogenous and endogenous NO in living cells. The y axis of each graph is relative tion of eNOS in COS cells. eNOS constructs fused with RFP include: WT-eNOS- fluorescent intensity (RFI) of DAF-2 fluorescence. (A) NO donor, NONOate, in- RFP, which is WT eNOS that primarily localizes at the Golgi apparatus, and creased DAF-2 fluorescence in living cells. Images of DAF-2 taken every minute ␮ RFP-eNOS-NLS to target the nucleus. (Scale bar: 10 m.) (B) eNOS expression after COS-7 cells were loaded with DAF-2DA. The fluorescent intensity of DAF-2T of eNOS constructs. (C) NO production in COS-7 cells transfected with eNOS was measured after the dye was loaded. The average Ϯ SEM in each time point constructs. Forty-eight hours after the transfection of eNOS constructs or was obtained from three independent experiments. (B) An inhibitor of NOS control plasmid (RFP only), the media were processed for the measurement of (L-NAME) blocked DAF-2 fluorescence in cells that expresses WT-eNOS-RFP. Be- nitrite, a stable breakdown product of NO in aqueous solution by chemilu- fore being stimulated with ATP (100 ␮M) for NO production, cells were treated minescence. (Left) NO release from samples collected from basal (4 h) accu- with or without L-NAME (100 ␮M). A stock solution of L-NAME (100 mM) was mulation of nitrite in serum-containing media is shown. The same cells were prepared in water and diluted in culture medium (1:1,000) to have a final then incubated with serum-free DMEM for 6 h and the fresh media containing concentration of 100 ␮M. The treatment of cells with ATP resulted in DAF-2T agonist ATP (100 ␮M) for 30 min. (Right) NO release as a result of ATP fluorescence (Upper), which was blocked by L-NAME (Lower). Control RFI values stimulation is shown. The cells were lysed to determine the total protein were obtained from COS cells expressing WT-eNOS-RFP treated without a pres- concentration and evaluate equal eNOS expression by Western blot. Data are ence of L-NAME. * and **, P Ͻ 0.05. (Scale bars: 100 ␮m.) presented as means Ϯ SEM; n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.05.

ing eNOS to nucleus reduces NOS activation presumably because ment for local S-nitrosylation of proteins in living cells (1–5). Thus, of insufficient access to calcium/calmodulin and impaired phos- the purpose of this study is to examine local NO production and phorylation (28). Thus, localization of eNOS in different subcellular function by using organelle-targeted eNOS constructs. organelles influences the amount of NO produced. Results and Discussion Because of the instability and low concentrations of NO in biological systems, it had been very difficult to directly visualize NO To examine whether Golgi-localized eNOS can generate a local NO production in living cells. Direct imaging of NO in biological pool, we imaged NO production and the source of NO (NOS) simultaneously in living cells and tested whether the localization of systems became available with the development of a series of NOS correlates with local S-nitrosylation of proteins and whether fluorescent indicators diaminofluoresceins (DAFs) in 1998 (29). In Golgi-directed NOS regulates cellular exocytosis. To place the the presence of oxygen, NO and NO related reactive nitrogen source of NO in two distinct subcellular compartments in cells, we species nitrosate 4,5-DAF by a second-order reaction to yield the generated WT eNOS (WT-eNOS-RFP), which localizes primarily highly fluorescent DAF-2 triazole (DAF-2T). To test whether Ј Ј on the Golgi apparatus in transfected cells and a nuclear-localized DAF-2DA (4-amino-5-methylamino-2 ,7 difluorescein) is sensitive eNOS [RFP-eNOS-NLS (nuclear localization signal)] as fusion to detect intracellular nitrosation (29), COS-7 cells were loaded proteins with monomeric red fluorescent protein (RFP) to monitor with DAF-2DA then treated with NONOate/AM, which releases their subcellular localization in living cells that do not express NO intracellularly (Fig. 2A Left for representative