* Centre for Microscopy and Microanalysis, Department of Physiology and Pharmacology, and Centre for Molecular and
Cellular Biology, University of Queensland, Brisbane, Queensland 4072, Australia; and European Molecular Biology
Laboratory, 69012 Heidelberg, Germany
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Abstract |
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Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32-amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway.
Key words: caveolae; caveolin; Golgi complex; targeting; palmitoylation ![]() |
Introduction |
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CAVEOLAE are a characteristic plasma membrane
feature of many mammalian cell types. Since their
first visualization by the pioneers of electron microscopy (Palade, 1953; Yamada, 1955
), caveolae have
been defined by their characteristic morphology appearing
as bulb-shaped uncoated invaginations of 55-80nm diameter (Severs, 1988
; Anderson, 1993
, 1998
; Parton, 1996
). They
are the major surface feature of many highly differentiated
mammalian cells such as adipocytes, smooth muscle cells,
and endothelial cells. In the latter, caveolae have been
shown to bud from the surface and transport solutes across
the endothelial monolayer (Simionescu and Simionescu,
1991
; Schnitzer et al., 1994
). In fibroblasts caveolae have
been proposed to represent an alternative endocytic pathway (Montesano et al., 1982
; Tran et al., 1987
; Parton et al.,
1994
) and recent evidence suggests caveolae are vehicles
for the entry of certain viruses into animal cells (Anderson et al., 1996
; Stang et al., 1997
; Parton and Lindsay, 1999
).
A significant advance in the caveolae field came with
the cloning and characterization of caveolins (VIP21/caveolin-1, caveolin-2, and caveolin-3 [cav-1, -2, -3])1, major
membrane proteins of caveolae (Glenney and Soppet,
1992; Kurzchalia et al., 1992
; Way and Parton, 1995
;
Scherer et al., 1996
; Tang et al., 1996
). Caveolins are integral membrane components which form a hairpin in
the membrane with both NH2 and COOH termini oriented
towards the cytoplasm (Dupree et al., 1993
; Monier et al., 1995
). Caveolins share a putative 33-amino acid intramembrane domain flanked by charged residues. Recent
evidence suggests that caveolins are structural components involved in caveolae formation (Parton, 1996
),
key regulators of signaling events (Okamoto et al., 1998
)
and involved in cholesterol transport and regulation (Fielding
and Fielding, 1997
). Expression of cav-1 in cells that lack
caveolae causes de novo formation of caveolae (Fra et al., 1995b
; Lipardi et al., 1998
). This property may be dependent on the self-association of caveolin to form oligomers
(Monier et al., 1995
; Sargiacomo et al., 1995
) and interaction with both cholesterol (Murata et al., 1995
) and glycosphingolipids (Fra et al., 1995a
) in detergent-insoluble
glycosphingolipid-enriched surface domains (DIGs; Parton and Simons, 1995
). We have also postulated that this
property has been exploited in the formation of other cell
surface domains such as the T-tubule system of muscle cells (Parton et al., 1997
).
Accumulating evidence in vitro and in vivo suggests a
key role for caveolins in signal transduction. Caveolins
show a functional interaction with the small GTP binding
protein ras (Song et al., 1996), src kinases (Li et al., 1996b
),
trimeric G protein subunits (Li et al., 1995
), and other signaling molecules (reviewed by Okamoto et al., 1998
). It
has been proposed that caveolin oligomers may form a
scaffold upon which signaling molecules are sequestered on the cytoplasmic side of the plasma membrane. Despite
these advances, the relationship of the highly conserved
structure of caveolae to their role in signaling remains unclear and some cell types are apparently able to mediate signaling events without invaginated caveolae or caveolins
(Fra et al., 1994
; Gorodinsky and Harris, 1995
).
