(Received for publication, December 2, 1994; and in revised form, January 24, 1995)
From the
Caveolae are subdomains of the plasma membrane which concentrate
cholesterol, glycosphingolipids, and
glycosylphosphatidylinositol-linked proteins. It has recently been
demonstrated that specific members of the Src family of protein
tyrosine kinases require palmitoylation of NH-terminal
cysteine residues to localize in caveolae. Here we report that
caveolin, an integral membrane protein which forms part of the coat of
caveolae, also incorporates palmitate through linkage to cysteine
residues. Caveolin contains only three cysteine residues which are all
located on the COOH-terminal side of the hydrophobic transmembrane
region. Immunofluorescent staining of cells transfected with caveolin
indicated that, like the NH
terminus, this COOH-terminal
region is located on the cytoplasmic side of the plasma membrane.
Studies of cysteine substitution mutants showed that all three
cysteines are capable of incorporating palmitate and that the
juxtamembrane Cys
residue is the predominant site of
palmitoylation. Simultaneous mutation of all three cysteine residues in
caveolin resulted in the loss of ability to incorporate palmitate;
however, this did not affect localization of the protein. Thus,
palmitoylation of cysteine residues in nonmembrane spanning Src family
protein tyrosine kinases has different consequences than in the
transmembrane protein caveolin.
The plasma membrane of many cell types contains
non-clathrin-coated invaginations referred to as caveolae or
plasmalemmal vesicles. These organelles are enriched in
glycosylphosphatidylinositol (GPI)()-linked proteins,
cholesterol, and glycosphingolipids compared with the bulk-phase
membrane(1, 2) . Despite their description 40 years
ago by Yamada (3) and Palade(4, 5) , caveolae
have only recently been implicated in a number of processes important
for inter- and intracellular communication. Folate, for example, is
thought to enter cells through GPI-linked receptors localized to
caveolae through a process referred to as potocytosis(6) .
Caveolae may also be important in regulating cellular calcium
concentration, since they have been shown to contain an inositol
1,4,5-triphosphate-sensitive calcium channel (7) and an
ATP-dependent calcium pump(8) .
Caveolae have been
implicated in a signal transduction pathway that is engaged by
cross-linking of GPI-linked proteins on T cells. A mode of signal
transduction was suggested by association of GPI-linked membrane
proteins with specific members of the Src family of protein tyrosine
kinases, p56 and
p59
(9, 10, 11) . We have
recently shown that this association can be explained by their common
inclusion in Triton X-100-insoluble domains which contain caveolae (12) . This study showed that inclusion of the GPI-anchored
protein decay accelerating factor (DAF) in caveolae was mediated by its
lipid anchor while inclusion of p59
was mediated by dual
acylation of an amino-terminal Met-Gly-Cys motif, Gly
being
required for cotranslational myristoylation and Cys
being
necessary for incorporation of palmitate. Thus, two different
structural motifs have been defined which localize proteins to
caveolae. It is not clear how other proteins, specifically those
anchored to the membrane by hydrophobic transmembrane domains, are
localized to this organelle.
Caveolin(13) , also referred to
as VIP21(14) , is a 22-kDa integral membrane protein originally
described as a tyrosine-phosphorylated substrate in Rous sarcoma
virus-transformed fibroblasts(15) . Caveolin is a component of
the striated coat of caveolae(16) . Its gene encodes a
178-amino acid protein with an unusually long stretch of 33-40
hydrophobic amino acids in a predicted -sheet
conformation(13) . Western blotting and metabolic labeling of
caveolin has revealed a low molecular weight doublet and various high
molecular weight forms attributed to
self-association(13, 17, 18) . Two distinct
membrane topologies have been reported for caveolin. Based on the
protein topology rules of Hartmann et al.(19) and the
accessibility of caveolin to cell surface labeling, Kurzchalia et
al.(14) and Lisanti et al.(20) concluded that the NH
terminus of
caveolin was cytoplasmic, and the COOH terminus was extracellular. On
the other hand, antibodies directed at separate NH
- and
COOH-terminal domains of caveolin stained BHK cells only after
permeabilization, indicating that both ends of the molecule are
cytoplasmic(17) . This disparity has not been resolved.
