Palmitoylation of Caveolin-1 Is Required for Cholesterol Binding, Chaperone Complex Formation, and Rapid Transport of Cholesterol to Caveolae*

We previously demonstrated that a caveolin-chaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae. Caveolin-1 has a 33-amino acid hydrophobic domain and three sites of palmitoylation in proximity to the hydrophobic domain. In the present study, we hypothesized that palmitoylation of caveolin-1 is necessary for binding of cholesterol, formation of a caveolin-chaperone transport complex, and rapid, direct transport of cholesterol to caveolae. To test this hypothesis, four caveolin-1 constructs were generated that substituted an alanine for a cysteine at position 133, 143, or 156 or all three sites (triple mutant). These mutated caveolins and wild type caveolin-1 were stably expressed in the lymphoid cell line, L1210-JF, which does not express caveolin-1, does not form a caveolin-chaperone complex, and does not transport newly synthesized cholesterol to caveolae. All of the caveolins were expressed and the proteins localized to plasma membrane caveolae. Wild type caveolin-1 and mutant 133 assembled into complete transport complexes and rapidly (10–20 min) transported cholesterol to caveolae. Caveolin mutants 143 and 156 did not assemble into complete transport complexes, weakly associated with cholesterol, and transported small amounts of cholesterol to caveolae. The triple mutant did not assemble into complete transport complexes and did not associate with cholesterol. We conclude that palmitoylation of caveolin-1 at positions 143 and 156 is required for cholesterol binding and transport complex formation.

Caveolae are cholesterol/sphingomyelin-rich plasma membrane microdomains that are present in most cells (1,2). Cholesterol is central to the structure and function of caveolae (2). The cytoplasmic surfaces of caveolae are covered with a characteristic coat structure that is disrupted and partially disassembled by cholesterol-binding drugs such as filipin and nystatin (3,4). Cholesterol-binding drugs also cause invaginated caveolae to flatten within the plane of the membrane (4). Studies with cholesterol-depleted cells demonstrated that the number of invaginated caveolae were dramatically reduced, whereas the number of invaginated caveolae returned to control levels when the cells were cholesterol replete (5). Proteins linked to the extracellular side of the plasma membrane by glycosylphosphatidylinositol can associate with caveolae in a cholesterol-dependent manner (5). In addition, palmitoylated proteins, such as endothelial nitric-oxide synthase, interact with caveolae in a cholesterol-dependent manner (6). Recently, Fielding and Fielding (7) demonstrated that low density lipoprotein-derived free cholesterol traffics to caveolae and that a caveola-associated protein, caveolin, is regulated by oxysterols. In addition, Babitt et al. (8) have demonstrated that for scavenger receptor class B, type I, a high density lipoprotein receptor that mediates the selective uptake of cholesterol esters, resides in caveolae. We recently demonstrated that caveolae were the sites of SR-BI-dependent selective cholesterol ester uptake (9). Also, Fielding and Fielding (10) have demonstrated that caveolae are involved in the efflux of free cholesterol to high density lipoprotein.
Caveolin, a 22-kDa protein, is a member of a multigene family and is often found associated with the cytoplasmic coat of caveolae. However, it is also associated with the Golgi and to a lesser extent the cytoplasm (2). Multiple functions have been proposed for the caveolins and include vesicle sorting (11,12), negative regulation of signal transduction (2,13), structural coat of caveolae (4), cellular transformation (14,15), and cholesterol metabolism (2,16). Multiple lines of evidence demonstrate that caveolin is involved in the intracellular trafficking of cholesterol. First, caveolin directly binds to cholesterol (17). Second, transfection of caveolin-1 cDNA into a lymphocyte cell line lacking morphological caveolae or an enrichment of cholesterol in caveolae-like domains induced characteristic caveolae invaginations and a 4-fold enrichment of cholesterol in caveolae with respect to total plasma membranes (18,19). Third, oxidation of cholesterol with cholesterol oxidase caused caveolin to translocate from caveolae to the endoplasmic reticulum where it moved through a Golgi-endoplasmic reticulum intermediate compartment and accumulated in the Golgi (20). Caveolin and non-oxidized cholesterol cycled back to caveolae in a microtubule-dependent manner upon removal of the cholesterol oxidase (21). Fourth, we recently demonstrated that caveolin forms a chaperone complex consisting of HSP56, cyclophilin 40, cyclophilin A, and cholesterol (22). This caveolinchaperone complex transports newly synthesized cholesterol from the endoplasmic reticulum through the cytoplasm to caveolae (22).
