From the
Transport by discrete vesicular carriers is well established at
least in part because of recent discoveries identifying key protein
mediators of vesicle formation, docking, and fusion. A general
mechanism sensitive to N-ethylmaleimide (NEM) is required for
the transport of a divergent group of vesicular carriers in all
eukaryotes. Many endothelia have an abundant population of noncoated
plasmalemmal vesicles or caveolae, which have been reported with
considerable controversy to function in transport. We recently have
shown that like other vesicular transport systems, caveolae-mediated
endocytosis and transcytosis are inhibited by NEM (Schnitzer, J. E.,
Allard, J., and Oh, P.(1995) Am. J. Physiol. 268,
H48-H55). Here, we continue this work by utilizing our recently
developed method for purifying endothelial caveolae from rat lung
tissue (Schnitzer, J. E., Oh, P., Jacobson, B. S., and Dvorak, A.
M.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1759-1763)
to show that these caveolae contain key proteins known to mediate
different aspects of vesicle formation, docking, and/or fusion
including the vSNARE VAMP-2, monomeric and trimeric GTPases, annexins
II and VI, and the NEM-sensitive fusion factor NSF along with its
attachment protein SNAP. Like neuronal VAMPs, this endothelial VAMP is
sensitive to cleavage by botulinum B and tetanus neurotoxins. Caveolae
in endothelium are indeed like other carrier vesicles and contain
similar NEM-sensitive molecular machinery for transport.
Vesicular transport plays a fundamental role in the existence
and function of cells. In many endothelia, the abundance of noncoated
plasmalemmal vesicles or caveolae has suggested, ever since their
discovery(1) , an important but controversial role in the
fluid-phase transport of molecules from the circulating blood across
the endothelial barrier to the interstitium. A variety of studies using
electron microscopy to detect tracers crossing the endothelium have
implicated caveolae in the transcytosis of macromolecules (for review,
see Ref. 2). By contrast, because serial sectioning of endothelium
revealed that 1% or less of the noncoated vesicles can be found free in
the cytoplasm unattached to other vesicles or the plasma membrane, some
investigators have concluded that endothelial caveolae cannot bud and
detach from the cell surface to form free discrete vesicles and
therefore cannot function in transport(3, 4) . In the
last few years, studies have shown that specific proteins on the
endothelial cell surface facilitate the select transendothelial
transport of insulin and blood carrier proteins such as albumin (5) and transferrin (for review, see Ref. 6). In addition,
endothelial caveolae can mediate scavenger endocytosis to endosomes and
lysosomes for degradation(7) .
New key evidence supporting a
functional role for caveolae in transport comes from very recent
studies identifying two different types of inhibitory
compounds(7, 8, 9) . Cholesterol binding agents
such as filipin inhibit both the endocytosis and transcytosis of select
ligands that preferentially bind within caveolae. By removing
cholesterol from plasma membranes, filipin selectively disassembles
caveolae in lung endothelium, which produces a significant reduction in
the scavenger endocytosis of modified albumins, transcytosis of insulin
and native albumin, and even transcapillary permeability of albumin in
the rat lung in situ(7) . Moreover, such transport by
caveolae is also sensitive to alkylation with N-ethylmaleimide
(NEM),
It is well established for a
variety of cell types that NEM inhibits transport by discrete vesicular
carriers between specific intracellular compartments including
endoplasmic reticulum and Golgi trafficking, granule exocytosis, and
clathrin-dependent endocytosis and transcytosis (see Ref. 8 for a more
complete discussion). In the last few years, there have been a rapid
series of advances in the understanding of the molecular mechanisms
involved in vesicular transport (for reviews, see Refs. 10 and 11).
Specific cytosolic and membrane molecules are required for the ability
of transport vesicles: (i) to form or pinch off from the membrane of
origin into discrete vesicles via a process called budding or fission,
(ii) to interact specifically with their target site membrane, a
process called docking, and (iii) to fuse with the target membrane for
discrete delivery of their contents. The molecules implicated in these
aspects of vesicle transport include various GTP-binding
proteins(12, 13, 14, 15, 16, 17, 18, 19, 20, 21) ,
annexins (22-24), and the SNARE
complexes(10, 11, 17, 25) . In the SNARE
model, NEM inhibits transport by modifying a specific ATPase called NSF
(NEM-sensitive fusion protein), which interacts with soluble NSF
attachment proteins called SNAPs(26, 27, 28) .
