palmitoylation: Implications for nitric oxide signaling

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

Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: Implications for nitric oxide signaling (endothelial nitric oxide synthase/signal transduction/vascular biology/N-myristoylation)

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,

tDepartment of Pathology, Harvard Medical School, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215

CT 06536; and

Communicated by Vincent T. Marchesi, Yale Univeristy, New Haven, CT, March 13, 1996 (received for review February 5, 1996)

insoluble membranes (TIM), suggesting that caveolae are signal processing centers (2-11). Additionally, caveolae have been implicated in other important cellular functions, including endocytosis, potocytosis, and transcytosis (12, 13). Endothelial nitric oxide synthase (eNOS) is a peripheral membrane protein that metabolizes L-arginine to nitric oxide (NO). NO is a short-lived free radical gas involved in diverse physiological and pathological processes. Endothelial-derived NO is an important paracrine mediator of vascular smooth muscle tone and is an inhibitor of leukocyte adhesion and platelet aggregation (14, 15). As an autocrine mediator, NO has been implicated in the modulation of growth factor signals and cellular proliferation (16-18). Regulation of NO signaling in endothelial cells occurs largely at the level of eNOS activity controlled by cofactors and targeting of eNOS to specific intracellular membranes (19). eNOS is dually acylated by cotranslational N-myristoylation and posttranslational cysteine palmitoylation (20-23). Nmyristoylation of eNOS is necessary for membrane association and for subsequent palmitoylation at cysteines 15 and/or 26 as determined in broken cell lysates (23). Functionally, in cultured aortic endothelial cells, intact blood vessels and heterologous expression systems, eNOS is expressed primarily in the Golgi region of cells and such localization is necessary for optimal stimulated release of NO from intact cells (24). The importance of eNOS cysteine palmitoylation is less clear because mutation of the palmitoylation sites inhibits protein palmitoylation but does not significantly influence eNOS enzyme activity, in vitro or partitioning into high speed membrane fractions (23), suggesting that palmitoylation may serve another function, such as specific membrane targeting. Here we show that eNOS is targeted to caveolae, in vivo and in vitro, via palmitoylation of the protein on cysteines 15 and 26. These observations provide molecular evidence for a novel consensus sequence that may be sufficient for localization of proteins to caveolae and suggest a role for NO in modulating signal transduction through these plasma membrane compartments.

ABSTRACT The membrane association of endothelial nitric oxide synthase (eNOS) plays an important role in the biosynthesis of nitric oxide (NO) in vascular endothelium. Previously, we have shown that in cultured endothelial cells and in intact blood vessels, eNOS is found primarily in the perinuclear region of the cells and in discrete regions of the plasma membrane, suggesting trafficking of the protein from the Golgi to specialized plasma membrane structures. Here, we show that eNOS is found in Triton X-100-insoluble membranes prepared from cultured bovine aortic endothelial cells and colocalizes with caveolin, a coat protein of caveolae, in cultured bovine lung microvascular endothelial cells as determined by confocal microscopy. To examine if eNOS is indeed in caveolae, we purified luminal endothelial cell plasma membranes and their caveolae directly from intact, perfused rat lungs. eNOS is found in the luminal plasma membranes and is markedly enriched in the purified caveolae. Because palmitoylation of eNOS does not significantly influence its membrane association, we next examined whether this modification can affect eNOS targeting to caveolae. Wild-type eNOS, but not the palmitoylation mutant form of the enzyme, colocalizes with caveolin on the cell surface in transfected NIH 3T3 cells, demonstrating that palmitoylation of eNOS is necessary for its targeting into caveolae. These data suggest that the subcellular targeting of eNOS to caveolae can restrict NO signaling to specific targets within a limited microenvironment at the cell surface and may influence signal transduction through caveolae.

