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INTRODUCTION |
Originally named to describe membrane invaginations at the cell
surface, caveolae are specialized plasmalemmal domains rich in
glycosphingolipids, cholesterol, and lipid-anchored membrane proteins.
In endothelial cells (EC)1,
caveolae have a striated coat, the major protein of which is caveolin-1 (1). Caveolin-1 is involved in cholesterol trafficking (2),
oligomerizes to form a scaffold for assembly of signaling molecules
including receptors, signal transducers, and effectors (3), and regulates the activation state
of these signaling complexes (4).
Lipid modification, including palmitoylation, is an important mechanism
targeting signaling proteins to caveolae. Among the palmitoylated
proteins in caveolae are heterotrimeric G-protein
-subunits
(e.g.
s,
i,
o,
and
q), G-protein linked receptors, regulator of
G-protein signaling proteins, p21ras, nonreceptor
tyrosine kinases, and EC-specific nitric-oxide synthase (eNOS) (5).
Palmitoylation of several of these proteins has been shown to be a
post-translational and reversible modification that is also subject to
regulation (6). The post-translational nature of palmitoylation has
been shown by cell culture experiments in which palmitate labeling
occurs even in the presence of protein synthesis inhibitors (7). These
experiments clearly distinguish protein palmitoylation from protein
myristoylation, which is cotranslational and blocked by protein
synthesis inhibitors. The reversibility of protein-palmitate bonds has
been demonstrated by the rapid turnover of the palmitoyl group and may
be caused by the relative lability of the thioester linkage (8-10).
Protein palmitoylation and depalmitoylation reactions are considered to
be primarily enzymatic and catalyzed by unidentified membrane-bound
palmitoyl acyltransferases (11, 12) and by cytoplasmic and lysosomal forms of palmitoyl thioesterases (13-15), respectively. Regulation of
the protein palmitoylation and depalmitoylation cycle is thought to be
important in regulating protein localization, conformation, protein-protein interaction, and activity (16-18).
Caveolin-1, the most abundant caveolin family member in EC, is a
21-24-kDa integral membrane protein with a hairpin-like structure and
with both N and C termini facing the cytoplasm (19, 20). Caveolin-1 is
palmitoylated on three cysteine residues located near the C terminus of
the protein (20). Two isoforms, the slower migrating caveolin-1
and
the faster migrating caveolin-1
, differ by 31 N-terminal amino acids
because of alternate translation initiation sites (21). Their C termini
are identical and it is likely that both isoforms are palmitoylated
similarly. The function of caveolin-1 palmitate chains is not well
understood. They may increase the stability of the oligomers and
therefore the scaffold structure of caveolae (22). In addition,
caveolin-1 palmitate residues may regulate its interaction with
other proteins. A palmitate-deficient mutant of caveolin-1 exhibits
normal caveolar localization (20); however, its interaction with
G
i1 is diminished greatly (23). A recent report
suggests that at least two palmitate chains on caveolin-1 are required
for binding and transport of cholesterol (24).
We here report results from a kinetic analysis that shows that
caveolin-1 exhibits unique characteristics of palmitate incorporation. Caveolin-1 palmitoylation is essentially irreversible and completely inhibited by cycloheximide. Nonetheless it is a post-translational event that requires protein transport from the endoplasmic reticulum to
the plasma membrane. This distinguishes caveolin-1 from other caveolar
palmitoylated proteins and suggests that caveolin-1 may be subject to
unique regulatory mechanisms of palmitoylation and/or depalmitoylation
that account for its distinctive palmitoylation characteristics.
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EXPERIMENTAL PROCEDURES |
Materials--
[9,10-3H]Palmitic acid (35 Ci/mmol)
and [9,10-3H]myristic acid (49 Ci/mmol) were obtained
from PerkinElmer Life Sciences. Trans-35S-label (1175 Ci/mmol, referred to here as [35S]methionine), was from
ICN (Irvine, CA). Polyclonal rabbit anti-human caveolin-1 antibody and
mouse monoclonal antibody against EC-specific eNOS were from
Transduction Laboratories (Lexington, KY). Nonimmune rabbit and mouse
IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Brefeldin A
was purchased from Epicentre Technologies (Madison, WI). Protein
G-Sepharose was from Zymed Laboratories Inc. Protein
A-Sepharose and other assay reagents were from Sigma.