images and Right endogenous NOS. As seen in Fig. 1A, WT-eNOS-RFP was local- for quantitative data), and time-lapsed images of DAF-2T were ized in a perinuclear crescent and colocalized with GM130 as taken every minute immediately after the addition of the NO donor described (22–27), whereas RFP-eNOS-NLS was confined to the by a confocal laser scanning microscope (LSM510 Imaging System; nucleus. Next, we examined the levels of protein expression and NO Zeiss, Thornwood, NY). Because COS cells do not contain NOS release from transfected cells. As seen in Fig. 1B, both constructs isoforms, the background fluorescence at time 0 was low. The expressed well in transfected cells and released NO (measured as dye-loaded cells showed an increase in fluorescence intensity Ϫ NO2 with NO-specific chemiluminescence) (Fig. 1C). Both WT- immediately after the NO donor was given and the fluorescent eNOS-RFP and RFP-eNOS-NLS released basal (Ͼ4h;Fig.1C signal was diffusely distributed throughout the cell, suggesting the Left) and stimulated (with ATP for 30 min; Fig. 1C Right) NO, with specificity of the dye for detecting NO and that DAF did not WT-eNOS-RFP releasing two to three times the amount of NO concentrate in specific organelles in COS cells, making it an compared with RFP-eNOS-NLS. Although the expression levels of appropriate tool to detect NO derived from eNOS. Similar results WT-eNOS-RFP were similar to those of RFP-eNOS-NLS, target- were seen when using GSNO and S-nitroso-N-acetyl-D-L-

19778 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605907103 Iwakiri et al. Downloaded by guest on September 30, 2021 CELL BIOLOGY

Fig. 3. NO is confined to the region where eNOS targeted. The xaxis of each graph refers to the distance of the laser line scan in the individual cells shown with numbers on a line with an arrow (1, 2, and 3), reflecting the different regions of interest monitored for colabeled DAF-2 fluorescence, and the y axis is the fluorescent intensity for DAF-2T (blue line) and RFP (red line). (A) Regions of nitrosation were formed in and around the Golgi complex where WT-eNOS-RFP is highly localized. Small increases in DAF-2T were also observed on the nuclear membrane and plasma membrane. COS-7 cells were transfected with WT-eNOS-RFP, and nitrosation was monitored at the indicated time after the addition of ATP (100 ␮M). (B) Nitrosation was confined to the nucleus in cells that express RFP-eNOS-NLS. (Scale bars: 10 ␮m.)

penicillamine (SNAP) as NO donors [100 ␮M of each for 30 min; RFP (magenta line). Cells were observed under baseline conditions see supporting information (SI) Fig. 6). We also quantified the and after administration of ATP to evoke NO release. As seen in responsiveness of the dye to detect changes in nitrosation in cells Fig. 3A, WT-eNOS-RFP was highly localized in region 1, with lesser tranfected with WT-eNOS-RFP (Fig. 2B). COS cells were trans- enzyme in regions 2 and 3. However, upon stimulation of cells with fected with WT-eNOS-RFP, loaded with DAF-2DA, then stimu- ATP, the highest amount of DAF-2 fluorescence (NO signal; Fig. lated with ATP in the absence or presence of the NOS inhibitor 3A Right) was detected in a peri-nuclear region overlapping with or L-nitroarginine methylester (L-NAME). Treatment of cells with adjacent to WT-eNOS-RFP, similar to what was observed in ATP resulted in an increase in DAF-2 fluorescence, which was endothelial cells (33). Because NO partitions rapidly into hydro- inhibited by L-NAME, showing that we can monitor NO production phobic environments such as biological membranes, this result may in single cells expressing transfected eNOS constructs (see quan- reflect the membranous nature of protein-NO adducts formed (34). titative data in Fig. 2B Right). The incomplete inhibition of DAF-2 Stated differently, if NO-mediated nitrosation was regulated by fluorescence by L-NAME may be caused by the production of other diffusion only, the imaged DAF-2 fluorescence changes derived DAF-reactive compounds not derived