Caveolin has a 33-amino acid hydrophobic domain and three cysteine residues in the C-terminal portion of the protein that are palmitoylated. Studies by Lublin and others (23)(24)(25) have demonstrated that palmitoylation can target proteins to caveolae. However, Dietzen et al. (23) clearly demonstrated that palmitoylation of caveolin is not necessary to target this protein to caveolae. The function of caveolin palmitoylation is not known. In the present study, we hypothesized that palmitoy-lation of caveolin is necessary for binding of cholesterol, formation of a caveolin-chaperone transport complex, and rapid, direct transport of cholesterol to caveolae. To test this hypothesis, we constructed palmitoylation-minus mutants of caveolin-1 and stably expressed these mutant proteins in caveolin-minus cells. We demonstrated that two of the palmitoylation sites in caveolae are necessary for cholesterol binding and formation of the caveolin-chaperone transport complex. These data provide a mechanistic basis for the interaction of caveolin and cholesterol.
Cell Culture-L1210-JF cells are a murine lymphocyte cell line that does not express caveolin (19). On day 0, 1 ϫ 10 5 cells were seeded onto 100-mm dishes in RPMI 1640 medium plus 0.5% (v/v) glutamine, 25 mM HEPES, pH 7.4, and 10% (v/v) calf serum. Transfected cell medium also contained 300 g/ml Geneticin. The cholesterol pool was labeled by changing the medium on day 3 to RPMI 1640 medium plus 20 mM HEPES, pH 7.4, adding [ 3 H]acetate (30 Ci/dish), and incubating for the indicated times.
Transfection with Caveolin cDNA-Caveolin-1 cDNA was subcloned into a pCI-NEO vector (Promega) using EcoRI sites. Twenty-four hours before transfection, about one million cells were plated per 100-mm dish. On the day of transfection, 5 g of plasmid DNA was diluted in 200 l of serum-free RPMI media. In a separate tube, 20 l of Lipo-fectAMINE was diluted in 200 l of serum-free RPMI media. The diluted DNA and LipofectAMINE were then gently mixed and incubated at 25°C for 30 min. After the incubation, 6 ml of serum-free RPMI media was added to the DNA/LipofectAMINE mixture, mixed, and placed onto cells rinsed with serum-free RPMI media. The cells were incubated for 5 h at 37°C. Without washing, 3.6 ml of RPMI media containing 20% calf serum (v/v) was added. The cells were grown for 24 h. The media was removed, and RPMI media containing 10% calf serum (v/v) and 1.5 mg/ml Geneticin was added. The cells were grown under constant selection in medium containing 300 g/ml Geneticin.
Palmitoylation-Biosynthetic labeling with [ 3 H]palmitate was carried out as described previously (26). For the acylation experiments, the gels were soaked in 1 M Tris, pH 7.5, or 1 M hydroxylamine, pH 7.5, for 14 h before being processed for fluorography. Palmitoyl groups are attached by a thioester linkage and are susceptible to hydrolysis by hydroxylamine, whereas myristate is insensitive to hydroxylamine.
Isolation of Caveolae-Caveolae membranes were isolated as described previously (22) with the modifications described below. This procedure generates a highly purified plasma membrane microdomain that is free from intracellular markers (data not shown) and bulk plasma membrane markers. This method has been used extensively to characterize caveolae membrane signaling events (for reviews see Refs. 1 and 2).
Electrophoresis and Immunoblots-Samples were concentrated by trichloroacetic acid precipitation and washed in acetone. Pellets were suspended in Buffer C that contained 1.2% (v/v) ␤-mercaptoethanol and heated at 95°C for 3 min before being loaded onto gels. Proteins were separated in a 12.5% SDS-polyacrylamide gel using the method of Laemmli (27). The separated proteins were then transferred to PVDF.
The PVDF was blocked in Buffer A that contained 5% dry milk for 1 h at room temperature. Primary antibodies were diluted in Buffer A that contained 1% dry milk and incubated with the PVDF for 1 h at room temperature. The PVDF was washed four times for 10 min each in Buffer A plus 1% dry milk. The secondary antibodies (all conjugated to horseradish peroxidase) were diluted 1:20,000 in Buffer A plus 1% dry milk and incubated with the PVDF for 1 h at room temperature. The PVDF was then washed and the bands visualized by chemiluminescence (Pierce).