The majority of the above studies have concentrated on
the plasma membrane role of caveolins, yet there is considerable evidence for a functional role of caveolin in intracellular compartments. In particular, caveolins have
been implicated in polarized vesicular traffic in epithelia
(Scheiffele et al., 1998) and in cholesterol transport (Smart
et al., 1994
, 1996
; Fielding and Fielding, 1997
). Both of
these functions appear to rely on a dynamic cycling of caveolin through the cell. Antibodies to the NH2 terminus of
cav-1 preferentially label surface caveolae (Dupree et al.,
1993
) but antibodies against the COOH terminus of the
same protein show a staining pattern characteristic of the
Golgi complex (Dupree et al., 1993
). This pattern was interpreted as representing the TGN on the basis of colocalization of caveolin with a viral protein accumulated in the
TGN at 20°C by confocal microscopy. These observations, together with the identification of cav-1 within post TGN
vesicles (Kurzchalia et al., 1992
), suggested that cav-1 constantly cycles between the TGN and the cell surface (Dupree et al., 1993
). Recent work showed that antibodies to
cav-1 specifically inhibit transport from the TGN to the
apical surface (Scheiffele et al., 1998
). These results, together with studies showing a role for cholesterol in apical
transport (Keller and Simons, 1998
), have led to a model in
which caveolin plays a role in the formation or stabilization of lipid domains within the TGN (Simons and Ikonen,
1997
). This model relies on rapid trafficking of caveolin
between the surface and the TGN. Other data provided
evidence for a less conventional cycling pathway for cav-1.
These studies showed that cav-1 redistributes to the lumen
of the ER in response to treatment with cholesterol oxidase in a reversible manner (Smart et al., 1994
). This cycle appears to occur constitutively even without cholesterol
oxidase treatment and can be disrupted by treatment with
microtubule-depolymerizing agents when caveolin accumulates within the endoplasmic reticulum/Golgi intermediate compartment (ERGIC; Conrad et al., 1995
). This
pathway was proposed to play a role in cholesterol transport; caveolin directly binds cholesterol (Murata et al.,
1995
) and caveolin expression increases cholesterol transport from the ER to the plasma membrane (Smart et al.,
1996
) apparently via a cytosolic intermediate (Uittenbogaard et al., 1998
). As cav-1 expression is regulated by
cholesterol (Fielding et al., 1997
; Hailstones et al., 1998
)
through cholesterol-responsive promoter elements (Bist et
al., 1997
) this points to a primary role for caveolin in cholesterol homeostasis.
In view of these apparently conflicting data, we have reassessed and further characterized the intracellular location and routing of caveolin using immunofluorescence and immunoelectron microscopy and a mutational approach. We show that antibodies to the COOH terminus of cav-1 or to the scaffolding domain preferentially label caveolin within the cis-Golgi and not the cell surface. Golgi targeting information is located within the COOH terminus of the caveolin molecule and this domain is sufficient to target a soluble protein to the cytoplasmic face of the Golgi apparatus.
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Materials and Methods |
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Materials
Polyclonal antibodies were raised against a peptide corresponding to a
highly conserved region of cav-3, with a COOH-terminal cysteine for coupling to the carrier (CGFEDVIAEPEGTYSFDE). Antibodies were affinity purified as described previously (Parton et al., 1997). Other domain-specific caveolin anti-peptide antibodies have been described previously
(Dupree et al., 1993
). Anti-chick caveolin (anti-cavFL) was purchased
from Zymed Laboratories. Affinity-purified rabbit antibodies (unconjugated and biotinylated) against p23 (Rojo et al., 1997
) were provided by
Dr. Manuel Rojo (University of Geneva, Switzerland). Antibodies against
the mammalian KDEL-receptor ERD2 (Griffiths et al., 1994
) and giantin
(Linstedt and Hauri, 1993
) were gifts of Dr. Hans-Peter Hauri (Biozentrum, Basel, Switzerland) and Dr. Hans-Dieter Soeling (Goettingen, Germany). Fluorescein-labeled anti-mouse IgG, Cy3-labeled anti-rabbit IgG
and Cy2-labeled streptavidin were from Jackson ImmunoResearch. The
hybridoma used to generate monoclonal mouse anti-HA was provided by
Professor David James (University of Queensland). The rabbit anti-HA
and the cDNA for myc-tagged sialotransferase were both kindly provided
by Dr. Tommy Nilsson (EMBL, Heidelberg, Germany). Nocodazole was
purchased from Sigma Chemical Co. and was kept at
20°C (stock solution 10 mM in DMSO).
Cell Culture
BHK cells were grown and maintained as described previously (Gruenberg et al., 1989). Primary human fibroblasts were a gift of Professor D. James and were maintained in RPMI supplemented with 10% FCS.
C2C12 cells were cultured as described previously (Way and Parton,
1995
).
