(Received for publication, October 10, 1995; and in revised form, November 6, 1995)
From the
Caveolin, a 22-24-kDa integral membrane protein, is a
principal component of caveolar membranes in vivo. Caveolin
has been proposed to function as a scaffolding protein to organize and
concentrate signaling molecules within caveolae. Because of its unusual
membrane topology, both the N- and C-terminal domains of caveolin
remain entirely cytoplasmic and are not subject to luminal
modifications that are accessible to other integral membrane proteins.
Under certain conditions, caveolin also exists in a soluble form as a
cytosolic protein in vivo. These properties make caveolin an
attractive candidate for recombinant expression in Escherichia
coli. Here, we successfully expressed recombinant full-length
caveolin in E. coli. A polyhistidine tag was placed at its
extreme C terminus for purification by
Ni-nitrilotriacetic acid affinity chromatography.
Specific antibody probes demonstrated that recombinant caveolin
contained a complete N and C terminus. Recombinant caveolin remained
soluble in solutions containing the detergent octyl glucoside and
formed high molecular mass oligomers like endogenous caveolin. By
electron microscopy, recombinant caveolin homo-oligomers appeared as
individual spherical particles that were indistinguishable from
endogenous caveolin homo-oligomers visualized by the same technique. As
recombinant caveolin behaved as expected for endogenous caveolin, this
provides an indication that recombinant caveolin can be used to dissect
the structural and functional interaction of caveolin with other
protein and lipid molecules in vitro. Recombinant caveolin was
efficiently incorporated into lipid membranes as assessed by floatation
in sucrose density gradients. This allowed us to use defined lipid
components to assess the possible requirements for insertion of
caveolin into membranes. Using a purified synthetic form of
phosphatidylcholine (1,2-dioleoylphosphorylcholine), we observed that
incorporation of caveolin into membranes was cholesterol-dependent; the
addition of cholesterol dramatically increased the incorporation of
caveolin into these phosphatidylcholine-based membranes by
25-30-fold. This fits well with in vivo studies
demonstrating that cholesterol plays an essential role in maintaining
the structure and function of caveolae. Further functional analysis of
these reconstituted caveolin-containing membranes showed that they were
capable of recruiting a soluble recombinant form of
G
. This is in accordance with previous studies
demonstrating that caveolin specifically interacts directly with
multiple G protein
-subunits. Thus, recombinant caveolin
incorporated into defined lipid membranes provides an experimental
system in which the structure, function, and biogenesis of
caveolin-rich membrane domains can be dissected in vitro.
Caveolae are specialized domains of the plasma membrane that are found in most cell types(1, 2) . However, they are most abundant in adipocytes, endothelial cells, fibroblasts, and muscle cells(3) . In adipocytes, caveolae represent up to 20% of the total plasma membrane surface area(4) . Functionally, caveolae have been implicated in endothelial transcytosis(5) , potocytosis(1) , and signal transduction (6, 7, 8, 9, 10, 11, 12) . The ``caveolae signaling hypothesis'' states that caveolar localization of certain lipid-modified signaling molecules could provide a means for integrating certain transmembrane signaling events (13, 14, 15) .
Caveolin, a 22-24-kDa
protein, is an integral membrane component of caveolar
membranes(16) . It has been proposed that caveolin may function
as a scaffolding protein for organizing and concentrating
caveolin-interacting molecules within caveolae(17) . In this
regard, caveolin appears to be very important for the formation of
caveolar membranes. Caveolin expression levels correlate very well with
the biochemical and morphological appearance of caveolae. For example,
(i) caveolin is most abundant in cell types that contain numerous
caveolae, i.e. adipocytes, endothelial cells, smooth muscle
cells, and fibroblasts(18) ; (ii) caveolin and caveolae are
both induced 10-25-fold during the differentiation of 3T3-L1
fibroblasts to the adipocyte form(4, 19) ; (iii)
caveolin levels are dramatically reduced, and caveolae are
morphologically absent in cells transformed by various activated
oncogenes (v-abl, activated ras, and
others)(20) ; and (iv) recombinant expression of caveolin in
caveolin-negative cell lines results in the correct targeting of
caveolin to caveolae-enriched membrane fractions and allows the de
novo formation of caveolae(21, 22) . These
results suggest that caveolin represents an important structural
protein for directing the formation of caveolar membranes.