The current study was undertaken to examine the molecular structure of caveolin and analyze its determinants for localization to caveolae. Our data show that cytoplasmically oriented cysteine residues in the COOH-terminal domain of caveolin are modified by covalent attachment of palmitate. Collective mutation of all three cysteines in caveolin to serines eliminated palmitoylation but did not affect targeting of caveolin to caveolae.
Figure 1:
Incorporation of
[H]palmitate into caveolin and its sensitivity to
neutral hydroxylamine. MDCK cells were incubated with
[
H]palmitate for one h and lysed as described
under ``Materials and Methods.'' Duplicate samples of
caveolin immunoprecipitates from the lysate were analyzed by SDS-PAGE
and soaked overnight in either 1 M Tris (pH 7.5) or 1 M hydroxylamine (pH 7.5) followed by
fluorography.
Figure 2:
Identification of fatty acid removed from
caveolin as palmitate. Following metabolic labeling with
[H]palmitate, caveolin was immunoprecipitated
from an MDCK lysate, separated by SDS-PAGE, transferred to polyvinyl
difluoride membrane, and subjected to alkaline hydrolysis. Fatty acid
extracted from the hydrolysate was analyzed by C-18 reversed phase TLC.
The graph shows counts/min from the lane containing the caveolin
hydrolysate at indicated distances from the origin. Migration of
[
H]myristate (C14:0) and
[
H]palmitate (C16:0) standards was determined by
autoradiography and is indicated above the
graph.
The COOH
Terminus of Caveolin is Cytosolic-The likely targets for
palmitoylation in caveolin are the three cysteine residues in the
protein, all located on the COOH-terminal side of the putative
transmembrane domain. Depending on the membrane orientation of
caveolin, these cysteines may be extracellular or intracellular. To
determine the orientation of this region, 293 cells were transfected
with cDNA encoding caveolin with HA and c-Myc epitopes located at the
extreme NH and COOH terminus, respectively. Presence of the
HA and c-Myc epitopes added approximately 5 kDa to the apparent
molecular mass of caveolin as determined by SDS-PAGE and Western blot.
The occurrence of a 30-kDa doublet was not the result of limited
proteolysis as each band was recognized by antibodies toward both
epitopes (Fig. 3). Both the epitope-tagged and wild-type version
of caveolin localized to caveolae as determined by Sepharose 4B column
chromatography (not shown). Orientation of the epitope-tagged caveolin
was determined by immunofluorescent staining of transfected cells by
antibodies to each epitope in unpermeabilized 293 cells or 293 cells
permeabilized with 0.1% Triton X-100. As shown in Fig. 4, the
extracellular DAF epitope was unaffected by the presence of Triton (A and B), whereas staining with anti-HA occurred in
the presence (D) but not the absence (C) of Triton.
Likewise, Fig. 5shows that staining with anti-Myc occurred only
in the presence (D) and not the absence (C) of Triton
with anti-DAF staining remaining unaffected (A and B). Analysis of the same transfected 293 cells with the
anti-epitope antibodies by fluoresence-activated cell sorting, in which
only externally oriented epitopes would be accessible to antibody, was
also negative (data not shown). Together, these results confirm that
caveolin exists with both the NH
and COOH termini located
intracellularly.
Figure 3:
Recognition of epitope-tagged caveolin by
anti-caveolin, anti-HA, and Anti-c-Myc antibodies. 293 cells expressing
the caveolin containing the HA and c-Myc epitopes at the NH and COOH termini, respectively, were lysed and analyzed by
SDS-PAGE followed by Western blotting. Replicate lanes were probed with
anti-caveolin antibody (lane 1), anti-HA (lane 2),
and anti-c-Myc (lane 3).
Figure 4:
Detection of the NH-terminal
HA epitope of caveolin by immunofluorescence requires prior
permeabilization of cells with Triton. 293 cells expressing
epitope-tagged caveolin were stained with rabbit anti-DAF and murine
anti-HA antibodies in the absence (A and C) or
presence (B and D) of 0.1% Triton X-100. Fields A and B were photographed under excitation light and filter
appropriate to detect anti-DAF staining, whereas fields C and D were photographed under conditions appropriate to detect
anti-HA staining.