The compartmentalization of plasma membrane proteins is critical for specificity of intracellular signaling pathways. Integral membrane proteins, such as hormone receptors, are anchored in biological membranes by hydrophobic stretches of amino acids comprising transmembrane domains, whereas other proteins, such as G-proteins, are associated and targeted to subcellular membranes by the co- or posttranslational lipid modifications N-myristoylation, palmitoylation, or prenylation (1). Signals initiated at the cell surface can propagate via soluble intracellular messengers, changes in protein phosphorylation, protein-protein interactions, and protein-lipid interactions. The specialization of plasma membrane microdomains is a potential mechanism for integrating extracellular signals into intracellular messages. For example, caveolae are plasma membrane invaginations composed primarily of glycosphingolipids, cholesterol, and the integral membrane protein, caveolin. Several proteins involved in signal transduction, such as inositol 1,4,5-triphosphate receptors, calcium ATPase, members of the src family of nonreceptor tyrosine kinases, G proteins, G protein-coupled membrane receptors, and gangliosides have been found in caveolae or in Triton X-100-

MATERIALS AND METHODS Materials and Antibodies. Dulbecco's modified Eagle's medium (DMEM), glutamine, trypsin-EDTA, penicillin/ streptomycin, and fetal bovine serum (FBS) were from GIBCO. Rabbit polyclonal antibodies to eNOS and caveolin were from Transduction Laboratories (Lexington, KY); eNOS monoclonal antibody (mAb) was provided by J. Pollock (Medical College of Georgia) and e-COP mAb was provided by M. Krieger (Massachusetts Institute of Technology). Abbreviations: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; BLMVEC, bovine lung microvascular endothelial cells; TIM, Triton X-100 insoluble membranes; BAEC, bovine aortic endothelial cells; WT, wild type; TGN, trans-Golgi network. ITo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6448

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Cell Culture. Bovine aortic endothelial cells (BAEC) and bovine lung microvascular endothelial cells (BLMVEC) were obtained and grown in tissue culture as described (21, 25). NIH 3T3 cells were grown in DMEM supplemented with 10% (vol/vol) FBS, L-glutamine (1 mM), penicillin (100 units/ml), streptomycin (100 ,ug/ml), and tetrahydrobiopterin (100 ,uM; ref. 26). Subconfluent NIH 3T3 cells were transiently transfected with bovine wild-type (WT) or C15/26S palmitoylation mutant eNOS cDNAs subcloned into the mammalian expression vector, pcDNA3 (23), according to the standard calcium phosphate precipitation method. After overnight transfection, cells were trypsinized and cultured onto gelatin coated coverslips for confocal, immunofluorescence microscopy. Triton X-100 Solubility of Endothelial Proteins. One 100-mm dish of confluent BAEC (passage 2-4) was used for each sample. Each dish was washed two times with cold phosphate-buffered saline (PBS) and cells were released by incubating in MBS (125 mM NaCl/20 mM Mes, pH 6.0) plus 0.02% EDTA for 10 min on ice. Cells were collected by centrifugation for 5 min at 1000 x g at 4°C. The pellets were 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 x g at 4°C (10). The supernatant fraction was collected and designated the Triton-soluble fraction. The pellet was resuspended in modified RIPA buffer (0.15 mM NaCl/0.05 mM Tris HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/ 0.1% NaDodSO4) (23) and designated TIM. Total eNOS in both fractions was concentrated quantitatively with the affinity resin 2'5'-ADP-Sepharose as described (21). Beads were then placed into Laemmli sample buffer and electrophoresed by SDS/PAGE. Proteins were separated on 7.5% SDS gels. Proteins were transferred to nitrocellulose and Western blotted with an eNOS mAb H32 as described (21, 27). Indirect Immunofluorescence. BLMVEC or transfected NIH 3T3 cells were grown on glass coverslips. Cells were fixed in acetone for 5 min at -20°C then rinsed with PBS for 10 min at room temperature. The cells were then incubated sequentially with PBS plus 5% goat serum for 30 min at room temperature, a mixture of anti-eNOS mAb H32 and anticaveolin polyclonal antibody diluted in PBS plus 2% goat serum for 4 h at room temperature, and finally with a mixture of BODIPY (Molecular Probes)-goat anti-rabbit and Texas Red-goat anti-mouse IgGs for 1 h at room temperature. Cells were washed two times with PBS after each incubation step. Cells were mounted in Slow-Fade (Molecular Probes) and observed with a Bio-Rad MRC 600 confocal inverted microscope. Isolation of Luminal Plasma Membranes from BLMVEC. Confluent BLMVEC grown in T175 flasks were placed at 4°C for 10 min, washed with ice cold MBS, incubated with colloidal, positively charged silica and luminal plasma membranes (P) isolated as described (28). In addition, the lighter density band containing residual membranes (OM or other membranes, 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 nitrocellulose 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 peroxidase-conjugated, goat, anti-rabbit IgG secondary antibody and detected by ECL. Protein assays were performed with the Bio-Rad BCA kit by using bovine serium albumin as a standard. Caveolae Purification from Rat Lungs. Endothelial caveolae were isolated to homogeneity from rat lung tissue by a recently developed procedure (4, 11). In brief, ventilated rat lungs were perfused in situ at 10-13°C with a solution of