Several structural properties of caveolin are also consistent with a role for caveolin in organizing caveolar domains. Both the N- and C-terminal domains of caveolin face the cytoplasm and are thus accessible for interactions with cytoplasmically oriented molecules (23) ; caveolin copurifies with cytoplasmic signaling molecules including heterotrimeric G proteins, Src family tyrosine kinases, and Ras-related GTPases(6, 8, 9, 11, 12, 21) ; caveolin interacts directly with heterotrimeric G proteins and can functionally regulate their GTPase activity, holding the G protein in the inactive conformation (24) ; and caveolin exists within caveolar membranes as a high molecular mass homo-oligomer(17, 25) . Thus, caveolin could serve as an oligomeric docking site for organizing and concentrating inactive signaling molecules within caveolar membranes(17) .
To study
the molecular interaction of caveolin with other molecules, we have
recently expressed full-length caveolin and portions of caveolin as GST ()fusion proteins in Escherichia
coli(17, 21, 24) . However, preparations
of GST-full-length caveolin also contained substantial amounts of GST
cleaved to its active core as the complete fusion was somewhat
unstable. The partial success of this approach prompted us to express
full-length caveolin in E. coli without the GST moiety.
Full-length caveolin without GST would more closely approximate the
natural state of the caveolin molecule in vivo. Here, we
report the purification and characterization of soluble recombinant
full-length caveolin without GST and its interaction with lipid
membranes of defined molecular composition. Despite that caveolin is an
integral membrane protein, it was expressed well using this approach.
This may be related to the observations that under certain conditions,
caveolin also exists in a soluble form as a cytosolic protein in
vivo(26) , and both caveolin N- and C-terminal domains
remain entirely cytoplasmic and are not subject to luminal
modifications(21, 23, 27) .
Figure 1:
Purification of
recombinant caveolin. A, schematic diagram summarizing the
construction of recombinant caveolin. A Myc epitope tag was placed at
its C terminus with a polyhistidine tag following the Myc tag for
affinity purification by Ni-NTA-agarose
chromatography. Note that mAb 2234 recognizes an epitope within
caveolin residues 1-21; mAb 9E10 recognizes the Myc epitope
(EQKLISEEDLN). B, characterization of recombinant caveolin by
immunoblot analysis. Recombinant caveolin was biotinylated in vitro to assess its purity; it appeared as a single band by streptavidin
blotting. In addition, the same band contains a complete N and C
terminus as evidenced by immunoblot analysis with specific antibody
probes (mAb 2234 and mAb 9E10).
Recombinant caveolin appeared relatively pure. As caveolin fails to stain with standard protein stains(21) , purified preparations of recombinant caveolin were biotinylated in solution. This technique is more sensitive than silver staining; in vitro biotinylation allows the detection of picogram quantities of protein(29) . Biotinylated proteins were detected after SDS-PAGE and transfer to nitrocellulose by blotting with iodinated streptavidin. Caveolin appeared as a single band without any major contaminants or degradation products (Fig. 1B). However, it should be noted that the molecular mass of recombinant caveolin was slightly larger than that of endogenous caveolin. This reflects the addition of the Myc epitope and polyhistidine tags.
To monitor the completeness of the N- and C-terminal ends of caveolin, two specific antibody probes were utilized: mAb 2234, which recognizes caveolin residues 1-21, and mAb 9E10, which detects the Myc epitope placed at the C-terminal end of caveolin. The single major band observed by in vitro biotinylation was immunoreactive with both these probes (Fig. 1B), indicating that recombinant caveolin contains a complete N and C terminus and therefore represents the full-length caveolin protein.
