Proc. Natl. Acad. Sci. USA Vol. 93, pp. 6448-6453, June 1996 Cell Biology

Targeting of to endothelial cell caveolae via palmitoylation: Implications for nitric oxide signaling (endothelial nitric oxide synthase/signal transduction/vascular biology/N-) GUILLERMO GARC1A-CARDENA*, PHIL OHt, JIANwEI LIu*, JAN E. SCHNITZERt, AND WILLIAM C. SESSA*t *Molecular Cardiobiology Program and Department of Pharmacology, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536; and tDepartment of Pathology, Harvard Medical School, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215 Communicated by Vincent T. Marchesi, Yale Univeristy, New Haven, CT, March 13, 1996 (received for review February 5, 1996)

ABSTRACT The membrane association of endothelial insoluble membranes (TIM), suggesting that caveolae are nitric oxide synthase (eNOS) plays an important role in the signal processing centers (2-11). Additionally, caveolae have biosynthesis of nitric oxide (NO) in vascular endothelium. been implicated in other important cellular functions, includ- Previously, we have shown that in cultured endothelial cells ing endocytosis, potocytosis, and transcytosis (12, 13). and in intact blood vessels, eNOS is found primarily in the Endothelial nitric oxide synthase (eNOS) is a peripheral perinuclear region of the cells and in discrete regions of the membrane protein that metabolizes L-arginine to nitric oxide plasma membrane, suggesting trafficking of the protein from (NO). NO is a short-lived free radical gas involved in diverse the Golgi to specialized plasma membrane structures. Here, physiological and pathological processes. Endothelial-derived we show that eNOS is found in Triton X-100-insoluble mem- NO is an important paracrine mediator of vascular smooth branes prepared from cultured bovine aortic endothelial cells muscle tone and is an inhibitor of leukocyte adhesion and and colocalizes with caveolin, a coat protein of caveolae, in platelet aggregation (14, 15). As an autocrine mediator, NO cultured bovine lung microvascular endothelial cells as de- has been implicated in the modulation of growth factor signals termined by confocal microscopy. To examine if eNOS is and indeed in caveolae, we purified luminal endothelial cell cellular proliferation (16-18). Regulation of NO signaling plasma membranes and their caveolae directly from intact, in endothelial cells occurs largely at the level of eNOS activity perfused rat lungs. eNOS is found in the luminal plasma controlled by cofactors and targeting of eNOS to specific membranes and is markedly enriched in the purified caveolae. intracellular membranes (19). Because palmitoylation of eNOS does not significantly influ- eNOS is dually acylated by cotranslational N-myristoylation ence its membrane association, we next examined whether this and posttranslational palmitoylation (20-23). N- modification can affect eNOS targeting to caveolae. Wild-type myristoylation of eNOS is necessary for membrane association eNOS, but not the palmitoylation mutant form of the , and for subsequent palmitoylation at 15 and/or 26 as colocalizes with caveolin on the cell surface in transfected NIH determined in broken cell lysates (23). Functionally, in cul- 3T3 cells, demonstrating that palmitoylation of eNOS is tured aortic endothelial cells, intact blood vessels and heter- necessary for its targeting into caveolae. These data suggest ologous expression systems, eNOS is expressed primarily in the that the subcellular targeting ofeNOS to caveolae can restrict Golgi region of cells and such localization is necessary for NO signaling to specific targets within a limited microenvi- optimal stimulated release of NO from intact cells (24). The ronment at the cell surface and may influence signal trans- importance of eNOS cysteine palmitoylation is less clear duction through caveolae. because mutation of the palmitoylation sites inhibits protein palmitoylation but does not significantly influence eNOS The compartmentalization of plasma membrane proteins is enzyme activity, in vitro or partitioning into high speed mem- critical for