Cells and Culture Conditions--
Bovine aortic EC were isolated
as described (25) and maintained in Dulbecco's modified Eagle's (DME)
medium and F12 medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified
atmosphere containing 5% CO2.
Metabolic Labeling of Endothelial Cells--
EC in 10-cm dishes
were washed with serum-free DME medium and then incubated for 2 h
in the same medium. For measurement of incorporation of palmitate into
caveolin-1 and other proteins, EC were incubated for 15-30 min with
250 µCi/ml [3H]palmitic acid in serum-free medium
containing 3.5 mg/ml fatty acid-free bovine serum albumin. For
pulse-chase experiments, the medium was removed and the cells were
rinsed three times with serum-free DME medium. The radiolabel was
chased by the addition of DME medium containing cycloheximide (28 µg/ml) and either unlabeled palmitate (15 µg/ml) bound to fatty
acid-free bovine serum albumin or 5% fetal bovine serum as indicated.
To measure protein synthesis, EC were incubated for 15-30 min with 60 µCi/ml [35S]methionine in cysteine- and methionine-free medium.
Immunoprecipitation of Caveolin-1 from Endothelial Cell
Lysates--
EC were washed twice in phosphate-buffered saline and
lysed at 4 °C in 0.6 ml of immunoprecipitation buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 2.5 mM MgCl2, 0.5% sodium deoxycholate, 1% Nonidet P-40, 20 µg/ml aprotinin, and 5 µg/ml leupeptin) for 90 min. Cells were scraped, and the lysate was cleared by centrifugation at 16,000 × g for 3 min. An aliquot of the lysate was
subjected to 12% SDS-PAGE under nonreducing conditions. The remainder
of the lysate was added to SDS (0.1% final concentration).
Anti-caveolin-1 antibody was added for 90 min followed by protein
A-Sepharose for 60 min. A control cell lysate was immunoprecipitated
with rabbit nonimmune IgG. Immunoprecipitated complexes were washed three times with immunoprecipitation buffer and analyzed by 15% SDS-PAGE. In some experiments, the lysates were subjected to a second
immunoprecipitation with anti-eNOS antibody or mouse nonimmune IgG as
described above except that the antibody was added for 15 h and
analyzed by SDS-PAGE (7.5% acrylamide). Gels were analyzed by fluorography.
Determination of [3H]Palmitate Incorporation into
Caveolin-1--
To verify that the radiolabeled fatty acid
incorporated into caveolin-1 was linked by a thioester bond,
susceptibility to hydroxylamine treatment was examined (20). EC were
incubated with 250 µCi/ml [3H]palmitate for 30 min. The
cells were lysed, and caveolin-1 was immunoprecipitated with
anti-caveolin-1 antibody. Aliquots of the immunoprecipitated material
were fractionated by SDS-PAGE. Gel lanes were separated and soaked in
either 1 M hydroxylamine, pH 7.0, or 1 M
Tris-HCl, pH 7.0 for 4 h and then subjected to fluorography.
The specific fatty acid esterified to caveolin-1 was identified by
reverse-phase thin layer chromatography essentially as described (26).
EC were incubated with 250 µCi/ml [3H]palmitate for 30 min. The cells were lysed, and caveolin-1 was immunoprecipitated with
polyclonal anti-caveolin-1 IgG and separated by SDS-PAGE. The
caveolin-1 band was excised and subjected to in-gel alkaline hydrolysis
by treatment with 0.1 M KOH for 30 min. After acidification
of the extract to pH 4.5, the free fatty acids were extracted with
hexane, dried under N2, and dissolved in
chloroform/methanol (2:1, v/v). The extracted lipids were fractionated by reverse-phase thin layer chromatography (RP-18, Whatman) in a
resolving solvent system of acetonitrile/acetic acid (1:1, v/v). Standards containing [3H]palmitate and
[3H]myristate were fractionated in parallel lanes. The
lanes were scraped at 1-cm intervals, and radioactivity was determined
by scintillation counting.