from eNOS (30–32). from eNOS should have been similar to that seen with NO donors Treatment of human endothelial cells with vascular endothelial (see Fig. 2A). To further confirm that eNOS-derived NO acts growth factor increases the detection of DAF-2 nitrosation prod- locally, we performed identical experiments in cells transfected with ucts in the peri-nuclear region of the cells (33). Thus, we simulta- RFP-eNOS-NLS. This mutant eNOS highly localized at nucleus neously monitored the targeted RFP constructs (for eNOS local- (Fig. 3B). NO concentration (imaged by DAF-2) was colocalized in ization) and DAF-2 fluorescence (for nitrosation) in transfected the nucleus (area 1) and peri-nuclear regions (area 2). This pattern COS-7 cells by confocal microscopy over a 30-min period. In Fig. of DAF-2 nitrosation in cells expressing RFP-eNOS-NLS was 3, the x axis of each graph refers to the distance of the laser line scan distinct from what has observed in those expressing WT-eNOS- in the individual cells shown with 1, 2, and 3 reflecting the different RFP. The increase in NO at the peri-nuclear region is probably regions of interest monitored for colabeled DAF-2 fluorescence, because NO produced inside of the nucleus could diffuse out of and the y axis is the fluorescent intensity for DAF-2 (blue line) and nucleus and form an NO reservoir in the membranous peri-nuclear

Iwakiri et al. PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19779 Downloaded by guest on September 30, 2021 (Fig. 4D Right), which hydrolyzes S–NO bonds, before immunostaining with the S-nitrocysteine mAb. Collectively, these data indicate that NOS compartmentalization can govern the subcellular distribution of S-nitrosylated proteins in cells. These data suggest that physiological and pathophysiological stimuli can stimulate endothelial cells to regulate eNOS-mediated NO binding to target proteins, a result that may explain different cellular responses induced by NO donors and activation of endogenous NO synthesis. The above results suggest that the localization of NOS can determine local protein S-nitrosylation. To test whether eNOS localization influences the function of NO, we investigated the transport of a temperature-sensitive vesicular stomatitis virus glycoprotein (VSVG) tagged with GFP (tsO45-VSVG-GFP labeled as VSVG-GFP) from the endoplasmic reticulum (ER) to the cell surface. This glycoprotein is retained in the ER at 40°C by its misfolding, but upon temperature reduction to 32°C, folds properly and moves out of ER and into the Golgi complex before being transported to the plasma membrane (35). BSC-1 cells were used for this study because they do not express NOS isoforms and are easier to microinject with plasmid DNA constructs than COS cells. BSC-1 cells were microinjected with VSVG-GFP and a control plasmid, WT-eNOS-RFP or RFP-eNOS-NLS, and incubated at 40°C for 2–3 h to trap VSVG-GFP in the ER and then shifted to 32°C in the presence of cycloheximide to block further protein synthesis. As shown in Fig. 5A, in cells expressing VSVG-GFP (after 40 min release from the ER) and injected with control plasmid, there was surface targeting of VSVG relative to the total amount of VSVG in the cells. In contrast, in cells injected with WT-eNOS- RFP, there was a marked delay in targeting of VSVG-GFP to the cell surface, whereas cells injected with RFP-eNOS-NLS exhibited similar transport properties to control-injected cells. To further determine whether the reduced rate of transport observed in cells with WT-eNOS-RFP depends on NO, cells expressing WT-eNOS- Fig. 4. Protein S-nitrosylation is restricted to regions of the cell where eNOS is RFP were treated with L-NAME to abrogate NO release and the localized. S-nitrosylation of endogenous proteins were detected by using an VSVG exocytosis was examined (Fig. 5B). Cells treated with S-nitrosocysteine mAb. COS cells were transfected with WT-eNOS-RFP or RFP- L eNOS-NLS, and DAPI was used to detect nuclei (blue). (A) WT-eNOS-RFP was -NAME restored