Together, they form a functional SNARE fusion complex by direct
association with complementary SNAP receptors, the SNAREs that are the
targeting receptors located on the vesicles (vSNARE) and the target
membranes (tSNARE)(17, 25) .
If caveolae are indeed
vesicular carriers in endothelium, a complete understanding of this
process requires information about the molecular events comprising
caveolae formation, movement, docking, and fusion. Current knowledge
indicates that the molecular machinery necessary for vesicular
transport is not only conserved from yeast to mammalian systems (10, 29, 30) but operable for a very divergent
group of vesicular carriers ranging from the specialized synaptic
vesicles involved in highly regulated neurotransmitter release to the
more common exocytic and endocytic carriers found in many different
cell types(10, 11, 17) . As our initial foray
into this area, a logical starting point is the identification of the
molecular machinery responsible for vesicular transport as it relates
to the caveolae. In this regard, we hypothesize that caveolae may
possess and utilize NEM-sensitive molecular machinery similar to other
vesicular carriers. Indeed, our previous functional studies have shown
that NEM impaired caveolae-mediated transport systems in
endothelium(8) . Recently, we have developed a method for
isolating pure caveolae from the luminal surface of microvascular
endothelium in rat lung tissue(31) . The studies presented here
show that these purified endothelial caveolae do have the typical
molecular machinery necessary for vesicle formation, docking, and
fusion as found in many other vesicular carriers.
We recently developed a method for selectively purifying
endothelial caveolae from rat lung tissue(31) . Briefly, rat
lungs were perfused in situ at 10-13 °C with a
solution of positively charged colloidal silica particles that coated
the intimal endothelial cell surface of the blood vessels and created a
stable silica pellicle that specifically marked this membrane and
allowed its purification from tissue homogenates by centrifugation. The
sedimented pellets (P) contained highly purified silica-coated
luminal endothelial cell plasma membranes with associated caveolae and
showed ample enrichment for various endothelial cell surface markers
relative to the starting whole lung homogenates (H). Little
contamination from other tissue components was detected. The caveolae
were stripped from P by shearing during homogenization at 4 °C in
the presence of Triton X-100. These homogenates were subjected to
sucrose density centrifugation to yield three collected fractions: a
low density fraction of intact small vesicles (V) well separated
from the Triton X-100-soluble fraction (T) and the pellet
containing resedimented silica-coated membranes stripped of the
caveolae (P-V). V contained caveolae by morphological and
biochemical criteria with little, if any, apparent contamination from
other membranes. The isolated homogeneous population of morphologically
distinct caveolae were specific microdomains of the cell surface with
their own unique molecular topography quite distinct from the rest of
the plasma membrane. As with caveolae found on the endothelial cell
surface in vivo, the highly purified caveolae (V) were
enriched in caveolin, plasmalemmal Ca
Very recently, we found that caveolae can
function in transport like other vesicular carriers by showing that NEM
effectively inhibits the endocytosis and transcytosis of select
macromolecules found in caveolae (8). Here, we continue this work and
examine the purified endothelial caveolae for the presence of key
proteins known to mediate particular stages of NEM-sensitive transport
of other vesicular carriers. In each section, we discuss the known
relevance of each molecule to vesicular transport.
The fractions collected from the rat lungs were examined for the
presence of vSNARE by testing antibodies recognizing members of the
VAMP family. First, we utilized a monoclonal antibody (C1 10.1) that
recognizes all known forms of VAMP, including cellubrevin, which appear
by SDS-PAGE as small proteins of about 15-20
kDa(36, 37, 38, 39, 40, 41) .
As shown in Fig. 1, this antibody recognized a single protein
band at 18 kDa, which was greatly enriched in P relative to H and even
more so in V. Upon subfractionation of P, almost all of the VAMP signal
(>95%) was found in V with little, if any, detected in P-V. When
isoform-specific antibodies were tested, anti-VAMP-2 IgG gave very
similar results to C1 10.1, whereas antibodies specific for VAMP-1 did
not recognize the 18-kDa protein (data not shown).