Recombinant Semliki Forest Virus
Recombinant Semliki Forest Virus (SFV)-cav-1 and SFV-cav-3 were prepared in BHK cells according to an established protocol (Liljestrom and
Garoff, 1991; Olkkonen et al., 1993
). In brief, canine cav-1 and mouse
cav-3 were PCR amplified from the original clones with introduced 5'
BamHI and 3' SmaI restriction sites. After sequencing of both strands, the
cDNAs were cloned into the appropriate sites of pSFV1 (Gibco, Grand
Island, NY). RNA was generated by in vitro transcription from pSFV-cav-1, pSFV-cav-3, and pSFV-Helper1 and electroporated into BHK cells.
Culture supernatant was harvested after 48 h and frozen at
70°C. The
BHK cells used to produce recombinant SFV were harvested and prepared for Western blotting. The expression level of cav-1 was >10-fold
higher than endogenous levels.
For immunofluorescence experiments cells were infected with undiluted recombinant SFV containing culture supernatant for 1 h at 37°C after which the infection medium was diluted 10-fold with normal culture medium and incubated for a further 12 h. The cav-2-SFV construct was kindly provided by Dr. Elina Ikonen (National Public Health Institute, Helsinki, Finland).
Subcellular Fractionation
BHK cell fractions were kindly provided by Professor Jean Gruenberg.
BHK cells were homogenized in isotonic sucrose solution and the membranes of the post-nuclear supernatant were fractionated using a step sucrose gradient to produce a fraction enriched in p23 and ERD2 exactly as
described (Rojo et al., 1997).
Cloning and Expression
A series of NH2-terminal truncation mutants of cav-3 were generated
from the original cDNA clone by PCR. Cav3DIH (residues 16-151),
Cav3NED (residues 33-151), Cav3DGV (residues 54-151), and Cav3KSY
(residues 108-151) were generated using the forward primers DIHfor 5'
CGGGGTACCACCATGGACATTCACTGCAAGGAG 3', NEDfor
5' CGGGGTACCATGAATGAGGACATTGTGAAG 3', DGVfor 5'
CGGGGTACCACCATGGACGGTGTATGGAAGGTG 3' and KSYfor 5' CGGGGTACCACCATGAAGAGCTACCTGATCGAG 3', respectively, and the reverse primer RORrev 5' CCGGAATTCTTAGCCTTCCCTTCGCAGCACCACCTT 3'. Similarly, truncations of cav-1
(residues 135-183) were generated with and without three putative
COOH-terminal palmitoylation site cysteines mutated to alanine. Wild-type (WT) cav-1 cDNA template was used to generate Cav1KSF and cav-1
(Cys-Ala) cDNA template was used to generate Cav1KSFp both using
the forward primer CAV1(KSF)for 5' CGGGGTACCACCATGAAGAGTTTCCTGATTGAGATTCAGTGC 3' and the reverse primer
CAV1HArev 5' ATAAGAATGCGGCCGCCTGTTTCTTTCTGCATGTTGATGCGG 3'. The resulting mutants were cloned into pCB6KXHA (Way and Parton, 1995
), a derivative of the eukaryotic expression
vector pCB6, containing the HA epitope tag (YPYDVPDYA), downstream of an in frame NotI site.
The Cav3KSY region was also amplified with the forward primer, KSYGFPfor 5' CCGGAATTCGAAGAGCTACCTGATCGAG 3', and the reverse primer, CAV3rev 5' TCCCCCCGGGTTAGCCTTCCCTTCGCAGCACC 3', for in frame insertion into the COOH-terminal green fluorescent protein (GFP) fusion vector, pEGFP-C1 (CLONTECH Laboratories).
Further truncations of Cav3KSY were similarly prepared using the following combinations of primers; Cav3IYS (residues 120-151) and Cav3IRT (residues 125-151) used the forward primers, IYSfor 5' GGAATTCCATCTACTCACTGTGTATCCGC 3' and IRTfor 5' GGAATTCCATCCGCACCTTCTGC 3' respectively, with the reverse primer CAV3rev.
Cav-3C, a COOH-terminal truncation mutant of cav-3 (residues 1-107)
was generated using the forward primer, CAV3GFPfor 5' CCGGAATTCAATGATGACCGAAGAGCACACGG 3', and the reverse primer,
CAV
Crev 5' TCCCCCGGGTTAAATGCAGGGCACCACGGC 3'.
Full-length cav-3 was similarly produced using the forward primer,
CAV3GFPfor, and the reverse primer, CAV3rev. The products were then
cloned into pBluescript (Stratagene) and subcloned into pEGFP-C1. The
sequences of all constructs were confirmed by sequencing of both strands
in pBluescript using the T3 and T7 primers.