Endogenous caveolin
exists as a 350-kDa homo-oligomer (containing
14-16
monomers/oligomer) as shown using several different
approaches(17, 25) . The oligomeric state of purified
recombinant caveolin was next assessed by employing an established
velocity gradient system developed previously to study homo-oligomers
of endogenous caveolin(17) . Several integral membrane proteins
have been previously shown to migrate at their expected monomeric
molecular mass in these same velocity gradients(17) . Fig. 2shows that purified recombinant caveolin migrated as
expected as a high molecular mass complex between 200- and 443-kDa
molecular mass standards. This provides an indication that recombinant
caveolin is correctly folded as it remains soluble in detergent
solutions and forms high molecular mass oligomers of the same relative
size as endogenous caveolin.
Figure 2: Velocity gradient centrifugation of recombinant caveolin. A, purified recombinant caveolin was loaded atop a 5-40% sucrose gradient containing octyl glucoside and subjected to centrifugation for 10 h. Fractions were analyzed on blots by incubation with anti-caveolin IgG (mAb 2234). Note that recombinant caveolin migrates as a distinct high molecular mass complex and peaks in fraction 8. No caveolin monomers were detectable. The migration of molecular mass standards is also shown for comparison. B, shown is a graphic representation of A quantitated by scanning densitometry as described previously(8, 44) .
Endogenous caveolin homo-oligomers
appear as individual globular particles (4-6 nm in diameter)
by electron microscopy using a low-angle platinum shadowing
technique(17) . The same approach was used to examine the
ultrastructure of recombinant caveolin. Fig. 3shows that
recombinant caveolin appeared as individual globular particles of the
same diameter previously reported for endogenous caveolin
homo-oligomers. Thus, recombinant caveolin behaves as expected for
endogenous caveolin in its solubility properties, oligomeric state, and
morphologic appearance.
Figure 3:
Low-angle platinum shadowing of
recombinant caveolin homo-oligomers. Homo-oligomers appear as
individual spherical particles with a diameter of 4-6 nm. Bar = 0.05 µm.
It is important to note that when the same construction used here was recombinantly expressed in mammalian cells, it cofractionated with endogenous caveolin (Fig. 4), an indication that the Myc epitope tag and the His tag do not interfere with the caveolar targeting of this engineered form of caveolin. Previous reports have also indicated that tagging of caveolin at either its N or C terminus with the Myc epitope does not affect its caveolar localization in vivo(21, 27, 30) .
Figure 4:
Polyhistidine-tagged caveolin
cofractionates with endogenous caveolin. Caveolin containing a
C-terminal polyhistidine tag (see Fig. 1;
caveolin-Myc-His) was stably expressed in Madin-Darby
canine kidney cells as described previously for other caveolin
constructs(21) . Madin-Darby canine kidney cells were then
fractionated using an established protocol that separates caveolin from
the bulk of cellular membranes and cytosolic proteins. This
fractionation scheme is based on the specific buoyant density of
caveolin-rich domains and their resistance to solubilization by the
non-ionic detergent Triton X-100 at low
temperatures(6, 8, 12, 15, 19, 26, 45) .
The distributions of polyhistidine-tagged caveolin
(caveolin-Myc-His
) and endogenous caveolin are shown.