We examined the rat lung fractions for the presence of
NSF and SNAP. As shown in Fig. 3, NSF and
It should be mentioned that we have attempted to identify tSNAREs
(syntaxin and SNAP-25) in the rat lung fractions. Unfortunately few
antibodies were available to the different family members. The
brain-specific tSNAREs (syntaxin-1 and SNAP-25) were not found in our
preparations. It will be interesting to determine in the future which
one of the many family members exists on the endothelial cell surface
for specific interaction with caveolar VAMP.
Here, utilizing a standard blotting
procedure for identifying these GTPases using
[
Like
the small GTPases (as discussed above), the heterotrimeric G proteins
were also recently found in isolated detergent-resistant membrane
fractions derived from various different
sources(47, 49, 50) . Here, we used specific
antibodies to examine whether any of these regulatory G proteins were
also associated with purified endothelial caveolae. Monospecific
peptide antibodies against various G protein subunits were used to
immunoblot the rat lung fractions. Fig. 4shows that all of these
proteins were enriched in P and V relative to H. The caveolae were rich
in G
Our rat lung fractions were tested using
antibodies specific for distinct members of the annexin family to
determine whether any annexins were located within caveolae. Fig. 3shows that both annexins II (36 kDa) and VI (78 kDa) were
detected easily in all of the fractions. They were present rather
equally in P and V and also remained in P-V. The residual presence of
annexin in P-V is consistent with its proposed bilateral function on
the vesicle and target membranes required not only to diminish local
repulsive forces between the membranes but quite possibly creating a
local perturbation or ``weakening'' in the lipid bilayer that
permits the blending or fusion of two distinct membranes found in close
apposition (22, 24).
We are greatly indebted to Drs. R. Jahn, W. Trimble,
and S. W. Whiteheart for their generous gifts of antibodies.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)suggesting by analogy with other
vesicular carrier systems a dependence on membrane fusion and specific
NEM-sensitive factors(8) .
Materials
Sources for antibodies against: NSF
(mouse monoclonal; 6E6) and -SNAP (rabbit) from Dr. S. W.
Whiteheart (University of Kentucky); peptides corresponding to the
first 15 amino acids of the N-terminal region of rat VAMP-1 and -2
(peptide affinity-purified rabbit) from Dr. W. Trimble (University of
Toronto); synaptobrevin (recognizes all known VAMPs including
cellubrevin) (mouse monoclonal; C1 10.1) and brain syntaxin (rabbit)
and SNAP-25 (rabbit) from Dr. R. Jahn (Yale University); annexins II
and VI from Biodesign International (Kennebunk, ME);
-actin
(monoclonal) from Sigma; rab5 (peptide affinity-purified rabbit) from
Santa Cruz Biotechnology (Santa Cruz, CA); and trimeric GTP-binding
protein subunits (rabbit) from Calbiochem. Botulinum neurotoxin B
(BoNT) and tetanus toxin (TeTx) were purchased from Calbiochem,
Dynabeads from Dynal (New Hyde Park, NY), and
[
-
P]GTP from DuPont NEN. Sources for other
reagents are as in our past work(5, 7, 8) .
SDS-PAGE and Immunoblotting
The proteins of
selected fractions were solubilized with cold solubilization buffer
containing 0.17 M Tris-HCl (pH 6.8), 3% (w/v) SDS, 1.2% (v/v)
-mercaptoethanol, 2 M urea, and 3 mM EDTA in
double distilled water. After incubation in boiling water for 10 min,
the proteins were separated by SDS-PAGE and electrotransferred onto
nitrocellulose or Immobilon filters for immunoblotting as described in
Ref. 5. Briefly, the whole filter or, to conserve reagents such as
antibodies, filter strips cut from each gel lane (3/lane) were probed
using a variety of primary antibodies followed by the appropriate
I-labeled reporter IgG. In other cases, the reporter IgG
was conjugated to horseradish peroxidase, and the binding was detected
on the filters using ECL chemiluminescent substrate as per the
manufacturer's instructions (Amersham Corp.). Band intensities
were quantified by PhosphorImager analysis (Molecular Dynamics Inc.,
Sunnyvale, CA), densitometry of autoradiograms, and/or direct counting
of
radioactivity. The detected signals in each lane or filter
strip were normalized to the total protein loaded onto the gels as
determined by BCA assay and/or silver staining of duplicate gels.