Immunoprecipitations-Protein A-Sepharose beads were first blocked by incubating them for 4 h at 4°C with the appropriate cell lysate (200 g/ml) plus 30 mg/ml bovine serum albumin in Buffer B or radioimmune precipitation buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Blocked beads were then used to preclear the experimental fractions that had been adjusted to 1% (v/v) Triton X-100, 60 mM octyl glucoside, and 0.1% (w/v) SDS. Precleared fractions were then incubated for 18 h at 4°C with the appropriate antibody before adding blocked, protein A-Sepharose beads and incubating an additional 2 h at 4°C. The beads were collected by centrifugation, washed five times in high salt radioimmune precipitation buffer (500 mM NaCl), and then placed in sample buffer or lipids extracted and analyzed by TLC (22). Immunoprecipitated proteins were detected by immunoblots or silver staining. Slight alterations in the protein to detergent ratio dramatically affected the solubility of caveolae components, in particular caveolin (data not shown). Consequently, SDS was used to ensure consistent and complete solubilization of membranes.
Radiolabeled Cholesterol Determination-Thin-layer chromatography and liquid scintillation counting was used to measure the amount of [ 3 H]sterol in each sample (22). Each sample was adjusted to a volume of 1 ml with distilled water. Methanol (1.2 ml) containing 2% (v/v) acetic acid was added to the sample before vortexing two times for 30 s each. Chloroform (1.2 ml) was then added and the sample vortexed two times for 30 s each. The organic and aqueous phases were separated in a Beckman clinical centrifuge at 3750 rpm for 15 min at room temperature. The organic phase was dried under nitrogen and then suspended in 50 l of the solvent system (80:20:1; petroleum ether:ethyl ether: acetic acid). Pure cholesterol was dissolved in the solvent system and used as a standard (5 g/spot). Lipids were visualized by charring with sulfuric acid/ethanol and heating at 180°C for 10 min. Unlabeled cholesterol was added to each fraction to facilitate visualization. The appropriate spots were scraped, and the amount of radiation was quantified by liquid scintillation counting.
Cholesterol Mass-To each 0.25-ml sample, 1 ml of hexane:isopropyl alcohol (3:2) was added and incubated at room temperature for 30 min. The organic phase was saved while the aqueous phase was reextracted. The organic phases were combined and dried to completeness with nitrogen. The sample was solubilized in 600 l of 1% Triton X-100 in chloroform and dried to completeness with nitrogen. The sample was then solubilized in 300 l of water, and the total cholesterol was determined as described previously (9).
Other Methods-Protein concentrations were determined using the Bio-Rad Bradford kit. OptiPrep interfered with other standard protein determination methods including the Bradford microassay.

Expression and Localization of Caveolin-Palmitoylation
Mutants-To examine the role of palmitoylation on caveolin function, each of the individual cysteine residues known to be palmitoylated (23) was converted to alanine (constructs 133, 143, and 156), or all of the cysteines were converted to alanines (triple mutant) (Fig. 1). Wild type caveolin cDNA and each mutant cDNA construct were confirmed by sequence analysis before being stably transfected into caveolin-negative, L1210-JF lymphocytes. Cells expressing each caveolin were processed to isolate caveolae, and the degree of caveolin expression and the subcellular location of each caveolin were determined by SDS-PAGE and immunoblot analysis (Fig. 2). Wild type caveolin, 133, 143, and 156 were expressed at comparable levels (Ϯ6%), whereas the triple mutant was expressed at 51 Ϯ 10% of that of wild type caveolin. All of the caveolins were preferentially localized in the caveolae fraction ( Fig. 2A, compare CM to PM). The non-caveola markers, clathrin and transferrin receptor, were excluded from the caveola fraction (Fig. 2B).
To confirm that palmitoylation of the mutant proteins was altered, the cells were labeled with [ 3 H]palmitate for 6 h and then lysed and caveolin-immunoprecipitated. The precipitated material was divided equally and resolved by SDS-PAGE. To confirm that the radioactivity associated with each caveolin was bona fide palmitate, one gel was soaked in Tris and one gel was soaked in hydroxylamine, which hydrolyzes the thioester linkage of palmitate. Fig. 3 demonstrates that each of the individual caveolin mutant proteins contained 30 Ϯ 8% less radioactivity than the wild type caveolin whereas the triple mutant did not contain any radioactivity. In addition, treatment with hydroxylamine removes the radioactivity associated with the caveolins. Association of Sterol with Caveolin-We next tested the ability of the mutant caveolins to bind to sterol. The cellular sterol pools were labeled by incubating cells for 16 h in the presence of [ 3 H]acetate at 37°C. The cells were lysed and the cytosol isolated by centrifugation at 250,000 ϫ g for 1 h. The cytosolic pool of caveolin was then immunoprecipitated with caveolin IgG (22). The precipitated material was extracted and the associated lipids resolved by thin-layer chromatography. All of the radioactivity was associated with the sterol fraction (data not shown). To account for potential differences in the efficiency of immunoprecipitation between each caveolin, the amount of caveolin precipitated was quantified by densitometry (data not shown) and used to normalize the amount of sterol associated with each caveolin. Wild type caveolin and 133 associated with similar amounts of sterol, whereas 143 was reduced by 50% and 156 was reduced by 75% (Fig. 4). The triple mutant did not associate with significant amounts of sterol (Fig. 4).