Figure 5: Detection of the COOH-terminal c-Myc epitope of caveolin by immunofluorescence requires prior permeabilization of cells with Triton. 293 cells expressing the epitope-tagged caveolin were stained with rabbit anti-DAF and murine anti-c-Myc antibodies in the absence (A and C) or presence (B and D) of 0.1% Triton X-100. Fields A and B were photographed under excitation light and filter appropriate to detect anti-DAF staining, whereas fields C and D were photographed under conditions appropriate to detect anti-c-Myc staining.
Figure 6:
All three cysteine residues in caveolin
are targets for palmitoylation. 293 cells expressing wild-type or
various mutated forms of epitope-tagged caveolin were labeled for 1 h
with [H]palmitate and lysed as described under
``Materials and Methods.'' A shows the effect of
mutating individual cysteine residues on palmitate incorporation.
Caveolin immunoprecipitates from each of three separate labeling
experiments were analyzed by SDS-PAGE and fluorography (shown above).
5% of the respective cell lysates were subjected to SDS-PAGE and
Western blotting with anti-HA antibody. The section of the Western blot
corresponding to the proper molecular mass of epitope-tagged caveolin
is shown below the fluorographs. B shows similar experiments
measuring the degree of palmitate incorporation following simultaneous
mutation of two specified cysteine
residues.
Thioester-linked
Palmitate Is Not Required for Localization of Caveolin to
Caveolae-We previously demonstrated that the presence of
Cys, which is required for palmitoylation of
p59
, was both necessary and sufficient within the context
of the Src family of kinases to determine localization to
caveolae(12) . Ultrastructural and biochemical evidence
indicates that membrane fractions enriched in caveolae can be isolated
as high molecular weight, Triton-insoluble
aggregates(26, 27) . These Triton-insoluble complexes
have been recovered with the void volume eluate of Sepharose 4B columns (24) and in a low density complex with glycosphingolipids
following equilibrium sucrose density gradient
centrifugation(1) . Caveolin expressed endogenously in MDCK
cells elutes with the void volume fraction following Sepharose 4B
column chromatography(12) . Thus, to determine if
palmitoylation of caveolin was necessary for its targeting to caveolae,
293 cells expressing the wild-type protein and the nonpalmitoylated
version of caveolin (Cys
) were lysed in 1% Triton
X-100 and subjected to Sepharose 4B column chromatography.
Epitope-tagged caveolin eluted primarily in fractions 2 and 3 with very
small amounts found in fractions 4 and 5, consistent with location in
caveolae (Fig. 7). The unknown modification which induces the
caveolin doublet did not affect localization of the molecule to
caveolae as both bands were recovered in the void volume. Most
importantly, the Cys
version of caveolin eluted with
a pattern that was indistinguishable from the wild type. Following
equilibrium sucrose density gradient centrifugation, both the wild-type
and Cys
versions of caveolin were also isolated from
a low density fraction which contains protein components of caveolae in
complex with glycosphingolipids and cholesterol (data not shown). Thus,
in contrast to p59
, caveolin does not require
modification with palmitate for partitioning into caveolae.
Figure 7:
Palmitoylation of caveolin does not affect
its localization to caveolae. 293 cells expressing the wild-type or
Cys versions of epitope-tagged caveolin were lysed in
Triton X-100 and subjected to Sepharose 4B column chromatography.
Column fractions 1-7 were analyzed by Western blotting with
anti-HA antibody. Only the section of the Western blot corresponding to
the proper molecular mass of epitope-tagged caveolin is shown. V
indicates the elution of void volume material
from the column in fraction 2 as measured with intact erythrocytes.
Soluble, nonaggregated proteins of 20 and 70 kDa ran with a peak in
fraction 5.
We report for the first time that caveolin incorporates
palmitate through a thioester linkage. This type of modification is
post-translational, reversible, and has been demonstrated for a number
of proteins which localize to caveolae including G protein
subunits (28, 29, 30) , and members of the
Src family of protein tyrosine kinases, p56
and
p59
(12, 25, 31, 32, 33) .
The pattern of palmitoylation in caveolin is unique in that acylation
occurs on three widely dispersed targets, Cys
,
Cys
, and Cys
. In contrast, G protein
subunits and Src family kinases are palmitoylated on either a single
cysteine residue or a pair of closely spaced cysteine residues.