Proteolytic Cleavage of VAMP
VAMP was
proteolytically cleaved by BoNT B and TeTx using a modification of the
method described in Ref. 32. Briefly, 30 µg of silica-coated
endothelial cell membranes (P) in 20 µl of 5 mM HEPES
containing 0.3 M glycine and 0.3 M NaCl were treated
for 60 min at 37 °C with BoNT B or TeTx (both activated by
reduction in 10 mM DTT). The cleavage was terminated by
addition of solubilization buffer followed by boiling for 4 min. As a
control, reduced toxin was preincubated with captopril to inactivate
endopeptidase activity of the toxins(33) .
-ATPase, and
inositol 1,4,5-trisphosphate receptor(31) . By contrast, other
cell membrane markers were nearly totally excluded from V despite being
present amply in P. Angiotensin-converting enzyme was found in T while
as expected the cytoskeletal proteins Band 4.1 and
-actin remained
primarily in P-V. (The abbreviations H, P, V, T, and P-V will be used
extensively below.)
Caveolae Are Enriched in Toxin-sensitive VAMP
The VAMPs
comprise a family of integral membrane proteins that appear to be well
conserved among organisms as diverse as yeast and
humans(29, 34, 35) . Various transport vesicles
including synaptic and exocytic vesicles contain members of the VAMP
family (vSNARE) for specific docking and/or
fusion(35, 36, 37, 38, 39) .
VAMP (also called synaptobrevin) was first identified as a 18-kDa
protein expressed in brain tissue and enriched in purified synaptic
vesicles(36, 37) . Currently, there are at least three
known mammalian isoforms with different tissue and cellular
distributions (40). VAMP-1 is expressed most abundantly in the nervous
system(36, 37) . Although VAMP-2 is also abundant in
brain, it may also be expressed in the exocytic vesicles of adipocytes
and pancreatic exocrine cells(38, 39) . The third VAMP
called cellubrevin has a broad tissue distribution(41) . Mutants
of the yeast homologues of VAMP are defective in secretion, suggesting
a role for VAMP in constitutive secretion(34) . Moreover,
selective removal of VAMPs from synaptic vesicles by zinc protease
neurotoxins inhibits neurotransmitter release and vesicle
fusion(33) . VAMP proteins interact with distinct isoforms of
the tSNARE syntaxin, a family of proteins residing on membranes with
which the carrier vesicles dock and fuse(42) . The interaction
of complementary SNAREs appears to ensure specificity and fidelity in
targeting of carrier vesicles to their appropriate acceptor membranes.
Figure 1:
VAMP is expressed on the endothelial
cell surface concentrated in the caveolae. Proteins of the indicated
fractions isolated from rat lungs (see text) were resolved by
5-15% SDS-PAGE and electrotransferred onto nitrocellulose. This
filter was immunoblotted with monoclonal antibody C1 10.1 using
chemiluminescence detection. 5 µg of protein were loaded onto each
gel lane from fractions: H (rat lung homogenate), P (silica-coated
endothelial plasmalemmal pellet), and P-V (silica-coated membranes
stripped of caveolae). For V (caveolae), only 1 µg was
used.
In order to
corroborate further that this 18-kDa protein is truly a VAMP protein,
we employed an additional test. It was reported that the SNAREs share a
common motif that renders them susceptible to proteolytic cleavage by
specific neurotoxins and that botulinum B and tetanus toxins
specifically cleaved VAMPs(43) . To examine whether the
immunoreactive 18-kDa protein on the endothelial cell surface shares
this distinctive property of the VAMPs, P was treated with BoNT (see
``Experimental Procedures''). As shown in Fig. 2, this
18-kDa protein, which was detected by the affinity-purified antibodies
specific for VAMP-2, was indeed sensitive to proteolytic cleavage by
BoNT in a dose-dependent manner. At the higher concentrations, BoNT
almost completely degraded this protein. By comparison, similar signals
for -actin were seen in all lanes, consistent with equal
protein loading for each gel lane and a lack of proteolysis of
-actin. In addition, BoNT required the customary proteolytic
activation with reduction in order to be effective. Fig. 2shows
that VAMP digestion occurred only with the toxin exposed to DTT.