Protein Identification by Mass Spectrometry--
The identity of
the proteins immunoprecipitated by the caveolin-1 antibody was verified
by mass spectrometric analysis. Lysates were prepared from EC in 2-4
10-cm dishes and subjected to immunoprecipitation using 10 µl of
polyclonal anti-caveolin-1 IgG and SDS-PAGE. The protein was excised
from the Coomassie Blue-stained gel and trypsin-digested in
situ. The digest was analyzed by capillary liquid
chromatography-electrospray mass spectrometer, and peptide sequences
were determined by collision-activated dissociation using a liquid
chromatography-electrospray-tandem mass spectrometer (Finnigan LCQ-Deca
ion trap with a Protana nanospray ion source interfaced to a Phenomenex
Jupiter C18 reversed-phase capillary column). Sequences
were determined by manual interpretation of collision-activated
dissociation spectra.
 |
RESULTS |
Incorporation of Palmitate into Caveolin-1 Requires de Novo Protein
Synthesis--
Protein palmitoylation is considered generally to be a
post-translational modification independent of de novo
protein synthesis (7, 8). This characteristic differentiates
palmitoylation from myristoylation, a cotranslational modification that
is blocked by protein synthesis inhibitors (27). To investigate the
cellular mechanism of caveolin-1 palmitoylation, bovine aortic EC were pretreated with cycloheximide for 60 min before incubation with [3H]palmitate for 20 min. Caveolin-1 was
immunoprecipitated from EC lysates using polyclonal anti-caveolin-1
IgG, and [3H]palmitate-labeled protein was detected by
fluorography. Surprisingly, cycloheximide treatment blocked essentially
all incorporation of [3H]palmitate into caveolin-1 (Fig.
1A, upper panel).
This inhibition was also seen after longer labeling periods in the
presence of cycloheximide up to 6 h (data not shown). Previous
results by Baker et al. (28) showed substantial labeling of
caveolin-1 by [3H]palmitate in cycloheximide-treated NIH
3T3 cells (28). We found that the complete inhibition of palmitoylation
of caveolin-1 in either EC (Fig. 1A, upper panel)
or mouse NIH 3T3 cells (data not shown) was observed only in cells
pretreated with cycloheximide. When cycloheximide was added only during
the labeling period, substantial incorporation of
[3H]palmitate into caveolin-1 was observed, which agreed
with the previous report (28). In parallel dishes to determine the
effectiveness of the inhibitor, cycloheximide completely blocked
de novo synthesis of caveolin-1 in EC metabolically labeled
with [35S]methionine (Fig. 1A, middle
panel). Interestingly, cycloheximide added without pretreatment
inhibited caveolin-1 synthesis completely but inhibited its
palmitoylation only partially. These findings suggest the existence of
a newly synthesized pool of caveolin-1 lacking palmitate chains and
that incorporation of palmitate into caveolin-1 in this pool is
post-translational but limited to a period 60 min or less after
synthesis. Coomassie Blue staining of the gel served as a loading
control and showed that the total amount of cellular caveolin-1 was not
changed by cycloheximide treatment (Fig. 1A, lower
panel).

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Fig. 1.
Effect of protein synthesis inhibition on
palmitate incorporation into caveolin-1. A, bovine
aortic EC were preincubated for 60 min with 28 µg/ml cycloheximide
(CHX) or with medium alone (Cont.) and then
incubated for an additional 20 min under the same conditions in the
presence of [3H]palmitate (upper panel) or
[35S]methionine (middle panel). In one
experiment EC was treated with cycloheximide during the labeling period
only (CHX (no pre-treat.)). Immunoprecipitation
(IP) of caveolin-1 from the cell lysates was done using
polyclonal anti-caveolin-1 antibody (Anti-Cav-1) or
nonimmune rabbit purified IgG (IgG) as control. The
immunoprecipitated material was subjected to SDS-PAGE and fluorography.