specificity of intracellular signaling pathways. In- brane fractions (23), suggesting that palmitoylation may serve tegral membrane proteins, such as hormone receptors, are another function, such as specific membrane targeting. Here anchored in biological membranes by hydrophobic stretches of we show that eNOS is targeted to caveolae, in vivo and in vitro, amino acids comprising transmembrane domains, whereas via palmitoylation of the protein on cysteines 15 and 26. These other proteins, such as G-proteins, are associated and targeted observations provide molecular evidence for a novel consensus to subcellular membranes by the co- or posttranslational lipid sequence that may be sufficient for localization of proteins to modifications N-myristoylation, palmitoylation, or prenylation caveolae and suggest a role for NO in modulating signal (1). Signals initiated at the cell surface can propagate via transduction through these plasma membrane compartments. soluble intracellular messengers, changes in protein phosphor- ylation, protein-protein interactions, and protein-lipid inter- actions. MATERIALS AND METHODS The specialization of plasma membrane microdomains is a Materials and Antibodies. Dulbecco's modified Eagle's potential mechanism for integrating extracellular signals into medium (DMEM), glutamine, trypsin-EDTA, penicillin/ intracellular messages. For example, caveolae are plasma streptomycin, and fetal bovine serum (FBS) were from membrane invaginations composed primarily of glycosphingo- GIBCO. Rabbit polyclonal antibodies to eNOS and caveolin lipids, , and the integral membrane protein, caveo- were from Transduction Laboratories (Lexington, KY); eNOS lin. Several proteins involved in signal transduction, such as monoclonal antibody (mAb) was provided by J. Pollock (Med- inositol 1,4,5-triphosphate receptors, calcium ATPase, mem- ical College of Georgia) and e-COP mAb was provided by M. bers of the src family of nonreceptor tyrosine kinases, G proteins, -coupled membrane receptors, and gan- Krieger (Massachusetts Institute of Technology). gliosides have been found in caveolae or in Triton X-100- Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; BLMVEC, bovine lung microvascular endothelial cells; TIM, The publication costs of this article were defrayed in part by page charge Triton X-100 insoluble membranes; BAEC, bovine aortic endothelial payment. This article must therefore be hereby marked "advertisement" in cells; WT, wild type; TGN, trans-Golgi network. accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed.

6448 Downloaded by guest on September 24, 2021 Cell Biology: Garcl'a-Cardefia et al. Proc. Natl. Acad. Sci. USA 93 (1996) 6449 Cell Culture. Bovine aortic endothelial cells (BAEC) and positively charged colloidal silica particles that coated the bovine lung microvascular endothelial cells (BLMVEC) were luminal endothelial cell surface of the pulmonary vasculature. obtained and grown in tissue culture as described (21, 25). NIH Silica coating followed by cross-linking created a stable silica 3T3 cells were grown in DMEM supplemented with 10% pellicle that specifically marked luminal plasma membranes (vol/vol) FBS, L-glutamine (1 mM), penicillin (100 units/ml), and allowed for purification of these membranes from tissue streptomycin (100 ,ug/ml), and tetrahydrobiopterin (100 ,uM; homogenates by centrifugation. The sedimented pellets (P) ref. 26). Subconfluent NIH 3T3 cells were transiently trans- contained highly purified endothelial cell luminal plasma fected with bovine wild-type (WT) or C15/26S palmitoylation membranes with associated caveolae and showed ample en- mutant eNOS cDNAs subcloned into the mammalian expres- richment ofvarious endothelial cell surface markers relative to sion vector, pcDNA3 (23), according to the standard calcium the starting whole lung homogenates (H; refs. 4 and 11). phosphate precipitation method. After overnight transfection, Caveolae were removed from P by shearing during homoge- cells were trypsinized and cultured onto gelatin coated cov- nization at 4°C in the absence of Triton