Proc. Natl. Acad. Sci. USA 93 (1996)

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positively charged colloidal silica particles that coated the luminal endothelial cell surface of the pulmonary vasculature. Silica coating followed by cross-linking created a stable silica pellicle that specifically marked luminal plasma membranes and allowed for purification of these membranes from tissue homogenates by centrifugation. The sedimented pellets (P) contained highly purified endothelial cell luminal plasma membranes with associated caveolae and showed ample enrichment of various endothelial cell surface markers relative to the starting whole lung homogenates (H; refs. 4 and 11). Caveolae were removed from P by shearing during homogenization at 4°C in the absence of Triton X-100. These homogenates were subjected to sucrose density centrifugation to yield two collected fractions; a low density fraction of a homogenous population of biochemically and morphologically distinct caveolar vesicles (V'). Protein samples from each fraction (5 ,ug) were separated by SDS/PAGE (5-15% gradient gels) and electrotransfered to nitrocellulose membranes for immunoblotting with antisera to eNOS and caveolin as described above.

RESULTS Triton X-100 insolubility was considered a hallmark of cytoskeletal proteins (30) and, more recently, of proteins residing in caveolae and in other glycolipid-rich domains (11, 31). To examine if eNOS resides in Triton X-100 insoluble fractions of cultured BAECs, Triton X-100 insoluble and soluble fractions were prepared and Western blotted with an eNOS mAb. As seen in Fig. 1, -10% of eNOS resided in TIM, with 90% being Triton soluble. Similar results were obtained using human umbilical vein endothelial cells and in HEK 293 cells stably transfected with the eNOS cDNA. Because large vessel endothelium in intact blood vessels have few caveolae relative to microvascular endothelium (25, 32) and culturing of endothelial cells causes a significant loss in caveolae (25), we examined the colocalization of eNOS and caveolin in BLMVEC. BLMVEC have significantly more caveolae than other cultured endothelia, including aortic and pulmonary artery endothelial cells (25). As seen in Fig. 2, eNOS was localized in the perinuclear region of the cell and in a specific microdomain of the plasma membrane (Fig. 2A). Caveolin was localized primarily at the leading edge with faint, perinuclear staining (Fig. 2B). The merged images of Fig. A and B demonstrated colocalization of eNOS and caveolin at

S

I

199 -

120 -

87 -

48 -

FIG. 1. Partitioning of eNOS into Triton X-100-soluble (S) and -insoluble (I) fractions of cultured BAEC. S and I fractions were prepared from BAEC lysates, ADP-Sepharose concentrated and electrophoresed on SDS/PAGE, and Western blotted with an eNOS mAb. Similar results were obtained in human umbilical vein endothelial cells and HEK 293 cells stably transfected with the eNOS cDNA.