The positions of caveolin-1 (upper arrow) and -1
(lower arrow) isoforms are indicated. The gel shown in the
upper panel was subjected to Coomassie Blue staining to show
total amount of caveolin-1 (lower panel). B, EC
were incubated with [3H]palmitate for 20 min, and
caveolin-1 was immunoprecipitated from the cell lysate. The
immunoprecipitated material was fractionated by SDS-PAGE, and the gels
were treated for 4 h with 1 M hydroxylamine, pH 7.0 (HA), or 1 M Tris-HCl, pH 7.0, as a control
(Cont.). Gel radioactivity was detected by fluorography.
C, EC were incubated with [3H]palmitate for 30 min. Caveolin-1 was immunoprecipitated from lysates separated by
SDS-PAGE and hydrolyzed in 0.1 M KOH. After acidification,
fatty acids were extracted and separated by reverse-phase thin layer
chromatography. Radioactivity in bands of 1.0-cm width was counted by
liquid scintillation. The mobility of authentic
[3H]palmitate (Palm.) and
[3H]myristate (Myr.) standards are indicated
by arrows. D, EC were preincubated with
cycloheximide for 2 h and then with [3H]palmitate
(upper panel) or [35S]methionine (lower
panel) as described in A. Lysates were
immunoprecipitated using an eNOS monoclonal antibody
(anti-eNOS) or a control IgG. The position of eNOS is
indicated by the arrow. E, EC were preincubated
with cycloheximide for 2 h and then with
[3H]palmitate (left panel) or
[35S]methionine (right panel) as described in
A. Total cell lysates were subjected to SDS-PAGE and
fluorography. F, EC were preincubated for 45 min in the
presence of 200 µM puromycin (Pur.) and then
incubated for 20 min under the same conditions with
[3H]palmitate (upper panel) or
[35S]methionine (lower panel). Caveolin-1 was
immunoprecipitated as in A and subjected to SDS-PAGE and
fluorography.
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The identity of the [3H]palmitate-labeled protein was
confirmed by mass spectrometric sequence analysis of proteolytic
fragments of the eluates. The sequence of fragments in the upper
band in Fig. 1A (upper panel) was identical
to that of bovine caveolin-1
(data not shown). The lower band
contained sequences found in bovine caveolin-1
and also a protein
with a sequence consistent with human caveolin-2 (the bovine sequence
has not been reported). It is likely that the observed
[3H]palmitate in the lower band is due to caveolin-1
because caveolin-2 does not contain the C-terminal Cys residues that
are palmitoylated in other caveolin types (and palmitate linked to
caveolin-2 has not been reported).
Because inhibition of protein acylation by cycloheximide is considered
generally to be evidence for cotranslational myristoylation rather than
post-translational palmitoylation, we investigated the identity of the
radiolabeled fatty acid and the nature of the linkage to caveolin-1.
Protein palmitoylation occurs on Cys residues through a thioester bond
sensitive to treatment with hydroxylamine (20). EC were incubated with
[3H]palmitate, and caveolin-1 was immunoprecipitated from
lysates using polyclonal anti-caveolin-1 IgG and subjected to
SDS-PAGE. Incubation of the gel in neutral hydroxylamine removed
all radioactivity, consistent with a palmitate thioester bond (Fig.
1B). A chromatographic method was used to identify more
precisely the fatty acid linked to caveolin-1 (26). Caveolin-1 was
immunoprecipitated from [3H]palmitate-labeled EC and
fractionated by SDS-PAGE. The fatty acids released by alkaline
hydrolysis were extracted and subjected to reverse-phase thin layer
chromatography. The mobility of the fatty acid released from caveolin-1
was the same as that of [3H]palmitate (Fig.
1C).
To examine the target specificity of cycloheximide we determined its
effect on incorporation of [3H]palmitate into a second
palmitoylated EC protein, eNOS. Palmitoylation of eNOS was not
decreased by cycloheximide (Fig. 1D). To evaluate the effect
of cycloheximide on an array of EC proteins, lysates not subjected to
immunoprecipitation were examined. Palmitate incorporation into
multiple proteins was diminished by cycloheximide but not inhibited
completely. Interestingly, modification of proteins with molecular
mass greater than 65 kDa was not decreased (Fig. 1E,
left). The effectiveness of the cycloheximide treatment was shown by metabolic labeling with [35S]methionine (Fig.