Moreover, cleavage was not evident in another control using activated
toxin pretreated with captopril, an antihypertensive drug that inhibits
zinc endopeptidase activity. Similar proteolysis of this protein was
also achieved using tetanus toxin (data not shown).
Figure 2:
Proteolysis of VAMP on the endothelial
cell surface by botulinum B toxin. Aliquots of P (30 µg) were
incubated with buffer alone or containing the indicated amount of
single-chain BoNT that was activated by reduction with DTT in most
cases or inhibited by pretreatment with captopril (see
``Experimental Procedures''). After treatment, the samples
were analyzed as in Fig. 1 by immunoblotting with affinity-purified
antibodies specific for VAMP-2 and a monoclonal antibody against
-actin. The signals detected for VAMP-2 were quantified by
densitometry and are presented graphically for each gel lane.
Together, these
studies provide strong evidence that toxin-sensitive VAMP-2 and
possibly cellubrevin (but clearly not VAMP-1) are expressed on the
endothelial cell surface in vivo almost exclusively within
caveolae. These findings agree well with the known predominant
distribution of VAMP-1 in brain tissue and the more widespread
expression of the other VAMPs in non-neuronal
tissues(36, 37, 38, 39, 40) . At
this time, the data do not rule out the possibility that this caveolar
VAMP protein is a novel member of the VAMP family with similar
molecular weight and toxin sensitivity to VAMP-2. The VAMPs found in
caveolae may function, as they do for other vesicular carriers, in
transport by specifically mediating membrane docking and/or fusion.
Caveolae Are Enriched in NSF and SNAP
Next, we
have tested for other components of the SNARE complex. NSF and SNAP are
known to bind together and mediate their effects on vesicle formation
and/or vesicle fusion through direct interactions with the
SNAREs(26, 28) . Originally identified as a
NEM-sensitive fusion factor by exploiting the ability of NEM to inhibit
vesicular transport, NSF appears to be a trimeric ATPase whose
hydrolysis of ATP may be required for vesicle fusion with target
membranes(44) . Although there is much evidence supporting a
role for NSF in membrane fusion, it has not been proven conclusively.
An alternative role for NSF and SNAP in vesicle formation and budding
has also been identified(45) . Regardless of the specific nature
of its function, it is quite clear in both mammalian and yeast systems (30) that NSF and SNAPs are required components for vesicular
transport.
-SNAP were amply
enriched in V relative to both P and H. Very little of NSF and SNAP
remained in P-V, indicating that almost all (>90%) of both molecules
found bound to the endothelial cell plasma membranes was concentrated
on the caveolae. Confirming our earlier results shown in Fig. 1,
VAMP was also amply enriched in these strips as detected by both the C1
10.1 and VAMP-2 isoform-specific antibodies. The presence of NSF and
-SNAP bound to the caveolae was a bit surprising in light of the
current belief that these important fusion factors are primarily
cytosolic except when associated with the SNARE complex, which forms
after the vesicle docks with its target site via direct binding of
vSNARE to tSNARE(17, 25) . The association of NSF and
SNAPs to purified vesicles was noted previously, and they could be
dissociated easily by high salt
concentrations(25, 26, 45) . Such binding was
especially evident in preparations where NSF appears to function in
vesicle formation(45) . To be certain that we were not
identifying a novel membrane form, we incubated purified caveolae in 1 M NaCl and observed salt- and time-dependent dissociation of
NSF from caveolae (data not shown).
Figure 3:
Caveolae are rich in VAMPs, NSF, SNAP, and
annexins. Filter strips of gel lanes loaded with proteins from
fractions H, P, V, and P-V were immunoblotted with antibodies specific
for the indicated proteins (see ``Experimental Procedures'').
The band shown in the strips labeled VAMPs was recognized by monoclonal
antibody C1 10.1. 5 µg of protein was loaded onto each gel lane for
SDS-PAGE except for V in the strips probed for VAMP where only 1 µg
was used.
VAMP, NSF, and SNAP were present
constitutively on the surface of the caveolae, which immediately
suggested that these vesicles might be not only in the process of
forming and budding but also ``primed'' for vesicle docking
and fusion once released from the plasma membrane. Under these
conditions, the transport process for caveolae would not be slowed by
the time necessary for the diffusion and binding of cytosolic NSF and
SNAP, as required for other vesicles to complete the formation of the
fusion complex, but only would require docking via binding to tSNARE
for more immediate fusion. This potentially more expeditious process
might provide an explanation for why few caveolae were found free in
the cytoplasm of endothelium(3, 4) . Moreover, the
presence of NSF as part of the caveolar machinery for transport was
consistent with studies documenting the inhibitory effects of NEM on
caveolae-mediated endocytosis and transcytosis(8, 9) .