Fractions were collected from the top, separated by SDS-PAGE (15%
acrylamide), and analyzed by immunoblotting. Immunoblot analysis was
carried out with mAb 9E10 to selectively detect caveolin-Myc-His
(not shown) and with anti-caveolin IgG (mAb 2297) to detect
caveolin-Myc-His
and endogenous caveolin. Note that both
- and
-isoforms of endogenous caveolin are detected (21 and
24 kDa) and that they cofractionate with caveolin-Myc-His
(29 kDa). The generation of
-caveolin results from an
alternate translation initiation site at amino acid 32 within the
full-length caveolin cDNA(21) . We have previously shown, using
this fractionation scheme, that endogenous caveolin (fractions 5 and 6)
is purified
2000-fold relative to total Madin-Darby canine kidney
cell lysates(15, 24) . In addition, caveolin is
separated from >99.95% of total Madin-Darby canine kidney cellular
proteins and compartment-specific markers for the endoplasmic
reticulum, Golgi apparatus, lysosomes, mitochondria, and non-caveolar
plasma membrane that remain in fractions 9-13 of these
bottom-loaded sucrose density
gradients(6, 8, 15) .
Figure 5:
Incorporation of recombinant caveolin
into lipid membranes. The incorporation of recombinant caveolin into
membranes was monitored by floatation in sucrose density gradients.
Recombinant caveolin (85 µg) was mixed with 1.8 mg of the
purified lipid extract dissolved in octyl glucoside (see
``Experimental Procedures''). The mixture was adjusted to 40%
sucrose and placed at the bottom of an ultracentrifuge tube. A
5-30% discontinuous sucrose gradient (lacking detergent) was
formed above and subjected to centrifugation for 20 h. A
light-scattering band confined to the 5-30% sucrose interface
(fractions 4 and 5) was observed and contained the associated lipids.
This band formed regardless of whether or not caveolin was added.
Fractions (1 ml) were collected from the top and subjected to
immunoblot analysis with anti-caveolin mAb 2234. Note that caveolin
pellets in the absence of lipids as it forms a high molecular mass
oligomer (caveolin alone). In the presence of lipids, caveolin
associates with membranes and attains buoyancy (caveolin plus
lipids). Note that the lipid extract does not contain endogenous
caveolin (lipids alone).
Additional control experiments were performed to rule out the possibility that caveolin is randomly trapped within these lipid membranes as they form. For this purpose, we also expressed and purified a recombinant cytosolic protein (GST) in E. coli. In contrast to recombinant caveolin, recombinant GST showed no incorporation into lipid membranes (data not shown). As a purified cytosolic protein is excluded during the process of membrane reconstitution, this indicates that caveolin is not simply trapped within the lumen of these lipid membranes.
The lipid extract used in our initial experiments contained a complex mixture of naturally occurring lipids. To simplify the system, we next used a chemically synthetic form of pure phosphatidylcholine that is fluid at 4 °C. This lipid assembled into membranes as expected and migrated to the 5-30% sucrose interface. However, caveolin was very inefficiently incorporated under these conditions; little or no caveolin attained buoyancy.
Several independent lines of evidence suggest that
cholesterol plays an essential role in maintaining both the structure
and function of caveolar membranes in
vivo(16, 26, 31, 32) . In
addition, it has been postulated that caveolin may interact
specifically with cholesterol, although no direct evidence has been
presented to support this assertion(26) . Thus, we supplemented
chemically pure phosphatidylcholine with cholesterol (in a 1:1 molar
ratio) and monitored the incorporation of caveolin into this
two-component lipid system. Under these conditions, the addition of
cholesterol dramatically increased the incorporation of caveolin into
these phosphatidylcholine-based membranes by 25-30-fold (Fig. 6). Thus, this model system (recombinant caveolin inserted
into purified or synthetic lipid membranes) provides an experimental
system in which the structure, function, and biogenesis of
caveolin-rich membranes can be dissected in vitro.
Figure 6:
Cholesterol-dependent incorporation of
recombinant caveolin into phosphatidylcholine-based membranes.