1E, right). Puromycin, a protein synthesis inhibitor with a mechanism different from cycloheximide, also blocked
palmitoylation of caveolin-1 (Fig. 1F) but did not inhibit palmitoylation of eNOS and only partially inhibited palmitoylation of
most visible cellular proteins (data not shown). These results suggest
that the process of palmitoylation of caveolin-1 differs from that of
most proteins studied so far in that it requires de novo
protein synthesis. Several mechanisms may be responsible for this
requirement, namely (i) caveolin-1 palmitoylation is cotranslational,
(ii) caveolin-1 palmitoylation occurs during trafficking to the plasma
membrane, (iii) caveolin-1 palmitoylation requires the synthesis of
accessory proteins, e.g. chaperones, or (iv) caveolin-1
palmitoylation occurs after synthesis and delivery to the plasma
membrane but is irreversible, and therefore incorporation of
[3H]palmitate is found only on newly synthesized protein.
Caveolin-1 Palmitoylation Is Not Cotranslational--
To examine
whether incorporation of palmitate into caveolin-1 is cotranslational,
we examined the effects of inhibitors of protein transport between
endoplasmic reticulum and plasma membrane. A lack of an effect would be
consistent with palmitate incorporation proximal to the inhibited
trafficking step and with cotranslational modification. Newly
synthesized caveolin-1 travels from the endoplasmic reticulum through
the Golgi apparatus, where it is incorporated into detergent-resistant
membranes (29, 30), and then to the cell surface (31). Brefeldin A
(BFA) reversibly blocks the exit of secretory and membrane proteins
from the endoplasmic reticulum by disassembling the Golgi apparatus and
mixing it into the endoplasmic reticulum (32). Incubation of EC with
BFA dramatically reduced [3H]palmitate labeling of
caveolin-1 (Fig. 2A,
upper panel). The much smaller decrease in
[35S]methionine labeling indicates that the effect of BFA
was not caused by decreased neosynthesis of caveolin-1 (Fig.
2A, lower panel). The BFA-induced retrograde
transport of vesicles from the Golgi to endoplasmic reticulum is
prevented by the addition of nocodazole, which depolymerizes
microtubules; thus in combination with BFA, nocodazole can distinguish
the effects of inhibition of anterograde and retrograde transport (33).
Nocodazole did not prevent the inhibition of caveolin-1 palmitoylation
by BFA, indicating that anterograde transport from the endoplasmic
reticulum to Golgi is necessary for palmitate incorporation (Fig.
2A). The inhibition of palmitoylation of caveolin-1 by BFA
was highly specific as shown by [3H]palmitate labeling in
cell lysates; palmitoylation of most visible proteins was not
inhibited, whereas palmitoylation of two proteins of ~60 and 70-75
kDa was increased markedly (Fig. 2B).

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Fig. 2.
Effect of inhibitors of intracellular
trafficking on palmitate incorporation into caveolin-1.
A, EC were preincubated for 15 min with 20 µM
nocodazole (NOC) and then for 30 min with nocodazole, 10 µM brefeldin A (BFA), both BFA and nocodazole,
or with medium alone as control (Cont.) in the presence of
[3H]palmitate (upper panel) or
[35S]methionine (lower panel). Caveolin-1 was
immunoprecipitated from the cell lysates, and incorporation of the
radiolabel was detected as in Fig. 1A. B, total
cell lysates were subjected to SDS-PAGE and fluorography. C,
EC were incubated for 30 min with 10 µM monensin
(Mon.) or medium alone (Cont.) in the presence of
[3H]palmitate (upper panel) or
[35S]methionine (lower panel). In one
treatment, the cells were preincubated with monensin (Mon.
pre-treat.) for 2 h before addition of the radiolabel.
Caveolin-1 was immunoprecipitated and detected by SDS-PAGE and
fluorography as in Fig. 1A.