Defining the precise functional role of these proteins in mediating
different mechanistic aspects of caveolar transport will probably
require the development of new in vitro transport assays.
Caveolae Are Rich in Both Small and Large GTP-binding
Proteins
At least six major groups of GTPases have been
implicated in vesicular trafficking including the heterotrimeric G
proteins and various members of the superfamily of monomeric Ras-like
GTP-binding proteins (for recent review, see Ref. 13). Through
conformational changes dependent on guanine nucleotide exchange and GTP
hydrolysis, GTPases act as molecular switches that control the
rate-limiting step in the formation of protein complexes mediating
various cellular functions. In the process of discrete vesicular
transport, their control of assembly and disassembly of protein
complexes appears to drive the budding, targeting, and/or fusion of
transport vesicles. The great diversity of GTP-binding proteins along
with the restricted localization of individual members within select
intracellular compartments (15) creates a specific mechanism for
regulating vectorial transport with high fidelity.
Small Ras-like GTPases
The role of Ras-like
GTP-binding proteins in vesicular transport has been studied the most
extensively of all of the GTPases. Generally in both mammalian and
yeast systems, specific small GTPases have been localized to specific
organelles and identified with specific functions such as Sar1
regulation of vesicle budding from endoplasmic reticulum, Arf control
of coat assembly and disassembly in the Golgi, and the multiplicity of
Rab family members mediating the targeting and fusion process, each one
specific for a given aspect of the exocytic and endocytic
pathways(13, 14, 15, 16, 17) .
Most recently, they have been implicated in the catalysis of SNARE
complex formation(18) .
-
P]GTP(46) , we found that the
purified caveolae do contain small GTP-binding proteins. As shown in Fig. 4, both P and V had a rather strong signal with two bands at
20 and 27 kDa, suggesting (based on size) potential members of the Arf
and Rab families, respectively. They were not exclusive to the caveolae
but were found also in other cell surface domains as indicated by their
continued presence in P-V. Densitometric scanning of the immunoblots
allowed the signal ratio between P and P-V to be quantified. The
normalized signal in V was 5-7-fold greater than in P-V. At this
time we are attempting to identify which of the more than 50 known
small GTPases are present within caveolae. It has been reported that
Rab5 is present within detergent-resistant membranes isolated from rat
lungs(47) . Unfortunately, the vesicles isolated in this type of
preparation are heterogeneous and do not represent purified caveolae
but also contain various other detergent-insoluble membrane regions,
including distinct noncaveolar microdomains from the cell surface and
the trans-Golgi, both enriched in glycosylphosphatidylinositol-anchored
proteins and glycolipids(31, 48) .
(
)Therefore, it was necessary to test our purified
endothelial caveolae to ascertain whether one of the monomeric GTPases
detected in V was Rab5. Using antibodies specific for Rab5, we found
little, if any, signal in V; it was present in T and P-V (Fig. 4). This result is quite consistent with past work (15) immunolocalizing Rab5 to endosomes and general plasmalemma
(but not caveolae). Because small GTP-binding proteins tend to localize
in specific cellular compartments(15) , it is possible that
novel GTPase(s) may exist specifically for the caveolae.
Figure 4:
Endothelial caveolae contain large and
small GTP-binding proteins. Filter strips with proteins from H, P, V,
and P-V were immunoblotted with peptide-specific antibodies recognizing
Rab5 or the indicated G protein subunits. In the lane labeled P-GTP, the strips were blotted directly
with [
-
P]GTP as in Ref.
46.