X-100. These homo- erslips for confocal, immunofluorescence microscopy. genates were subjected to sucrose density centrifugation to Triton X-100 Solubility of Endothelial Proteins. One yield two collected fractions; a low density fraction of a 100-mm dish of confluent BAEC (passage 2-4) was used for homogenous population of biochemically and morphologically each sample. Each dish was washed two times with cold distinct caveolar vesicles (V'). Protein samples from each phosphate-buffered saline (PBS) and cells were released by fraction (5 ,ug) were separated by SDS/PAGE (5-15% gradi- incubating in MBS (125 mM NaCl/20 mM Mes, pH 6.0) plus ent gels) and electrotransfered to nitrocellulose membranes 0.02% EDTA for 10 min on ice. Cells were collected by for immunoblotting with antisera to eNOS and caveolin as centrifugation for 5 min at 1000 x g at 4°C. The pellets were described above. suspended in 0.5 ml of MBS plus 1% Triton X-100 and incubated on ice for 30 min. The samples were Dounce homogenized (20 strokes) and centrifuged for 5 min at 16,000 RESULTS x g at 4°C (10). The supernatant fraction was collected and Triton X-100 insolubility was considered a hallmark of cy- designated the Triton-soluble fraction. The pellet was resus- toskeletal proteins (30) and, more recently, ofproteins residing pended in modified RIPA buffer (0.15 mM NaCl/0.05 mM in caveolae and in other glycolipid-rich domains (11, 31). To Tris HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/ examine if eNOS resides in Triton X-100 insoluble fractions of 0.1% NaDodSO4) (23) and designated TIM. Total eNOS in cultured BAECs, Triton X-100 insoluble and soluble fractions both fractions was concentrated quantitatively with the affinity were prepared and Western blotted with an eNOS mAb. As resin 2'5'-ADP-Sepharose as described (21). Beads were then seen in Fig. 1, -10% of eNOS resided in TIM, with 90% being placed into Laemmli sample buffer and electrophoresed by Triton soluble. Similar results were obtained using human SDS/PAGE. Proteins were separated on 7.5% SDS gels. umbilical vein endothelial cells and in HEK 293 cells stably Proteins were transferred to nitrocellulose and Western blot- transfected with the eNOS cDNA. ted with an eNOS mAb H32 as described (21, 27). Because large vessel endothelium in intact blood vessels Indirect Immunofluorescence. BLMVEC or transfected have few caveolae relative to microvascular endothelium (25, NIH 3T3 cells were grown on glass coverslips. Cells were fixed 32) and culturing of endothelial cells causes a significant loss in acetone for 5 min at -20°C then rinsed with PBS for 10 min in caveolae (25), we examined the colocalization of eNOS and at room temperature. The cells were then incubated sequen- caveolin in BLMVEC. BLMVEC have significantly more tially with PBS plus 5% goat serum for 30 min at room caveolae than other cultured endothelia, including aortic and temperature, a mixture of anti-eNOS mAb H32 and anti- pulmonary artery endothelial cells (25). As seen in Fig. 2, caveolin polyclonal antibody diluted in PBS plus 2% goat eNOS was localized in the perinuclear region of the cell and in serum for 4 h at room temperature, and finally with a mixture a specific microdomain of the plasma membrane (Fig. 2A). of BODIPY (Molecular Probes)-goat anti-rabbit and Texas Caveolin was localized primarily at the leading edge with faint, Red-goat anti-mouse IgGs for 1 h at room temperature. Cells perinuclear staining (Fig. 2B). The merged images of Fig. A were washed two times with PBS after each incubation step. and B demonstrated colocalization of eNOS and caveolin at Cells were mounted in Slow-Fade (Molecular Probes) and observed with a Bio-Rad MRC 600 confocal inverted micro- S I scope. Isolation of Luminal Plasma Membranes from BLMVEC. 