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To evaluate the role of more distal trafficking in palmitate
incorporation into caveolin-1, we incubated EC with monensin. Monensin
equilibrates the monovalent cations Na+, K+,
and H+ across biological membranes, which perturbs protein
transport from medial- to trans-Golgi compartments (34) and also
increases intralysosomal pH. Pretreatment of EC with monensin
substantially reduced [3H]palmitate labeling of
caveolin-1 (Fig. 2C, upper panel) without affecting caveolin-1 neosynthesis (Fig. 2C, lower
panel). The inhibition was not due to the lysosomotropic property
of monensin because chloroquine, which also increases intralysosomal
pH, did not inhibit caveolin-1 palmitoylation (data not shown).
Together these results show that an intact trafficking pathway is
necessary for caveolin-1 palmitoylation.
Disruption of Caveolin-Chaperone Complexes Does Not Inhibit
Caveolin-1 Palmitoylation--
Protein synthesis inhibitors may block
palmitoylation of caveolin-1 by an indirect mechanism, e.g.
by inhibition of chaperones or other accessory proteins required for
caveolin-1 trafficking rather than by a direct effect on caveolin-1
itself. Caveolin-1 is present in the cytoplasm as a complex with the
immunophilins FK506-binding protein 52, cyclophilin A, and cyclophilin
40 (35). These chaperones could be the target of cycloheximide either
because their neosynthesis is necessary for caveolin-1 trafficking or because cycloheximide competitively binds immunophilins that may disrupt complex formation with caveolin-1 (35, 36). To test this
mechanism, we examined the effect of agents that specifically bind
immunophilins on [3H]palmitate labeling of caveolin-1.
Cyclosporin A and rapamycin were used to bind cyclophilins and
FK506-binding protein 52, respectively, at concentrations known to
disrupt the caveolin-chaperone complex (35). Immunophilin binding did
not diminish caveolin-1 palmitoylation, suggesting that the synthesis
and activity of these chaperones are not critical for palmitoylation of
newly synthesized caveolin-1 (Fig.
3).

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Fig. 3.
Effect of immunophilin-binding drugs on
palmitate incorporation into caveolin-1. EC were preincubated for
60 min with 1 µM cyclosporin A (CSA), 100 nM rapamycin (Rap.), or medium as control
(Cont.) and then for 30 min under the same conditions but in
the presence of [3H]palmitate (upper panel) or
[35S]methionine (lower panel). Caveolin-1 was
immunoprecipitated and detected as in Fig. 1A.
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Palmitate Modification of Caveolin-1 Is Essentially
Irreversible--
An alternative explanation for our observations is
that the modification of caveolin-1 by palmitate is essentially
irreversible. If this is the case, the apparent dependence of
palmitoylation on new protein synthesis would be due to the fact that
[3H]palmitate cannot be incorporated into caveolin-1 that
has been palmitoylated already but instead is incorporated
preferentially into newly synthesized, and thus nonpalmitoylated,
caveolin-1. To test this mechanism, the rate of caveolin-1
depalmitoylation was measured in a pulse-chase experiment. EC were
incubated with [3H]palmitate or
[35S]methionine for 30 min, and the radiolabel was chased
for up to 24 h with medium in the presence of cycloheximide and
excess unlabeled palmitate. The release of [3H]palmitate
from caveolin-1 was negligible during the 24-h chase period (Fig.
4A, upper panel),
as was turnover of the caveolin-1 polypeptide chain (Fig.
4A, lower panel). This result was in marked contrast to the rapid release of [3H]palmitate from eNOS,
which was essentially complete after 3 h (Fig. 4B,
upper panel); the high rate of palmitate turnover was not
caused by eNOS protein turnover, which had a half-life of about 9 h (Fig. 4B, lower panel). Analysis of total
[3H]palmitate-labeled proteins in cell lysates shows that
the rate of release of [3H]palmitate from EC proteins is
rapid with apparent half-lives of less than 6 h for most
detectable proteins (Fig. 4C). These results indicate that
the process of palmitoylation of caveolin-1 is essentially irreversible
in EC and that this characteristic distinguishes it from most other
palmitoylated EC proteins.