Heterotrimeric GTP-binding Proteins
Unlike the
smaller monomeric Ras-like GTPases, the heterotrimeric G proteins
consisting of ,
, and
subunits were thought until
recently to function only as signal transducers for ligand binding to
cell surface receptors. In the last few years, a new important function
for them has been identified in vesicular trafficking including
endoplasmic reticulum-to-Golgi and intra-Golgi transport, exocytosis,
endosome fusion, vesicle budding, endocytosis, and
transcytosis(12, 19, 20, 21) .
and various G
subunits (but not
enriched relative to P). None of them resided exclusively within the
caveolar microdomains of the endothelial plasma membranes, as indicated
by the signal remaining in P-V. The G
form was
partially solubilized by the Triton X-100. As quantified by
densitometric scanning of the immunoblots, a stronger signal for many
of these proteins was seen in V relative to P-V with signal ratios
ranging from 2 to nearly 9. It would appear that caveolae are like
other transport vesicles and are rich in several types of
heterotrimeric G proteins(21) . Establishing the exact function
of these GTP-binding proteins in regulating caveolar transport and/or
signaling awaits further investigation.
Caveolae Contain Annexins II and VI
The annexins
are a widely expressed family of at least 10 distinct proteins that
exhibit interdependent binding to phospholipid bilayers and
Ca (24). They have been implicated in numerous
cellular functions including membrane trafficking, primarily at the
step of vesicle aggregation and fusion, and can strongly influence the
exocytosis of secretory granules in neutrophils, chromaffin cells, and
possibly nerve and plant cells(22, 24) . Annexins also
play a major role in the mechanism of endocytosis by affecting vesicle
formation, membrane aggregation, and even endosomal fusion(23) .
Consistent with their apparent function in membrane budding and fusion,
annexins upon binding lipid bilayers reduce lateral lipid diffusion,
change the fluid phase structure of the membrane, and disrupt local
lipid structure sufficiently to induce transmembrane ion
transport(24) .
The Same Caveolae Can Contain Both VAMP-2 and
Caveolin
In order to be certain that the transport or fusion
machinery proteins such as VAMP are not contained in separate
non-caveolar vesicles or complexes that happen to co-fractionate with
the ``true'' caveolae, we isolated the VAMP-2 containing
vesicles by immunoadsorption and magnetic separation. By Western
blotting, we detected caveolin in vesicles immunoisolated with
anti-VAMP-2 but not nonimmune serum (see Fig. 5). This analysis
revealed that: (i) anti-VAMP-2 serum interacts with the intact
caveolae, (ii) caveolae can be isolated by immunoadsorption with
anti-VAMP-2 but not nonimmune serum, and (iii) these specifically
immunoisolated vesicles are indeed caveolae as indicated by the
presence of caveolin. Furthermore, in a similar experiment, we found
that purified caveolae interact with antibodies to albondin, an
endothelial cell surface protein mediating albumin transcytosis via
caveolae (5) and that these immunoisolated caveolae also contain
caveolin.(
)It would appear that caveolin and the
transport/fusion machinery can all be found in the same caveolae.
Figure 5:
Immunoisolated caveolae contain both
VAMP-2 and caveolin. Dynabeads conjugated with anti-rabbit IgG (100
µl) were incubated for 4-5 h with either anti-VAMP-2 or
nonimmune (NI) rabbit serum and then washed by resuspension
and magnetic separation. A starting material of 2 µg of purified
caveolae (V) was split in half and incubated overnight with these two
sets of beads. The V and any V bound to nonimmune or anti-VAMP-2
Immunobeads after washing were analyzed by Western blotting with mouse
monoclonal antibodies to caveolin.
Conclusions
By utilizing the caveolae that have
been purified to homogeneity directly from luminal endothelial cell
plasmalemma derived from rat lungs, we have demonstrated that
endothelial caveolae represent specific microdomains on the endothelial
cell surface that have their own unique molecular topography consisting
of certain preferentially distributed proteins but not other cell
surface proteins. This ``molecular fingerprint'' provides
critical information disclosing for the first time that endothelial
caveolae are indeed like other vesicular carriers and contain similar
molecular machinery known to function in vesicle formation, docking,
and fusion. These key proteins include family members of the VAMPs,
GTPases, and annexins along with NSF and SNAPs. As vesicles mediating
transport into and across endothelium, caveolae provide a specific
mechanism necessary for facilitating the transport of select
blood-borne nutrients to the underlying tissue cells. The
identification of this molecular machinery in caveolae provides not
only strong direct molecular evidence for a role of caveolae in
transport but also the basis for future systematic investigations into
the functions of these proteins in different aspects of caveolar
transport.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.