199 - Confluent BLMVEC grown in T175 flasks were placed at 4°C for 10 min, washed with ice cold MBS, incubated with colloi- dal, positively charged silica and luminal plasma membranes (P) isolated as described (28). In addition, the lighter density - band containing residual membranes (OM or other mem- 120 branes, containing basolateral and intracelluar membranes) was collected from the top of the gradient for analysis. Protein samples from each fraction (5 jig) were separated by SDS/ PAGE (5-15% gradient gels) and electrotransfered to nitro- 87 - cellulose membranes for immunoblotting by using primary polyclonal antibodies for eNOS, caveolin, and s-COP. s-COP is a specific marker for Golgi and post-Golgi vesicles (29). Immunoreactive proteins were labeled with a horseradish 48 - peroxidase-conjugated, goat, anti-rabbit IgG secondary anti- body and detected by ECL. Protein assays were performed with the Bio-Rad BCA kit bovine serium albumin as by using FIG. 1. Partitioning of eNOS into Triton X-100-soluble (S) and a standard. -insoluble (I) fractions of cultured BAEC. S and I fractions were Caveolae Purification from Rat Lungs. Endothelial caveo- prepared from BAEC lysates, ADP-Sepharose concentrated and lae were isolated to homogeneity from rat lung tissue by a electrophoresed on SDS/PAGE, and Western blotted with an eNOS recently developed procedure (4, 11). In brief, ventilated rat mAb. Similar results were obtained in human umbilical vein endothe- lungs were perfused in situ at 10-13°C with a solution of lial cells and HEK 293 cells stably transfected with the eNOS cDNA. Downloaded by guest on September 24, 2021 6450 Cell Biology: Garcia-Cardefia et al. Proc. Natl. Acad. Sci. USA 93 (1996)

FIG. 2. eNOS colocalizes with caveolin in cultured microvascular endothelial cells. BLMVEC were fixed and double immunolabeled for eNOS (A) and caveolin (B) as described. C represents merged images of A and B with yellow marking regions in which the signals overlap. Note in A the intense labeling of eNOS in both perinuclear and plasma membrane regions of the cell. the leading edge of BLMVEC (Fig. 2C). To biochemically luminal plasma membranes suggesting that eNOS is in caveo- show that eNOS resided on the cell surface of BLMVEC, we lae. However, recent studies demonstrated that TIM are not purified luminal plasmalemmal endothelial membranes (P) equivalent to purified caveolae because they contain other from the other cellular membranes (OM, inclusive of intra- distinct microdomains, including those rich in GPI-anchored cellular and basolateral membranes) and examined the abun- proteins and cytoskeletal proteins (11). Thus, to more defin- dance of eNOS, caveolin, and --COP, a specific marker for itively identify the plasma membrane domains wherein eNOS Golgi and post-Golgi vesicles, in each fraction. As seen in Fig. resides, we first purified luminal endothelial cell plasma mem- 3, with equivalent loading of protein from each fraction, eNOS branes from intact rat lungs and examined the presence of was found in H, P and OM. This is in contrast to caveolin, eNOS protein in various subfractions. As seen in Fig. 5, eNOS which was enriched in P relative to H and OM and s-COP was detected in whole rat lung homogenates (H) and in the which was detected only in OM. This confirms that mature luminal plasma membrane fraction (P), demonstrating that eNOS resides in both plasmalemma and intracellular mem- eNOS was indeed present on the cell surface of endothelium, branes. in vivo. In the absence of Triton X-100, these silica-coated To examine if palmitoylation influences the subcellular plasma membranes were subfractionated by shearing followed targeting of eNOS to caveolin-rich plasmalemmal microdo- by sucrose density centrifugation. As reported recently, this mains, we transiently transfected NIH 3T3 cells with WT or the procedure yielded a homogeneous population of caveolae (11). palmitoylation mutant of eNOS (C15/26S) cDNAs and exam- Here, eNOS was markedly enriched in the purified caveolae ined the localization of eNOS and caveolin by confocal mi- (V') relative to P. Caveolin, a marker for caveolae, was also croscopy. As seen in Fig. 4, WT eNOS was found lightly enriched in the V' fraction relative to P. eNOS was not distributed throughout the perinuclear region and strongly detectable in luminal plasma membranes stripped of caveolae displayed in the plasma