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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 show that eNOS resided on the cell surface of BLMVEC, we purified luminal plasmalemmal endothelial membranes (P) from the other cellular membranes (OM, inclusive of intracellular and basolateral membranes) and examined the abundance of eNOS, caveolin, and --COP, a specific marker for Golgi and post-Golgi vesicles, in each fraction. As seen in Fig. 3, with equivalent loading of protein from each fraction, eNOS was found in H, P and OM. This is in contrast to caveolin, which was enriched in P relative to H and OM and s-COP which was detected only in OM. This confirms that mature eNOS resides in both plasmalemma and intracellular membranes. To examine if palmitoylation influences the subcellular targeting of eNOS to caveolin-rich plasmalemmal microdomains, we transiently transfected NIH 3T3 cells with WT or the palmitoylation mutant of eNOS (C15/26S) cDNAs and examined the localization of eNOS and caveolin by confocal microscopy. As seen in Fig. 4, WT eNOS was found lightly distributed throughout the perinuclear region and strongly displayed in the plasma membrane, as seen in BLMVEC, whereas the C15/26S eNOS was detected in the perinuclear region and diffusely throughout the cytoplasm, but not on the cell surface or leading edge (Fig. 4 Left). In a majority of the transfected cells, eNOS antigen in the plasma membrane of 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, colocalized with caveolin on the cell surface and found in

H

luminal plasma membranes suggesting that eNOS is in caveolae. However, recent studies demonstrated that TIM are not equivalent to purified caveolae because they contain other distinct microdomains, including those rich in GPI-anchored proteins and cytoskeletal proteins (11). Thus, to more definitively identify the plasma membrane domains wherein eNOS resides, we first purified luminal endothelial cell plasma membranes from intact rat lungs and examined the presence of eNOS protein in various subfractions. As seen in Fig. 5, eNOS was detected in whole rat lung homogenates (H) and in the luminal plasma membrane fraction (P), demonstrating that eNOS was indeed present on the cell surface of endothelium, in vivo. In the absence of Triton X-100, these silica-coated plasma membranes were subfractionated by shearing followed by sucrose density centrifugation. As reported recently, this procedure yielded a homogeneous population of caveolae (11). Here, eNOS was markedly enriched in the purified caveolae (V') relative to P. Caveolin, a marker for caveolae, was also enriched in the V' fraction relative to P. eNOS was not detectable in luminal plasma membranes stripped of caveolae (data not shown) whereas caveolin was present in smaller amounts relative to V', as previously described (11). Thus, eNOS resides on the cell surface primarily in caveolae. eNOS

Caveolin

WT

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 indicated fractions isolated from BLMVEC were resolved by SDS/ 5-15% PAGE and Western blotted with eNOS, caveolin, and E-COP mAbs as described. Proteins (5 jig) were loaded into each lane from the following fractions: H (BLMVEC homogenate), P (silica-coated luminal endothelial cell plasma membrane), and OM (other intracellular and basolateral membranes).

FIG. 4. Palmitoylation is necessary for targeting of eNOS into caveolin-rich domains of the plasma membrane. NIH 3T3 cells were transiently transfected with either WT or palmitoylation mutant (C15/26S) eNOS cDNAs. Cells were fixed and double immunolabeled for eNOS and caveolin as described. Arrowheads denote colocalization of eNOS and caveolin (Top) and arrows denote a lack of colocalization (Bottom).

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Proc. Natl. Acad. Sci. USA 93 (1996) V