the rate of VSVG-GFP transport to that ob- concentrated in a peri-nuclear pattern (red; Left). Labeling with an isotype served in control or RFP-eNOS-NLS-expressing cells, demonstrat- control antibody for the S-nitrosocysteine mAb showed low background levels ing control of basal membrane transport by NO, perhaps via (Center). Thus, there was no overlap of red and green (Right). (B) WT-eNOS-RFP S-nitrosylation of proteins important for secretion. colocalized with cellular S-nitrosylated proteins (green) around the peri-nuclear One protein implicated in secretion and a target for S- area of cells. (C) RFP-eNOS-NLS (red) was concentrated in nucleus and colocalized nitrosylation is the N-ethylmaleimide-sensitive factor (NSF). NSF with S-nitrosylated proteins (green). (D) The secondary antibody (Alexa 488- was first identified as a cytosolic protein necessary for in vitro conjugated goat anti-mouse) for S-nitrosocysteine mouse monoclonal antibody reconstitution of intercisternal Golgi transport, and subsequently did not cause nonspeciﬁc binding to COS-7 cells (Left). The NO donor, SNAP (100 shown to regulate vesicle trafficking and exocytosis by mediating ␮M), increases S-nitrosylated proteins in COS cells, and the pattern of S- nitrosylated proteins were distinctive from cells transfected with WT-eNOS-RFP membrane fusion through its ATPase activity (36). Indeed, the S-nitrosylation of NSF by eNOS inhibits the stimulated exocytosis and RFP-eNOS-NLS (Center). Incubation with 0.2%-HgCl2 abolishes S-nitrosylation caused by the treatment with 100 ␮M-SNAP (Right). (Scale bars: 10 ␮m.) of Von Willebrand’s factor in endothelial cells (37). Moreover, S-nitrosylation of NSF has been shown to inhibit NSF disassembly of soluble NSF attachment protein receptor (SNARE) complexes, region. Collectively, these data show that NO is preferentially resulting in the inhibition of exocytosis in endothelial cells (37). channeled to sites in proximity to the source of eNOS activity. Based on these findings, we tested whether the delayed transport If NO is formed at the primary site of eNOS localization in cells observed in cells expressing WT-eNOS-RFP is associated with (Fig. 3), then it is tenable that S-nitrosylation of proteins may also S-nitrosylation of NSF by using a biotin-switch method to label be localized as suggested (10, 21). To test this idea, we performed S-nitrosylated proteins as described (Fig. 5C). As seen in Fig. 5C, immunofluoresence analysis for protein S-nitrosylation by using an transfection of both WT-eNOS-RFP or RFP-eNOS-NLS results in S-nitrosocysteine-specific antibody in COS cells transfected with the S-nitrosylation of NSF via a HgCl2-sensitive bond. However, the WT-eNOS-RFP or RFP-eNOS-NLS (Fig. 4). As shown in Fig. 4A level of NSF S-nitrosylation is markedly greater in cells expressing Left (red channel), WT-eNOS-RFP is concentrated in a peri- WT-eNOS-RFP compared with cells expressing RFP-eNOS-NLS, nuclear pattern. Labeling with an isotype control Ab for the suggesting that the decreased VSVG-GFP trafficking in cells S-nitrocysteine mAb (mouse IgG) demonstrates low background expressing WT-eNOS-RFP may be caused, in part, by increased levels (Fig. 4A Center) with little merging of the two patterns. NSF S-nitrosylation and inhibition of NSF activity as described (37). Imaging of cells expressing WT-eNOS-RFP (Fig. 4B Center)or These observations clearly indicate that localization of eNOS RFP-eNOS-NLS (Fig. 4C Center) shows that the pattern of S- influences protein S-nitrosylation and subsequent NO-dependent nitrosylated proteins coregisters with the targeted eNOS con- cellular functions. structs. In contrast, cells treated with the NO donor drug, SNAP, Our data support the idea that NOS generates NO locally to show a markedly different pattern of S-nitrosylated protein immu- regulate compartmentalized protein S-nitrosylation and signaling nreactivity (Fig. 4D Center). Moreover, the levels of S-nitrosylated akin to calcium-calmodulin or protein phosphorylation being reg- protein detection can be eliminated by preincubation with HgCl2 ulated by subcellular targeting of a calmodulin target or a kinase