Formation of a Functional Chaperone Complex-We previously demonstrated that the cytosolic pool of caveolin-1 was associated with HSP56, cyclophilin 40, and cyclophilin A (22). To determine whether palmitoylation was required for the formation of the caveolin-chaperone transport complex, we isolated cytosolic fractions and immunoprecipitated caveolin-1 using previously described conditions (22) (Fig. 5). The immunoprecipitated material was separated by SDS-PAGE and immunoblotted for caveolin, HSP56, cyclophilin 40, and cyclophilin A. Wild type caveolin-1 and 133 coimmunoprecipitated HSP56, cyclophilin 40, and cyclophilin A. Mutant 143 coimmunoprecipitated HSP56 and cyclophilin 40 but not cyclophilin A. Mutant 156 coimmunoprecipitated cyclophilin 40 and cyclophilin A but not HSP56. The triple mutant only coimmunoprecipitated cyclophilin 40.
We previously demonstrated that the caveolin-chaperone transport complex will transport newly synthesized cholesterol from the endoplasmic reticulum to caveolin within 10 -20 min (22). To determine whether the mutant caveolins are capable of transporting cholesterol to caveolae, we used a temperature shift assay (22). Cholesterol can be synthesized at 14°C in the endoplasmic reticulum, but little of the sterol can traffic through the cell (28). Shifting the temperature to 37°C permits a bolus of labeled sterol to move through intracellular trafficking routes. Cells were chilled to 14°C and then incubated with [ 3 H]acetate for 2 h before adding excess unlabeled acetate and shifting the temperature to 37°C for various times. The cells were then subfractionated and the amount of sterol in intracellular membranes (Fig. 6, open square) and caveolae (open circle) was determined by thin-layer chromatography and scintillation counting. As shown previously (19,22), wild type caveolin transports radiolabeled sterol to caveolae within 10 -20 min (Fig. 6A). Mutant 133 also transports similar amounts of radiolabeled sterol to caveolae within 10 -20 min (Fig. 6B). However, mutants 143 and 156 and the triple mutant did not translocate radiolabeled sterol to caveolae by 60 min (Fig. 6, C-E). Even after 4 h, mutants 143 and 156 and the triple mutant did not translocate radiolabeled sterol to caveolae (data not shown).
We next determined if the inability of the caveolin mutants to transport cholesterol affected the mass of cholesterol associated with caveolae. To determine this, caveolae were isolated from each mutant cell line, and the mass of cholesterol in each caveolae fraction was determined with a commercially available kit (9). Cells expressing wild type caveolin and 133 were highly enriched in cholesterol, whereas mutants 143 and 156 and the triple mutant were only slightly enriched in cholesterol (Fig. 7). DISCUSSION Caveolin has an unusually long hydrophobic region of approximately 33 amino acids followed by three cysteine residues that are known to be palmitoylated (23). We hypothesized that this hydrophobic region along with the palmitoylated cysteine residues could form a hydrophobic binding pocket to sequester and transport cholesterol through the cytosol. To test this hypothesis, caveolin mutants were generated that selectively removed one palmitoylation site each or all three sites. The present data suggest that the palmitoylation of caveolin plays FIG. 4. The association of sterol with caveolin. The ability of the mutant caveolins to bind sterol was tested by radiolabeling cells for 16 h in the presence of [ 3 H]acetate at 37°C. The cells were lysed and the cytosol isolated by centrifugation at 250,000 ϫ g for 1 h. The cytosolic pool of caveolin was then immunoprecipitated with caveolin IgG. The precipitated material was extracted and the associated lipids resolved by thin-layer chromatography. To account for potential differences in the efficiency of immunoprecipitation between each caveolin cell line, the amount of caveolin precipitated was quantified by densitometry (data not shown) and used to normalize the amount of sterol associated with each caveolin. The data are from three independent experiments (mean Ϯ S.E., n ϭ 3). WT, wild type.

FIG. 5. Formation of the caveolin-chaperone complex.