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Fig. 4.
Turnover of [3H]palmitate in
caveolin-1 and other EC proteins. EC were incubated with
[3H]palmitate or [35S]methionine for 30 min, washed thoroughly with DME medium, and chased in DME medium
containing 28 µg/ml cycloheximide and either 15 µg/ml unlabeled
palmitate bound to fatty acid-free bovine serum albumin (for palmitate
incorporation) or 5% fetal bovine serum (for methionine incorporation)
for up to 24 h. A, caveolin-1 was immunoprecipitated
from cell lysates, and [3H]palmitate (upper
panel) or [35S]methionine (lower panel)
was detected as in Fig. 1A. B, eNOS was
immunoprecipitated from cell lysates as in Fig. 1B.
C, EC were incubated with [3H]palmitate and
then chased with cycloheximide in 5% fetal bovine serum as in
A. Total cell lysates were resolved by SDS-PAGE, and
radioactivity was detected by fluorography (left
panel). B, densitometric scans were done on each
lane (right panel).
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 |
DISCUSSION |
Our results show that incorporation of [3H]palmitate
into caveolin-1 requires de novo synthesis of the protein.
This requirement is not due to cotranslational modification but rather
results from the absence of a detectable
palmitoylation/depalmitoylation cycle. Our data also show that
palmitate incorporation occurs within 60 min of caveolin-1 synthesis
and that an intact trafficking pathway is required, indicating that
palmitoylation occurs distal to the Golgi apparatus, most likely in the
caveolae or plasma membrane compartments. These findings are not
limited to cells of a single type or from a single species because
similar results were obtained using murine NIH 3T3 fibroblastic cells
(data not shown).
The observed low rate of depalmitoylation does not imply that the
palmitate linkage to caveolin-1, or its pathway of formation, is unique
but rather that the enzymatic or nonenzymatic depalmitoylation reaction
is extremely slow. The in vitro sensitivity to neutral hydroxylamine (our results and Ref. 20) indicates that palmitate is
linked to caveolin-1 by a transesterification-labile thioester bond.
The resistance to cleavage of the palmitate linkage in caveolin-1 in EC
may be caused by the inaccessibility of caveolin-1 to the depalmitoylating enzyme(s) or to the possibility that caveolin-1 is a
weak substrate for these enzymes. Little is known about the substrate
specificities of the palmitoyl protein thioesterases. Palmitoyl protein
thioesterase-1 and its homologue, palmitoyl protein thioesterase-2, are
localized in the lysosomes and are thus unlikely to be responsible for
caveolin-1 deacylation. However, acyl protein thioesterase-1, which is
located primarily in the cytoplasm and increases the turnover rate of
palmitate on G
s and eNOS, is a candidate for regulation
of caveolin-1 deacylation (14, 15). In agreement with our results, Yeh
et al. (14) recently showed that caveolin-1 is not a
substrate for acyl protein thioesterase-1. It is not known whether
caveolin-1 lacks a specific sequence required for thioesterase
recognition or whether the orientation of the palmitate groups within
the membrane renders them inaccessible to the active site of the
enzyme. An alternate possibility is that our findings on caveolin-1
palmitoylation are restricted to cells under basal conditions, and the
palmitoylation/depalmitoylation cycle is activated by agonists.
However, in preliminary studies using thrombin, bradykinin, and several
cholinergic and adrenergic ligands, we have not observed
agonist-dependent caveolin-1 increases in the rate of
palmitoylation (data not shown).
Our results show that [3H]palmitate labeling of newly
synthesized caveolin-1 requires an intact trafficking pathway as shown by inhibition by BFA and monensin. Other investigators have reported that palmitoylation of SNAP-25 and GAP-43 is also inhibited by brefeldin A (33). Interestingly, both proteins are modified by
palmitate but not by other acyl groups (SNAP-25 and GAP-43 are modified
by four and two palmitate groups, respectively). In contrast,
palmitoylation of several proteins that are both myristoylated and
palmitoylated, e.g. G
o, G
i,
and p59fyn, is insensitive to brefeldin A (33, 37). Thus it is
possible that the specific acyl modifications define not just
susceptibility to acylation-deacylation cycling but also specific
intracellular trafficking pathways.