membrane, as seen in BLMVEC, (data not shown) whereas caveolin was present in smaller whereas the C15/26S eNOS was detected in the perinuclear amounts to as described region and diffusely throughout the cytoplasm, but not on the relative V', previously (11). Thus, cell surface or leading edge (Fig. 4 Left). In a majority of the eNOS resides on the cell surface primarily in caveolae. transfected cells, eNOS antigen in the plasma membrane of eNOS Caveolin WT-transfected cells colocalized with caveolin; a pattern absent in cells transfected with the palmitoylation mutant (Fig. 4 Right). Similar results were obtained in NIH 3T3 cells stably transfected with WT or C15/26S eNOS (data not shown). The above data showed that WT eNOS was in TIM, WT colocalized with caveolin on the cell surface and found in H P OM _m'm _. eNOS

...... _ _: Caveolin C15/26S

-COP

FIG. 3. eNOS is found in luminal plasma membranes and other intracellular membranes isolated from BLMVEC. Proteins of the FIG. 4. Palmitoylation is necessary for targeting of eNOS into indicated fractions isolated from BLMVEC were resolved by SDS/ caveolin-rich domains of the plasma membrane. NIH 3T3 cells were 5-15% PAGE and Western blotted with eNOS, caveolin, and E-COP transiently transfected with either WT or palmitoylation mutant mAbs as described. Proteins (5 jig) were loaded into each lane from (C15/26S) eNOS cDNAs. Cells were fixed and double immunolabeled the following fractions: H (BLMVEC homogenate), P (silica-coated for eNOS and caveolin as described. Arrowheads denote colocaliza- luminal endothelial cell plasma membrane), and OM (other intracel- tion of eNOS and caveolin (Top) and arrows denote a lack of lular and basolateral membranes). colocalization (Bottom). Downloaded by guest on September 24, 2021 Cell Biology: Garcl'a-Cardefia et al. Proc. Natl. Acad. Sci. USA 93 (1996) 6451 in luminal membranes. Further sub- H P V highly purified plasma fractionation reveals that eNOS may reside exclusively within the caveolar microdomain of this plasma membrane fraction. eNOS This in situ method allows for accurate determination of protein localization in luminal plasma membranes and specific subdomains such as the caveolae. For example, when caveolae ...... ~~~~~...... are purified from luminal plasma membranes, it is clear that Caveolin other plasmalemmal domains remain behind. In fact, microdo- Caveolin ~~~~~. mains rich in GPI-linked proteins can be isolated separately FIG. 5. eNOS is present on the endothelial cell surface and is from the residual plasmalemma after isolation of caveolae enriched in caveolae. Proteins of the indicated fractions isolated from (11). Distinction between these two domains is not possible rat lungs were resolved by SDS/5-15% PAGE and Western blotted with other currently available methodologies for isolating with eNOS and caveolin mAbs as described. Protein (5 ,ug) were caveolae. Other methodologies (37, 38), rely primarily on loaded into each lane from fractions: H (rat lung homogenate), P isolation of caveolae-containing fractions from cultured cells. (silica-coated luminal endothelial cell plasma membrane), and V' In vitro, endothelial cells have fewer caveolae, so that the (endothelial cell caveolae). amount of protein in the caveolar subdomain of the plasma DISCUSSION membrane will be underestimated. More importantly, these other methods cannot focus specifically on the endothelial cell The present study demonstrates that eNOS is enriched in surface purified from its biologically active state in tissue. highly purified caveolae isolated from intact rat lungs and eNOS is dually acylated via cotranslational N-myristoylation colocalizes with caveolin, a marker for caveolae, in cultured on Gly-2 and posttranslational palmitoylation of cysteines 15 microvascular endothelial cells and in fibroblasts transiently and/or 26. By analogy to Src family members and G protein a transfected with the eNOS cDNA. Palmitoylation at cysteine subunits, N-myristoylation of Gly-2 is necessary for membrane residues 15 and 26 is required for directing eNOS to