©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endothelial Caveolae Have the Molecular Transport Machinery for Vesicle Budding, Docking, and Fusion Including VAMP, NSF, SNAP, Annexins, and GTPases (*)

Jan E. Schnitzer (§) , Jun Liu , Phil Oh

From the (1)Department of Pathology, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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),()suggesting by analogy with other vesicular carrier systems a dependence on membrane fusion and specific NEM-sensitive factors(8) .

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.


EXPERIMENTAL PROCEDURES

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) .


RESULTS AND DISCUSSION

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-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.)

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.

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.

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).


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.

We examined the rat lung fractions for the presence of NSF and SNAP. As shown in Fig. 3, NSF and -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.

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.

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) .

Here, utilizing a standard blotting procedure for identifying these GTPases using [-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) .

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 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) .

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).

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL43278 and HL52766 (to J. E. S.) and was done during the tenure of an Established Investigatorship Award from the American Heart Association and Genentech (to J. E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Harvard Medical School, Research North-Beth Israel, 99 Brookline Ave., Boston, MA 02215. Tel.: 617-667-3577; Fax: 617-667-3591; E-mail: jschnitz@BIH.HARVARD.EDU.

The abbreviations used are: NEM, N-ethylmaleimide; NSF, NEM-sensitive fusion protein; BoNT, botulinum neurotoxin B; TeTx, tetanus toxin; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; P, pellet; H, homogenate; V, vesicle; T, Triton X-100-soluble fraction; P-V, pellet containing resedimented silica-coated membranes stripped of the caveolae; VAMP, vesicle-associated membrane protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; tSNARE, target-associated SNAP receptor; vSNARE, vesicle-associated SNAP receptor.

J. E. Schnitzer, D. McIntosh, A. M. Dvorak, J. Liu, and P. Oh, submitted for publication.

J. E. Schnitzer and P. Oh, manuscript in preparation.


ACKNOWLEDGEMENTS

We are greatly indebted to Drs. R. Jahn, W. Trimble, and S. W. Whiteheart for their generous gifts of antibodies.


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