Because there are not other reports of proteins with similar
palmitoylation kinetics, we can only speculate on the cellular consequences of the essentially irreversible nature of the palmitate linkage on caveolin-1. The fact that caveolin-1 remains in a continuous palmitoylated state suggests a tight and possibly irreversible interaction with membranes. The "two-signal model" for membrane binding (6, 38, 39) proposes that the first signal (e.g. lipid modification, poly-basic stretch of amino acids, interaction with
a membrane-bound protein) permits transient interaction of a protein
with membranes, whereas the second signal (often a palmitate chain)
provides a more persistent association with a specific membrane.
According to this model, the presence in caveolin-1 of a 33-amino acid
hydrophobic domain as well as three palmitate chains may constitute the
equivalent of four membrane-binding signals and promote essential
irreversibility of the membrane interaction. Thus the persistent
palmitate chains may be important for a structural role of caveolin-1
rather than a kinetic role. Indeed, this idea is consistent with the
finding (using palmitoylation-deficient mutants of caveolin-1) that
palmitate modification is required for optimal stability of the
caveolin-1 oligomers (22), which may contribute to the rigid structure
of caveolae (40). Likewise, caveolin-1 palmitate residues are required
for efficient binding and sequestration of signaling molecules in
caveolae (23). Thus unlike most caveolar proteins that require cycles
of palmitoylation and depalmitoylation for normal function,
e.g. translocation between cellular compartments (17) and
regulation of protein interaction (41), caveolin-1 function may require
persistent palmitate linkages.
In contrast to its potentially static role in caveolar structure and in
protein-protein interactions, caveolin-1 and caveolae have been
implicated in dynamic cellular processes including transcytotic and
potocytotic endothelial transport (42), polarized vesicular trafficking in epithelial cells (43), and two-way transport of
cholesterol (and other lipids) between the endoplasmic reticulum/Golgi system and the plasma membrane (44-46). These transport processes suggest that caveolar structures are dynamic and also that caveolin-1 traverses intracellular compartments (47). In fact, cycling of
caveolin-1 between the plasma membrane caveolae and the endoplasmic reticulum/Golgi system has been shown (45), as well as redistribution in several situations including phosphatase inhibition (48), heat shock
(49), and after formation of cell-cell contacts (50). An important role
of caveolin-1 palmitate residues in these dynamic processes is just
becoming clear. The presence of palmitate on at least two of the three
Cys residues is necessary for cholesterol binding, chaperone complex
formation, and cholesterol transport to caveolae (24). Although
palmitoylation and depalmitoylation cycling may be required for
intercompartmental translocation of multiple proteins (6, 17, 38, 39),
in the absence of a detectable palmitate cycle it is unlikely that this
process regulates caveolin-1 translocation. An intriguing alternate
mechanism for regulation of caveolin-1 movement is suggested by recent
x-ray crystallographic studies of the release of Cdc42 from membranes by its regulator, rhoGDI (51). A hydrophobic pocket in rhoGDI surrounds
the membrane-binding geranylgeranyl moiety of Cdc42, thus leading to
membrane release. The identification of caveolin-1 in cytosolic
complexes in association with chaperones is consistent with this
mechanism (24, 35). However, the presence of a two-spanning integral
membrane region in caveolin-1, in addition to the palmitate residues,
may introduce steric and energetic obstacles to this potential
releasing mechanism. The requirement for palmitate for cholesterol
binding is consistent with a direct interaction of the fatty acids with
membrane lipids, and cytosolic caveolin-1 complexes may contain bound
lipids. Additional structural and biochemical studies will be necessary
to determine whether palmitate is involved directly or indirectly in
binding either cholesterol or chaperones. In either case, palmitate
cycling is unlikely to be required for regulation of caveolin-1
activity, but rather the absolute presence of palmitate residues may be
critical for normal caveolin-1 function in cells.