caveolae, association, whereas the role for cysteine palmitoylation is less as mutation of these sites inhibits this targeting of the protein. clear. In certain proteins, mutation of the palmitoylation sites Thus, eNOS joins the list of dually acylated proteins (Src inhibits [3H]palmitate incorporation and modestly increases kinase family members and G protein a subunits) that are protein solubility, suggesting that palmitate enhances protein found in the caveolar-signal processing center of cells (4). hydrophobicity and stable membrane association (39). How- Functionally, the targeting of eNOS to caveolae likely provides ever, this effect has not been seen for all palmitoylated endothelial cells with an efficient mechanism to locally pro- proteins, including eNOS (23). As recently described, cysteine duce NO in response to hemodynamic forces and to activation palmitoylation at position 3 in the amino terminal consensus of cell surface receptors. motif MGCXXC/S observed in the Src family members The findings that eNOS is found in purified caveolae and in (p56Ick, p59hck and p59fyn) is required for targeting to TIM both caveolin-containing plasmalemmal regions and Golgi containing caveolin and GPI-anchored proteins (5-7). In the regions of BLMVEC, unify the previous reports demonstrat- present study, we show that a significant fraction of eNOS ing that eNOS is found in the plasma membrane and/or on the colocalizes with plasmalemmal caveolin in transfected fibro- Golgi complex of cultured aortic endothelial cells and endo- blasts and that mutation of cysteines 15 and 26 prevents thelial cells lining intact blood vessels (24, 27, 32-36). Al- eNOS/caveolin colocalization. These data suggest that palmi- though found on the plasma membrane in some studies, the toylation of eNOS is necessary for Golgi association and/or paucity of cell surface associated eNOS in cultured large vessel retention by specific Golgi derived vesicles and subsequent endothelium and in intact large blood vessels is most likely targeting to caveolae. Thus, the dual acylation motif for related to the loss of caveolae due to culture conditions (25) N-myristoylation and cysteine palmitoylation of eNOS, and the relative lack of the organelle in the endothelium of M'GXXXS6...C5(GL)5 C26, is a novel caveolae targeting large vessels, respectively (25, 32). As seen in cultured BLM- motif distinct from that found in Src family members and G VEC, cells that retain 5-10 times more caveolae than BAECs protein a subunits (6). (25), a significant portion of eNOS appears in the caveolin-rich As we and others have previously described, a majority of plasma membrane domain, whereas in cultured BAECs, much eNOS in cultured endothelial cells and in cells stably trans- less plasma membrane associated eNOS immunoreactivity is fected with eNOS resides in the Golgi region and in discrete seen under identical culture and immunocytochemical staining plasma membrane domains (24, 27, 33-36), now shown here to conditions (24). Consistent with the immunocytochemical be the caveolae. A majority of the Golgi staining (-70%) localization of eNOS on the cell surface and in intracellular colocalizes with mannosidase II, a cis-medial Golgi marker, compartments is the presence of immunoreactive eNOS in with the residual Golgi staining representing eNOS most likely purified luminal plasma membranes and intracellular mem- in vesicles associated with the trans-Golgi network (TGN). branes prepared from BLMVEC. Thus, it is likely that the More importantly, Golgi targeting of eNOS is necessary for amount of eNOS in caveolae, visualized by microscopic tech- stimulated NO release from cells, as expression of cytosolic, niques or biochemically assayed in lysates prepared from nonacylated eNOS (G2A, myristoylation mutant) results in cultured endothelial cells, will be underestimated relative to markedly less NO release (24). In the present study (Figs. 2 and that seen in vivo and will be determined by the nature of the 3), eNOS in BLMVEC is clearly demonstrable by both immu- vessel from which the endothelial cells are isolated. More