19780 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605907103 Iwakiri et al. Downloaded by guest on September 30, 2021 regulated by the balance between the protein S-nitrosylation initi- ated by NOS and the reduction in S-nitrosylated proteins by GSNO reductase (GSNOR) or an equivalent enzyme (38, 39). GSNOR uses GSNO as a substrate (40). As GSNO exists in equilibrium with S-nitrosylated proteins, GSNOR indirectly regulates the cellular level of S-nitrosylated proteins. In fact, mice lacking the GSNOR gene have increased basal levels of total S-nitrosylated proteins (41). It is tempting to speculate that eNOS and GSNOR may colocalize and regulate the turnover of the local NO pool and thus NO-mediated signaling and cellular functions. As mentioned before, a high NO concentration (in the presence of oxygen to form N2O3) is thought to be required for the S-nitrosylation of proteins (20, 21). We demonstrate in this study that the compartmentalization of NOS, which forms a relatively high local concentration of NO equivalents, creates a favorable environment for S-nitrosylation of proteins. However, a high concentration of NO may not be a strict requirement if S-nitrosylation involves reactions between NO and thiyl radicals or that catalyzed by transition metals reactions (15, 42). In conclusion, we directly demonstrate that eNOS channels NO locally despite the highly diffusible nature of NO. Local NO at the Golgi apparatus created by eNOS is associated with the formation of regionally confined protein S-nitrosylation and restricted actions of NO on the rate of secretion of a model protein traveling through the Golgi apparatus. Considering that the Golgi apparatus is constantly being remodeled during the cell cycle and is the site of several important posttranslational modifications, the local regula-

tion of protein function via S-nitrosylation of proteins within the CELL BIOLOGY Golgi is an exciting avenue to explore. Methods Cells and Materials. We obtained COS-7 and BSC-1 cells from ATCC (Manassas, VA). All cells were grown in DMEM sup- Fig. 5. NO confined near the Golgi apparatus by WT-eNOS-RFP delays the plemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 exocytotic pathway of VSVG trafficking. (A) VSVG transport to the plasma mem- ␮ brane was impaired in cells expressing WT-eNOS-RFP. BSC-1 cells, grown on g/ml streptomycin, and 10% (vol/vol) FBS. DAF-2DA and 12-mm coverslips in a 12-well dish, were microinjected with a mixture of tsO45- DAF-2FM were from Calbiochem (San Diego, CA). Monoclonal VSVG-GFP plasmid DNA and WT-eNOS-RFP, RFP-eNOS-NLS, or RFP alone (con- antibodies against eNOS and Hsp90 were from BD Biosciences trol). After the microinjection, cells were incubated at 40°C for 2–3 h for the (San Jose, CA). S-nitrosocysteine was from A.G. Scientific, Inc. expression, then incubated at 4°C for 15 min for the protein folding in the (San Diego, CA), and Myc was from Cell Signaling Technology, presence of cyclohexamide (100 ␮g/ml). At this point, tsO45-VSVG-GFP protein is (Beverly, MA). Polyclonal antibody against NSF was from Santa exclusively localized at ER. Then, incubation of cells at 32°C was started to chase Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor 488 anti- the transport of tsO45-VSVG-GFP from ER to Golgi and to the surface of plasma mouse IgG and Alexa Fluor 680 anti-mouse IgG secondary membrane (40 min). After the incubation at 32°C for 40 min, cells were fixed in antibodies were from Invitrogen/Molecular Probes (Carlsbad, 4% paraformaldehyde and processed for the immunofluorescence of cell surface VSVG protein, using a mAb to the luminal/cell surface VSVG that was labeled with CA). IRDye800-conjugated anti-rabbit IgG secondary antibody Alexa 647. The quantification of the surface VSVG protein was performed by was from Rockland Immunochemical Inc. (Gilbertsville, PA). measuring