Recombinant caveolin (85 µg) was mixed with 2.5 mg of
synthetic phosphatidylcholine dissolved in octyl glucoside and treated
as described in the legend of Fig. 5. In a second condition,
phosphatidylcholine and cholesterol were mixed in a 1:1 molar ratio
before the addition of caveolin. After floatation in sucrose density
gradients, fractions 4 and 5 of each gradient (corresponding to the
light-scattering band at the 5-30% sucrose interface) were
collected, pooled, and subjected to immunoblot analysis with
anti-caveolin IgG. Note that cholesterol addition increased the
incorporation of caveolin into these phosphatidylcholine-based
membranes by
25-30-fold.
To test this idea more directly, we evaluated
whether recombinant caveolin could recruit soluble G protein
-subunits onto membranes in vitro. For this purpose, we
expressed and purified a soluble recombinant form of G
in bacteria (GST-G
). Reconstituted lipid
membranes containing recombinant caveolin were then briefly incubated
with soluble recombinant GST-G
. To stringently remove
unbound GST-G
, caveolin-containing lipid membranes
were then reisolated by floatation in sucrose density gradients. Thus,
bound GST-G
must attain the buoyancy of lipid
membranes in this system. As a control for nonspecific association of
GST-G
with membranes, an equivalent amount of
reconstituted lipid membranes lacking recombinant caveolin was
processed in parallel and subjected to the same binding and
refloatation after incubation with soluble recombinant
GST-G
. Fig. 7shows that GST-G
bound to reconstituted caveolin-rich membranes, but not to
caveolin-deficient membranes. This indicates that caveolin is
sufficient to recruit GST-G
onto lipid membranes. As
G
normally undergoes dual acylation (myristoylation
and palmitoylation) when expressed in mammalian cells, our results
indicate that these lipid modifications that do not occur in bacteria
are not absolutely necessary for the caveolin-dependent recruitment of
G
onto membranes. However, such lipid modifications
may still serve to modulate these interactions in vivo.
Figure 7:
Recruitment of a purified soluble form of
G onto reconstituted caveolin-rich membranes.
Caveolin was incorporated into membranes as described under
``Experimental Procedures'' and in the legend of Fig. 5. Equivalent amounts of reconstituted caveolin-rich
membranes and caveolin-deficient membranes were then incubated in
solution with soluble recombinant GST-G
. To remove
unbound GST-G
, membranes were then isolated by
refloatation in sucrose density gradients. After refloatation,
fractions 4 and 5 of each gradient (corresponding to the
light-scattering band at the 5-30% sucrose interface) were
collected, pooled, and subjected to immunoblot analysis with
anti-G
IgG. Note that the binding of soluble
GST-G
to membranes is caveolin-dependent.
GST-G
migrates at
68 kDa, as expected, as it
should have the cumulative molecular mass of GST (26-27 kDa) plus
G
(40 kDa).
To
rule out the unlikely possibility that the interaction of
GST-G with reconstituted caveolin-rich membranes is
simply due to nonspecific protein-protein adsorption, we performed
additional control experiments. For this purpose, we evaluated whether
another soluble recombinant protein, GST, could interact with these
membranes in a caveolin-dependent manner. No binding of GST to either
caveolin-containing membranes or caveolin-deficient membranes was
observed (data not shown). This is consistent with previous studies
demonstrating that the interaction of GST-caveolin with
baculovirus-expressed G protein
-subunits is highly
specific(24) .
Caveolin was first identified as a major v-Src substrate in
Rous sarcoma virus-transformed cells and later as a caveolar marker
protein (16, 18, 30, 34, 35) .
Cholesterol is thought to provide the ``lipid glue'' that
holds caveolin within caveolar membranes and is essential for the
proper biological functioning of
caveolae(16, 26, 31) . Increasing evidence
indicates that caveolin may function within caveolar membranes as a
scaffolding protein to organize and concentrate caveolin-interacting
molecules(17) . One class of caveolin-interacting proteins thus
far identified is G protein -subunits(24) .