The
protein sequence motifs and enzymes which putatively mediate protein
S-acylation have not been defined. Camp and Hofmann (34) have
purified a bovine brain thioesterase which depalmitoylates H-Ras.
Molecular cloning of this thioesterase, however, revealed that the
protein was primarily secreted (35) and, therefore, not likely
to act on caveolin or p59 as these are palmitoylated
intracellularly. Membrane proximity appears to be a common attribute of
palmitoylated cysteine residues. Cys
of caveolin lies at
the COOH-terminal end of the hydrophobic transmembrane region and is
followed by hydrophobic residues at three of the next six positions. An
examination of 6 amino acids both upstream and downstream of
Cys
and Cys
reveals that 50% of these
residues are also hydrophobic giving these regions an affinity for the
plasma membrane. Likewise, p59
requires the
membrane-anchoring influence of myristate at Gly
for
palmitoylation(12) . Quesnel and Silvius (36) have
recently shown that acylation of membrane proximal cysteine residues
occurs in a cell-free environment, suggesting that membrane affinity
may not only be necessary, but also sufficient to effect protein
S-acylation. On the other hand, the specificity of acyl chain
incorporation and rapid isoproterenol-mediated turnover of
G
-bound palmitate (37, 38, 39) implies that palmitoylation is
catalytically regulated. Further work is required to clarify the
mechanism(s) of attachment of thioester-linked fatty acids.
Thioester-linked palmitate is a component of one of the two
independent signals thus far defined for localization of proteins to
caveolae. In the case of p59, we have shown that
palmitoylation of Cys
in conjunction with myristoylation of
Gly
is both necessary and sufficient for its localization
to caveolae(12) . Palmitoylation of p59
modestly
strengthens its membrane association, but it is not yet clear how
palmitoylation specifically targets p59
to caveolae.
Palmitoylation alone is not sufficient to localize proteins to caveolae
as a chimeric, type I transmembrane protein consisting of the
ectodomain of DAF and the transmembrane sequence of an HLA class I
molecule and p59
as the cytoplasmic domain, incorporates
palmitate but does not localize to caveolae. (
)The second
signal for caveolar localization is the GPI anchor which has also been
implicated in apical sorting in polarized cells. Replacement of the GPI
anchor of DAF with a membrane-spanning domain results in a protein
which does not partition into caveolae. The coordinate influence of
these two signaling motifs results in the common inclusion of
GPI-linked proteins and Src protein tyrosine kinases in caveolae,
forming a basis for signal transduction through GPI-linked proteins.
Furthermore, these signals apply to ``single-leaflet''
proteins that do not fully traverse the plasma membrane, that is,
GPI-linked proteins insert only in the exoplasmic leaflet and Src
family protein tyrosine kinases insert into the cytoplasmic leaflet of
the plasma membrane. When and where the GPI anchor and the dually
acylated Met-Gly-Cys motif of p59
exert their influence
is only poorly understood. DAF becomes part of a Triton insoluble
complex consisting of glycosphingolipids in the Golgi apparatus on its
way to the plasma membrane(1) . Src family protein tyrosine
kinases, however, possess no signal sequence and are excluded from this
vesicular pathway from the Golgi to the membrane. It is not clear at
what point molecules like p59
are included in caveolae.
Signals which target transmembrane proteins to caveolae are not
known. Post-translational modification by palmitate was an intriguing
possibility considering the increasing abundance of palmitoylated
proteins in caveolae and its role in targeting Src kinases. Data
presented here clearly indicate that palmitoylation does not play the
same role in the targeting of caveolin as in the localization of
p59 to caveolae. The precise role played by covalent
attachment of palmitate in the function of caveolin must wait a further
definition of that function. Caveolin is located on the cytoplasmic
face of caveolae where it may be important in communicating with the
intracellular environment, possibly through interaction with the
cytoskeleton(16) . Fischer rat thyroid cells do not express
caveolin, missort GPI-linked proteins to the basolateral surface, and
do not form clusters between GPI-linked proteins and
glycosphingolipids, suggesting the possible involvement of caveolin in
these processes(40, 41) . Dissection of the domains
involved in the targeting and function of caveolin will define new
molecular signals important in the biogenesis and function of caveolae.