nocytochemistry and biochemical purification, in two immu- importantly, the segmental differentiation of eNOS localiza- noreactive pools; the perinuclear region (enriched in Golgi tion in caveolae throughout the vascular tree suggests that NO membranes) and in caveolin-rich, cell surface domains (caveo- may subserve different roles in the macro- and microcircula- lae). tions. Perhaps, in large vessels, NO acts primarily as a relaxing The presence of eNOS in these two cellular compartments factor, whereas in smaller-diameter vessels where the role of suggests anterograde transport of eNOS from the cytosol to NO in vascular control is less prominant, NO may act more the Golgi and then to caveolae, in addition to a possible efficiently as an inhibitor of leukocyte adhesion and platelet recycling pathway between the cell surface and the TGN. aggregation and a modulator of cell growth and vascular Based on our findings, the following biosynthetic pathway is permeability. proposed (Fig. 6). eNOS is translated on a cytoplasmic ribo- We also show that eNOS is expressed amply on the luminal some and is co-translationally myristoylated by N-myristoyl cell surface of microvascular endothelium, in vivo, as detected transferase, a cytosolic protein (1). The nascent N- Downloaded by guest on September 24, 2021 6452 Cell Biology: Garcia-Cardefia et al. Proc. Natl. Acad. Sci. USA 93 (1996) dynamic perturbations of the plasma membrane (via shear stress or cyclic strain) in endothelial cells can stimulate the production of an important second messenger, NO. Identifi- cation of the molecular machinery necessary for eNOS tar- geting and elucidation of the signals that determine the differential localization of eNOS in macro- and microvascular endothelial cells will undoubtedly shed more light on the functions of NO in the cardiovascular system. We thank Dr. Jennifer S. Pollock for the generous supply of eNOS mAb and Dr. Rudi Busse for advice and encouragement. This work is supported by grants from the National Institutes of Health (HL 51948 to W.C.S., F32-HL09224 to J.L., and HL43278 and HL52766 to J.E.S.), an Established Investigator Award from the American Heart Associ- ation/Genentech (J.E.S.), the Patrick and Catherine Weldon Dona- ghue Medical Research Foundation (W.C.S.), and the Government of Mexico/Yale University (G.G;-C.). The Molecular Cardiobiology Program at Yale is supported by a grant from American Cyanamid. 1. Wedegaertner, P. B., Wilson, P. T. & Bourne, H. R. (1995) FIG. 6. Proposed model of eNOS biosynthesis and targeting. In J. Bio. Chem. 270, 503-506. brief, eNOS is cotranslationally N-myristoylated (1); palmitoylated in 2. Fujimoto, T., Nakade, S., Miyawaki, A. Mikoshiba, K. & Ogawa, the Golgi network (2), and targeted to caveolae from the TGN (3). K. (1992) J. Cell Biol. 119, 1507-1513. eNOS can then be depalmitoylated and return to the Golgi network (4) 3. Fujimoto, I. (1993) J. Cell Biol. 120, 1147-1157. for another round of palmitoylation and caveolar targeting. The 4. Schnitzer, J. E., Oh, P., Jacobson, B. S. & Dvorak, A. N. (1995) trafficking to and from the Golgi is presumably vesicle mediated, Proc. Natl. Acad. Sci USA 92, 1759-1763. however supporting data is lacking. An alternative pathway leading 5. Rodgers, W., Crise, B. & Rose, J. K. (1994) Mol. Cell. Biol. 14, from a cytosolic N-myristoylated protein to a dually acylated, caveolar 5384-91. form of eNOS is possible (5). 6. Robbins, S. M., Quintrell, N. A. & Bishop, J. M. (1995) Moi. Cell. Bio. 15, 3507-3515. myristoylated protein may associate to a higher-order struc- 7. Shenoy-Scaria, A. M., Sietzen, D. J., Kwong, J., Link, D. C. & ture and target to the cytoplasmic face of the Golgi (2). The Lublin, D. M. (1995) J. Cell Biol. 126, 353-363. molecular mechanisms of Golgi targeting are not known, but 8. Li, S., Okamoto, T., Chun, M., Sargiacomo, M., Casanova, J. E., of eNOS is Hansen, S. H., Nishimoto, I. & Lisanti, M. P. (1995) J. Biol. N-myristoylation required for the enzyme to get Chem. 270, 15693-15701. into this compartment (24). eNOS can then be palmitoylated 