P

eNOS .......................~~~~~................... Caveolin Caveolin

~~~~~.

FIG. 5. eNOS is present on the endothelial cell surface and is enriched in caveolae. Proteins of the indicated fractions isolated from rat lungs were resolved by SDS/5-15% PAGE and Western blotted with eNOS and caveolin mAbs as described. Protein (5 ,ug) were loaded into each lane from fractions: H (rat lung homogenate), P (silica-coated luminal endothelial cell plasma membrane), and V' (endothelial cell caveolae).

DISCUSSION The present study demonstrates that eNOS is enriched in highly purified caveolae isolated from intact rat lungs and colocalizes with caveolin, a marker for caveolae, in cultured microvascular endothelial cells and in fibroblasts transiently transfected with the eNOS cDNA. Palmitoylation at cysteine residues 15 and 26 is required for directing eNOS to caveolae, as mutation of these sites inhibits this targeting of the protein. Thus, eNOS joins the list of dually acylated proteins (Src kinase family members and G protein subunits) that are found in the caveolar-signal processing center of cells (4). Functionally, the targeting of eNOS to caveolae likely provides endothelial cells with an efficient mechanism to locally produce NO in response to hemodynamic forces and to activation of cell surface receptors. The findings that eNOS is found in purified caveolae and in both caveolin-containing plasmalemmal regions and Golgi regions of BLMVEC, unify the previous reports demonstrating that eNOS is found in the plasma membrane and/or on the Golgi complex of cultured aortic endothelial cells and endothelial cells lining intact blood vessels (24, 27, 32-36). Although found on the plasma membrane in some studies, the paucity of cell surface associated eNOS in cultured large vessel endothelium and in intact large blood vessels is most likely related to the loss of caveolae due to culture conditions (25) and the relative lack of the organelle in the endothelium of large vessels, respectively (25, 32). As seen in cultured BLMVEC, cells that retain 5-10 times more caveolae than BAECs (25), a significant portion of eNOS appears in the caveolin-rich plasma membrane domain, whereas in cultured BAECs, much less plasma membrane associated eNOS immunoreactivity is seen under identical culture and immunocytochemical staining conditions (24). Consistent with the immunocytochemical localization of eNOS on the cell surface and in intracellular compartments is the presence of immunoreactive eNOS in purified luminal plasma membranes and intracellular membranes prepared from BLMVEC. Thus, it is likely that the amount of eNOS in caveolae, visualized by microscopic techniques or biochemically assayed in lysates prepared from cultured endothelial cells, will be underestimated relative to that seen in vivo and will be determined by the nature of the vessel from which the endothelial cells are isolated. More importantly, the segmental differentiation of eNOS localization in caveolae throughout the vascular tree suggests that NO may subserve different roles in the macro- and microcirculations. Perhaps, in large vessels, NO acts primarily as a relaxing factor, whereas in smaller-diameter vessels where the role of NO in vascular control is less prominant, NO may act more efficiently as an inhibitor of leukocyte adhesion and platelet aggregation and a modulator of cell growth and vascular permeability. We also show that eNOS is expressed amply on the luminal cell surface of microvascular endothelium, in vivo, as detected a

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in highly purified luminal plasma membranes. Further subfractionation reveals that eNOS may reside exclusively within the caveolar microdomain of this plasma membrane fraction. 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 other plasmalemmal domains remain behind. In fact, microdomains rich in GPI-linked proteins can be isolated separately from the residual plasmalemma after isolation of caveolae (11). Distinction between these two domains is not possible with other currently available methodologies for isolating caveolae. Other methodologies (37, 38), rely primarily on isolation of caveolae-containing fractions from cultured cells. In vitro, endothelial cells have fewer caveolae, so that the amount of protein in the caveolar subdomain of the plasma membrane will be underestimated. More importantly, these other methods cannot focus specifically on the endothelial cell surface purified from its biologically active state in tissue. eNOS is dually acylated via cotranslational N-myristoylation on Gly-2 and posttranslational palmitoylation of cysteines 15 and/or 26. By analogy to Src family members and G protein a subunits, N-myristoylation of Gly-2 is necessary for membrane association, whereas the role for cysteine palmitoylation is less clear. In certain proteins, mutation of the palmitoylation sites inhibits [3H]palmitate incorporation and modestly increases protein solubility, suggesting that palmitate enhances protein hydrophobicity and stable membrane association (39). However, this effect has not been seen for all palmitoylated proteins, including eNOS (23). As recently described, cysteine palmitoylation at position 3 in the amino terminal consensus motif MGCXXC/S observed in the Src family members (p56Ick, p59hck and p59fyn) is required for targeting to TIM containing caveolin and GPI-anchored proteins (5-7). In the present study, we show that a significant fraction of eNOS colocalizes with plasmalemmal caveolin in transfected fibroblasts and that mutation of cysteines 15 and 26 prevents eNOS/caveolin colocalization. These data suggest that palmitoylation of eNOS is necessary for Golgi association and/or retention by specific Golgi derived vesicles and subsequent targeting to caveolae. Thus, the dual acylation motif for N-myristoylation and cysteine palmitoylation of eNOS, M'GXXXS6.. .C5(GL)5 C26, is a novel caveolae targeting motif distinct from that found in Src family members and G protein a subunits (6). As we and others have previously described, a majority of eNOS in cultured endothelial cells and in cells stably transfected with eNOS resides in the Golgi region and in discrete plasma membrane domains (24, 27, 33-36), now shown here to be the caveolae. A majority of the Golgi staining (-70%) colocalizes with mannosidase II, a cis-medial Golgi marker, with the residual Golgi staining representing eNOS most likely in vesicles associated with the trans-Golgi network (TGN). More importantly, Golgi targeting of eNOS is necessary for stimulated NO release from cells, as expression of cytosolic, nonacylated eNOS (G2A, myristoylation mutant) results in markedly less NO release (24). In the present study (Figs. 2 and 3), eNOS in BLMVEC is clearly demonstrable by both immunocytochemistry and biochemical purification, in two immunoreactive pools; the perinuclear region (enriched in Golgi membranes) and in caveolin-rich, cell surface domains (caveo-

lae). The presence of eNOS in these two cellular compartments suggests anterograde transport of eNOS from the cytosol to the Golgi and then to caveolae, in addition to a possible recycling pathway between the cell surface and the TGN. Based on our findings, the following biosynthetic pathway is proposed (Fig. 6). eNOS is translated on a cytoplasmic ribosome and is co-translationally myristoylated by N-myristoyl transferase, a cytosolic protein (1). The nascent N-

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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. Identification of the molecular machinery necessary for eNOS targeting 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 Association/Genentech (J.E.S.), the Patrick and Catherine Weldon Donaghue 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. FIG. 6. Proposed model of eNOS biosynthesis and targeting. In brief, eNOS is cotranslationally N-myristoylated (1); palmitoylated in the Golgi network (2), and targeted to caveolae from the TGN (3). eNOS can then be depalmitoylated and return to the Golgi network (4) for another round of palmitoylation and caveolar targeting. The trafficking to and from the Golgi is presumably vesicle mediated, however supporting data is lacking. An alternative pathway leading from a cytosolic N-myristoylated protein to a dually acylated, caveolar form of eNOS is possible (5).

myristoylated protein

may associate to a higher-order structure and target to the cytoplasmic face of the Golgi (2). The molecular mechanisms of Golgi targeting are not known, but N-myristoylation of eNOS is required for the enzyme to get into this compartment (24). eNOS can then be palmitoylated on the Golgi by a putative palmitoyl-transferase and the palmitoylated protein then targets most likely to the TGN and then to caveolae (3). Once in caveolae, eNOS can be recycled back to the TGN (4), possibly via a route recently described for the caveolar coat protein, caveolin (40). The rapid turnover of palmitate (45 min) relative to the half-life of myristate and the eNOS polypeptide backbone (18 hr; ref. 23) suggests that the cycle of palmitoylation and depalmitoylation can regulate the amount of eNOS residing in the caveolae and the Golgi, respectively. Depalmitoylation of eNOS by a yet undescribed cell surface associated cysteine palmitoyl-thioesterase would facilitate this process. Alternatively, N-myristoylated eNOS may be targeted directly to the plasma membrane (5) where it can be palmitoylated. When myristoylated, depalmitoylated eNOS returns to the Golgi network, it then can be rapidly palmitoylated once again and targeted to caveolae. The signals

and mechanisms that stimulate the movement of eNOS or other dually acylated proteins through the Golgi to caveolae and from caveolae back to the Golgi are not known, but are likely mediated by specific vesicles and are presumably different in micro- and macrovascular endothelial cells due the dramatic differences in eNOS localization in these cell types. Moreover, the pool of eNOS necessary for basal or stimulated release of NO are not known. Both membrane-enriched and membrane-free, cytosolic fractions prepared from endothelial cell lysates contain catalytically active eNOS (34, 41), whereas more NO is released from intact cells expressing enzyme in the Golgi region of cells compared with cells expressing equivalent amounts of cytosolic eNOS (24). These data support the concept that biologically active eNOS resides in different subcellular compartments and suggest that each pool can be differentially regulated and responsive to different forms of stimulation (i.e., shear stress versus calcium mobilizing ago-

nists). The presence of eNOS in caveolae, in vivo and in vitro, suggests that receptor-G protein mediated signaling or hemo-

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