Here, we have begun to systematically reconstitute caveolin-rich membrane domains in vitro using purified recombinant proteins and synthetic lipid components. We expressed and purified a recombinant full-length form of caveolin from E. coli. Soluble recombinant caveolin contained a complete N and C terminus, formed high molecular mass oligomers of the correct size and shape as seen by velocity gradient centrifugation and electron microscopy, and could be inserted into lipid membranes after removal of the detergent octyl glucoside. Insertion of caveolin into lipid membranes appeared to depend on the presence of cholesterol, as shown using purified and synthetic lipid components.
How does cholesterol facilitate the incorporation of
caveolin into membranes? One possibility is that cholesterol adjusts
membrane fluidity to provide the proper lipid environment for insertion
of caveolin into membranes. Alternatively, cholesterol might interact
directly with caveolin as caveolin contains an unusual 33-amino acid
membrane-spanning region that is predicted to assume a
-conformation (35) . In support of the latter possibility,
caveolin is similar in many respects to a 293-amino acid bacterial
toxin known as
-hemolysin or staphylococcal
-toxin. Like
caveolin,
-hemolysin contains a membrane-spanning region that
assumes a
-conformation, inserts into target membranes in a
cholesterol-dependent fashion, forms SDS-resistant homo-oligomers, and
exists both as a soluble protein that is secreted and as an integral
membrane protein after insertion into target
membranes(36, 37, 38, 39, 40) .
Our results are also consistent with previous in vivo studies
demonstrating that oxidation of plasma membrane cholesterol can
transiently convert endogenous caveolin from an integral membrane
protein to a soluble cytosolic protein(26) . The in vivo process is also reversible; as cholesterol recovers and is reduced
to the
-OH form, caveolin is again converted to an integral
membrane protein and reassociates with caveolar membranes(26) .
Caveolin interacts directly and specifically with G protein
-subunits(24) . In this report, we used this established
interaction to examine whether reconstituted caveolin-rich membranes
are competent to recruit caveolin-interacting molecules onto membranes.
Reconstituted caveolin-rich membranes, but not caveolin-deficient
membranes, bound a soluble recombinant form of G
.
This interaction conferred buoyancy upon bound G
as
assessed by floatation in sucrose density gradients. This is the first
direct evidence that membrane-bound caveolin can interact with G
protein
-subunits as previous studies were performed using an
agarose-bound form of GST-caveolin. This also indicates that lipid
modification of G
may not be necessary for this
protein-protein interaction. However, this does not rule out the
possibility that lipid modifications or other molecules may facilitate
or regulate these interactions in vivo. We have previously
postulated that this type of interaction between caveolin and other
molecules could provide a mechanism for the recruitment of
caveolin-interacting molecules onto caveolar membranes in
vivo(13, 17, 24) .
The availability of soluble recombinant full-length caveolin should help in the reconstitution of other caveolin-related processes as well. Endogenous caveolin undergoes regulatable cytoplasmic modifications such as phosphorylation and palmitoylation in vivo(19, 27, 34, 41, 42) . As such, recombinant caveolin could serve as a valuable substrate for the identification and purification of the relevant caveolin-modifying enzymes, i.e. serine and tyrosine protein kinases and a palmitoyltransferase.
Currently, there are two opposing views regarding the role of caveolin in the formation of caveolar membranes: (i) that caveolin expression can lead to the formation of functional caveolae in caveolin-deficient cells (22) and (ii) that caveolin is not absolutely essential for the morphological formation of caveolar membranes(26) . Perhaps, not surprisingly, both of these views could be correct if caveolin were a gene family of immunologically distinct but functionally related molecules, like G proteins and Src family tyrosine kinases. Our most recent results suggest that other caveolin-related molecules exist and that multiple members of the caveolin gene family can be cloned and are coexpressed within a single cell(43) . In summary, the ability to biochemically reconstitute caveolin-rich membranes using purified and recombinant components provides an experimental system in which the structure and function of caveolin-rich membrane domains can be dissected in vitro.