9. Schnitzer, J. E., Liu, J. & Oh, P. (1995) J. Biol. Chem. 270, on the Golgi by a putative palmitoyl-transferase and the 14399-14404. palmitoylated protein then targets most likely to the TGN and 10. Chun, M., Liyanage, U. K., Lisanti, M. P. & Lodish, H. F. (1994) then to caveolae (3). Once in caveolae, eNOS can be recycled Proc. Natl. Acad. Sci. USA 91, 11728-11732. back to the TGN (4), possibly via a route recently described for 11. Schnitzer, J. E., McIntosh, D. P., Dvorak, A. M., Liu, J. & Oh, P. the caveolar coat protein, caveolin (40). The rapid turnover of (1995) Science 269, 1435-1439. palmitate (45 min) relative to the half-life of myristate and the 12. Anderson, R. G. W. (1993) Proc. Natl. Acad. Sci. USA 90, eNOS polypeptide backbone ref. that the 10909-10913. (18 hr; 23) suggests 13. Schnitzer, J. E. (1993) Trends Cardiovasc. Med. 3, 124-129. cycle of palmitoylation and depalmitoylation can regulate the 14. Moncada, S., Palmer, R. M. J. & Higgs, E. A. (1991) Pharmacol. amount of eNOS residing in the caveolae and the Golgi, Rev. 43, 109-142. respectively. Depalmitoylation of eNOS by a yet undescribed 15. Griffith, 0. W. & Stuehr, D. J. (1995) Annu. Rev. Physiol. 57, cell surface associated cysteine palmitoyl-thioesterase would 707-736. facilitate this process. Alternatively, N-myristoylated eNOS 16. Tsukahara, H., Gordienko, D. B., Tonshoff, B., Gelato, M. C. & may be targeted directly to the plasma membrane (5) where it Goligorsky, M. S. (1994) Kidney Int. 45, 598-604. can be palmitoylated. When myristoylated, depalmitoylated 17. Ziche, M., Morbidelli, L., Masini, E., Amerini, S., Granger, H. J., eNOS returns to the it then can be Maggi, C. A., Geppetti, P. & Ledda, F. (1994) J. Clin. Invest. 94, Golgi network, rapidly 2036-2044. palmitoylated once again and targeted to caveolae. The signals 18. Peunova, N. & Enikolopov, G. (1995) Nature (London) 375, and mechanisms that stimulate the movement of eNOS or 68-72. other dually acylated proteins through the Golgi to caveolae 19. Sessa, W. C. (1994) J. Vasc. Res. 131, 131-143. and from caveolae back to the Golgi are not known, but are 20. Pollock, J. S., Klinghofer, V., Forstermann, U. & Murad, F. likely mediated by specific vesicles and are presumably differ- (1992) FEBS Lett. 309, 402-404. ent in micro- and macrovascular endothelial cells due the 21. Liu, J. & Sessa, W. C. (1994) J. Biol. Chem. 269, 11691-11694. dramatic differences in eNOS localization in these cell types. 22. Robinson, L. J., Busconi, L. & Michel, T. (1995) J. Biol. Chem. Moreover, the pool of eNOS necessary for basal or stimulated 270, 995-998. 23. Liu, J., Garcia-Cardenia, G., & Sessa, W. C. (1995) Biochemistry release of NO are not known. Both membrane-enriched and 34, 12333-12340. membrane-free, cytosolic fractions prepared from endothelial 24. Sessa, W. C., Garcia-Cardefia, G., Liu. J., Keh, A., Pollock, J. S., cell lysates contain catalytically active eNOS (34, 41), whereas Bradley, J., Thiru, S., Braverman, I. M. & Desai, K. M. (1995) more NO is released from intact cells expressing enzyme in the J. Biol. Chem. 270, 17641-17644. Golgi region of cells compared with cells expressing equivalent 25. Schnitzer, J. E., Siflinger, A., DelVecchio, P. J. & Malik, A. B. amounts of cytosolic eNOS (24). These data support the (1994) Biochem. Biophys. Res. Commun. 199, 11-19. concept that biologically active eNOS resides in different 26. Tzeng, E., Billiar, T. R., Robbins, P. D., Loftus, M. & Stuehr, subcellular compartments and suggest that each pool can be D. J. (1995) Proc. Natl. Acad. Sci. USA 92, 11771-11775. and to different forms of 27. Pollock, J. S., Nakane, M., Buttery, L. D., Martinez, A., Springall, differentially regulated responsive D., Polak, J. M., Forstermann, U. & Murad, F. (1993) Am. J. stimulation (i.e., shear stress versus calcium mobilizing ago- Physiol. 265, C1379-C1387. nists). 28. Schnitzer, J. S. & Oh, P. (1996) Am. J. Physiol. 270, 416-422. The presence of eNOS in caveolae, in vivo and in vitro, 29. Guo, Q., Vasile, E. & Krieger, M. (1994) J. 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