the fluorescent intensity of Alexa 647, which was normalized by total The cDNA of monomeric RFP was a gift from Roger Tsien at expression of VSVG in cells by measuring fluorescent intensity of GFP. *, P Ͻ 0.01. the University of California at San Diego (La Jolla, CA). (B) The impaired VSVG transport in WT-eNOS-RFP-expressing cells was NO- dependent. To determine whether the decreased VSVG transport in cells express- eNOS Constructs. WT eNOS and eNOS-NLS fused with monomeric ing WT-eNOS-RFP is NO-dependent, cells microinjected with WT-eNOS-RFP and RFP in pCDNA3 were made to monitor the subcellular localization tsO45-VSVG-GFP were incubated at 40°C for3hinthepresence or absence of of eNOS, WT-eNOS-RFP, and RFP-eNOS-NLS, respectively. For ␮ L-NAME (100 M). Temperature was then shifted to 32°C for 40 min to chase VSVG WT-eNOS-RFP, RFP was inserted into the bovine eNOS cDNA in transport to the cell surface. *, P Ͻ 0.01. (C) S-nitrosylation of NSF was increased in cells expressing WT-eNOS-RFP. COS-7 cells, transfected with myc-NSF and pCDNA3 at the C terminus. eNOS construct that targets nucleus WT-eNOS-RFP or RFP-eNOS-NLS by transient transfection, were used 48 h after was made uby sing a modified eNOS with a mutation on the transfection. As a control, cells were transfected with myc-NSF alone to check for myristoylation site on eNOS (eNOS G2A, thereby called ‘‘mu- endogenous S-nitrosylation. Cells were incubated in DMEM without serum for tant’’), which therefore stays in cytosol by preventing N- 6 h, followed by the incubation with DMEM containing ATP (100 ␮M) with or myristoylation and cysteine palmitoylation of eNOS, modifications without 0.2% HgCl2 for 1 h. To detect S-nitrosylation of NSF, the biotin-switch that are required for targeting eNOS to the Golgi and plasma method developed by Jaffrey and colleagues (46, 47) was used. (Top) Purified membrane (23, 26). Three repeats of NLSs (PKKKRKVD) was S-nitrosylated protein was subjected to 10% SDS/PAGE and Western blot analysis fused at the C terminus of the cDNA of eNOS (G2A). RFP was by using myc antibody to detect NSF. (Middle and Bottom) eNOS expression and fused with eNOS (G2A)-NLS at the N terminus. NSF expression were also determined in the starting lysate before the isolation of S-nitrosylated proteins. Below the blot is the densitometric ratio of nitrosylated NSF to total NSF in the starting lysates. Transfection. For the real-time confocal imaging of NO in living cells, COS-7 cells were seeded on coverslips and transfected the next day with 1–2 ␮g of RFP-eNOS constructs by using Lipo- substrate. The accumulation of a local NO pool and S-nitrosylation fectAmine 2000 (Invitrogen) in OptiMEM media (Invitrogen), may regulate normal physiological processes or exert nitrosative according to the manufacturer’s instructions. Cells were used 48–72 stress (21). It is probable that the turnover of the NO pool is h after the transfection.

Iwakiri et al. PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19781 Downloaded by guest on September 30, 2021 NO Release. COS-7 cells were plated in six-well plates. Forty-eight Alexa 647, which was normalized by total expression of VSVG in hours after the transfection of eNOS constructs or control cells by measuring the fluorescent intensity of GFP. plasmid, the media were processed for the measurement of nitrite, a stable breakdown product of NO in aqueous solution S-Nitrosocysteine Immunocytochemistry. Immunostaining of S- by chemiluminescence (33). Nitrite accumulation was measured nitrosylated proteins was performed as described by Gow et al. by using a NO analyzer (Sievers Instruments, Boulder, CO). For (45) with some modifications. In brief, cells were transfected more details see SI Text. with WT-eNOS-RFP or RFP-eNOS-NLS as described. Twenty- four hours after transfection, cells were trypsinized and plated on 12-mm cover glasses with Ϸ30% confluency. After the Fluorescence Imaging. For real-time detection of NO production in attachment, cells were incubated in Hepes