We previously demonstrated that the cytosolic pool of caveolin was associated with HSP56, cyclophilin 40, and cyclophilin A. To determine whether palmitoylation was required for the formation of the caveolin-chaperone transport complex, we isolated cytosolic fractions by centrifugation at 250,000 ϫ g for 60 min. The cytosolic fractions were then immunoprecipitated with caveolin-1 IgG. The immunoprecipitated material was separated by SDS-PAGE and immunoblotted with IgGs for caveolin-1, HSP56, cyclophilin 40, and cyclophilin A. Representative data from four independent experiments are shown. HSP56, heat-shock protein 56; Cyp40, cyclophilin 40; CypA, cyclophilin A; WT, wild type. a role in cholesterol binding and in the assembly of the chaperone complex. Cholesterol binding to caveolin requires palmitoylation at residues 143 and 156 but not at residue 133. Furthermore, cyclophilin 40 coimmunoprecipitated with caveolin regardless of the palmitoylation state of caveolin, suggesting that palmitoylation is not necessary for caveolin-cyclophilin 40 interactions. Lack of palmitoylation at position 133 did not affect the formation of a functional chaperone complex. However, lack of palmitoylation at 143 prevented the binding of cyclophilin A, whereas lack of palmitoylation at 156 prevented the binding of HSP56. In addition, the triple mutant and mutants 143 and 156 did not traffic cholesterol to caveolae.
We previously demonstrated a role for caveolin in intracellular cholesterol trafficking by using a lymphocyte cell line, L1210-JF, that does not express caveolin. The critical experiments directly followed the protocol of Kaplan and Simoni (28). In brief, cells are incubated with [ 3 H]acetate at 14°C to label newly synthesized cholesterol in the endoplasmic reticulum. Upon warming to 37°C, labeled cholesterol rapidly (10 -20 min) translocates to the plasma membrane without moving through the Golgi (19,22). When these same experiments were repeated with human fibroblasts and coupled with subcellular fractionation to isolate caveolae, it was demonstrated that the newly synthesized cholesterol moved directly to caveolae (19). Surprisingly, the labeled sterol did not remain in caveolae but flowed into the bulk plasma membrane. When identical experiments were conducted with L1210-JF lymphocytes the newly synthesized sterol did not move to caveolae and only slowly (Ͼ60 min) moved to the bulk membrane (19). The small amount of transport to the bulk plasma membrane was inhibited by brefeldin A, which suggested that this was cholesterol moving through the classic membrane secretion pathway (19). L1210-JF cells expressing caveolin had dramatically different cholesterol trafficking kinetics. The radiolabeled cholesterol was rapidly (10 -20 min) and specifically transported to caveolae. The transport was not inhibited by nocodazole or brefeldin A, suggesting a direct transport mechanism.
Two possible models can explain the mechanism of caveolindependent cholesterol transport: vesicle-dependent and vesicleindependent. Previous work by Kaplan and Simoni (28) and others (29,30) has suggested that the direct transport of cholesterol to plasma membrane is dependent on a low density transfer intermediate. The identity of the transfer intermediate is still unknown. We recently used NIH 3T3 cells to demonstrate that approximately 10% of the total cellular pool of caveolin is present in the cytosol (22). Immunoprecipitation with caveolin IgG under stringent conditions consistently coimmunoprecipitated three additional proteins. These proteins were identified as known chaperone proteins: HSP56, cyclophilin 40, and cyclophilin A. Formation of the chaperone complex depended on the presence of caveolin because L1210-JF cells that did not express caveolin did not have the complex even though all the components (except caveolin) were present in the cells. Expression of caveolin-1 in L1210-JF cells promoted the formation of the chaperone complex (22).
Previous work by Murata et al. (17) demonstrated that caveolin binds at least 1 mol of cholesterol per mol of protein. Two approaches were used to demonstrate this interaction. First, cholesterol co-purified with caveolin in a sucrose gradient containing 0.2% SDS, which should have removed associated lip-ids. The second approach involved the association of purified caveolin with cholesterol-containing mixtures. One surprising result from this study was that Escherichia coli-expressed caveolin could not reconstitute into proteoliposomes without the addition of exogenous cholesterol. In addition, recent work by Fielding et al. (31) has demonstrated that caveolin and caveolae play a critical role in the efflux of free cholesterol from cells.
We have speculated that caveolin-mediated regulation of cellular cholesterol is involved in multiple disease processes such as atherosclerosis and hypertension (2). Our present data demonstrate that palmitoylation at amino acid residues 143 and 156 is necessary for caveolin to bind cholesterol, form a chaperone complex, and transport cholesterol directly to caveolae. These mechanistic studies are critical for future studies examining the cellular physiology of caveolin.