buffer containing 10 living cells, the membrane-permeable fluorescent indicator DAF- ␮M oxy-hemoglobin for2htoblock the NO released to the 2DA was used (29). Once inside cells, it is deacetylated by intra- neighboring cells. Cells were fixed with 4% paraformaldehyde cellular esterases to become DAF-2 and can be detected with for 10 min at room temperature. After permeabilization in goat excitation/emission maxima of 495/515 nm, respectively. The dye- serum containing 0.3% Triton X-100, 0.1 mM neocuproine, and loaded cells on coverslips were incubated at 37°C for 10 min in a 1 mM EDTA, cells were incubated with a mAb against S- Hepes-buffered solution (concentrations: 130 mmol/liter NaCl, 5 nitrosocysteine (2 ␮g/ml) overnight at 4°C. After washing, cells mmol/liter KCl, 1.25 mmol/liter CaCl2, 1.2 mmol/liter KH2PO4,1 were incubated with secondary antibody, Alexa 488-conjugated mmol/liter MgSO4, 19.7 mmol/liter Hepes, and 5 mmol/liter glu- goat anti-mouse (1:100), in 100% goat serum at room temper- cose; pH 7.4) or PBS followed by an incubation with 10 ␮M ature in the dark for 1 h. In a separate experiment, cells, not DAF-2DA. Dye-loaded cells were excited with the 488 nm of a transfected, were incubated with 100 ␮M SNAP for 30 min, and krypton/argon laser for DAF-2 and the 543 nm of a helium/neon immunostaining for S-nitrosocysteine was performed. As a ␮ laser for RFP, and increases in DAF-2 (for NO) and RFP (for negative control, cells treated with SNAP (100 M) were further eNOS localization) fluorescence were monitored simultaneously. incubated with 0.8% HgCl2 for 1 h at 37°C, which selectively The Zeiss LSM 510 Confocal Imaging System was used for the displaced NO from S–NO bonds on cysteine residue. S- nitrosylated proteins were visualized by confocal microscopy. detection. For more details see SI Text. Determination of S-Nitrosylation of NSF. COS-7 cells, transfected VSVG–GFP Transport Assay. BSC-1 cells, grown on 12-mm coverslips, with myc-NSF and WT-eNOS-RFP or RFP-eNOS-NLS by were microinjected with a mixture of tsO45-VSVG-GFP plasmid transient transfection as described, were used 48 h after the DNA (43) and WT-eNOS-RFP, RFP-eNOS-NLS, or RFP alone. transfection. Cells were incubated in DMEM without serum for After the microinjection, cells were incubated at 40°C for 2–3 h for 6 h, followed by the incubation with DMEM containing ATP the expression, then incubated at 4°C for 15 min for protein folding (100 ␮M) with or without 0.2% HgCl2 for 1 h. To detect in the presence of cyclohexamide (100 ␮g/ml). At this point, S-nitrosylation of proteins, the ‘‘biotin-switch’’ method devel- tsO45-VSVG-GFP protein was exclusively localized at the ER. oped by Jaffrey and colleagues was used (46, 47). Purified Then, incubation of cells at 32°C was started to chase the transport S-nitrosylated proteins were subjected to 10% SDS/PAGE (20 of tsO45-VSVG-GFP from the ER to the Golgi and the surface of ␮g protein per lane) and Western blot analysis. NSF was plasma membrane (40 min). After incubation at 32°C for 40 min, detected by using both anti-myc and anti-NSF antibodies. For cells were fixed in 4% paraformaldehyde for 15 min and processed more details see SI Text. for the immunofluorescence of cell surface VSVG protein, using a mAb VG (44), a kind gift from Ira Mellman (Yale University), We thank Dr. Graham Warren for helpful comments. This work was supported by National Institutes of Health Grants R01 HL64793, R01 which was labeled with Alexa 647, according to the manufacturer’s HL 61371, R01 HL 57665, and P01 HL 70295 (to W.C.S.), National instructions (Invitrogen). The quantification of the surface VSVG Institutes of Health Award K01 DK067933-01 (to Y.I.), and New protein was performed by measuring the fluorescent intensity of Investigator Award P30 DK34989 from the Yale Liver Center (to Y.I.).

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19782 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605907103 Iwakiri et al. Downloaded by guest on September 30, 2021