BHK, FRT, C2C12, and CV-1 cell lines were transiently transfected using Lipofectamine (GIBCO BRL) according to the manufacturer's instructions. In brief, cells were grown to ~50% confluence on coverslips and 10-cm dishes for immunofluorescence and electron microscopy, respectively, or 90% confluence on 10-cm dishes for biochemical analysis. The cells were washed twice with serum-free media before being transfected with a ratio of 1 µg DNA to 5 µl Lipofectamine per 1 ml of Opti-MEM (GIBCO BRL). The transfection mixture was left on the cells for 6 h before being washed with, and changed to, normal growth media lacking antibiotics and incubated for a further 18 h before fixation or harvesting. Nocodazole treatment was at a concentration of 10 µM for 2 h at 37°C before fixation.
Preparation of Crude Fractions
Confluent dishes of cells were washed twice with cold PBS before being
scraped into ice-cold HES homogenization buffer (0.25 M sucrose, 1 mM
EDTA, and 20 mM Hepes, pH 7.4) containing protease inhibitors. Cells
were homogenized by passaging through a 27-G syringe and nuclei and
unbroken cells were removed by centrifugation at 1,000 g for 5 min at 4°C.
The resulting supernatant was then centrifuged at 100,000 g for 30 min at
4°C to separate cytosol (supernatant) from cellular membranes (pellet).
For Western analysis the pellet was extracted with a volume of 1% SDS
HES or 1% Triton X-100 HES equal to the volume of supernatant for 10 min at ambient temperature followed by centrifugation at 10,000 g for 5 min at to remove insoluble material. Similarly, salt extractions were performed on the pellet with volumes of 1 M KCl in 50 mM Tris, pH 8.0, or
0.1 M Na2CO3, pH 11.5, equal to the supernatant. Triton X-114 phase separation was achieved using the method of Bordier (1981), with the exception that membranes (100,000-g pellet) were resuspended in the initial solution.
Western Blotting
Equal volumes of cytosol (100,000-g supernatant) and the detergent extracted microsomal membranes (100,000-g pellet) were boiled in SDS PAGE sample buffer. After electrophoresis proteins were transferred to Immobilon membrane (Millipore Corp.) using a Bio Rad trans blot semidry transfer cell. Membranes were blocked with 5% nonfat dry milk TBS-T (0.15 M NaCl, 0.1% Tween 20, and 20 mM Tris, pH 7.4) followed by incubation in specific antibody diluted in 1% non fat dry milk TBS-T. After washing with TBS-T membranes were incubated with a second antibody diluted in 0.2% BSA TBS-T. Bound antibody was detected using the enhanced chemiluminescence detection system (Amersham Corp.).
Immunofluorescence Microscopy
Cells were grown on glass coverslips and fixed with either methanol
(20°C,
5 min) or 3% paraformaldehyde (PFA, 20°C,
20 min). PFA-fixed cells were permeabilized for 5 min with 0.1% (wt/vol) saponin in
PBS and labeled as described previously (Parton et al., 1997
). For double
labeling with mouse and rabbit antibodies, cells were incubated with the
mixture of primary antibodies for 30 min, washed three times with PBS,
and incubated with a mixture of fluorescein- or Cy3-labeled anti-mouse
IgG and Cy3- or fluorescein-labeled anti-rabbit IgG for 20 min. In some
experiments, cells were double labeled with two rabbit antibodies (p23
and anti-concav). This was perfomed using the following sequence: anti-concav, Cy3 goat anti-rabbit, a blocking irrelevant rabbit antibody (anti-cholera toxin), then biotinylated rabbit anti-p23 followed by streptavidin-Cy2 (Monarch Medical). Control experiments omitting the primary
antibodies showed the specificity of the labeling. Samples were analyzed with a confocal laser scanning microscope (Bio Rad Laboratories) equipped
with an argon and a helium/neon laser for double fluorescence at 488 and
543 nm. Fluorescein/Cy2 and Cy3 signals were recorded sequentially
(emission filters: BP510-525 and LP590) using 63 or 100× plan-APOCHROMAT oil immersion objectives. For overlay, fluorescein and Cy3
images were adjusted to similar output intensities and merged with Adobe
Photoshop 3.0.5 into a composite RGB image using a Power Macintosh
7500/100 computer. Figures were arranged with Microsoft PowerPoint.
Colocalization was quantitated by analysis of confocal images of double-labeled nocodazole-treated cells in which individual puncta were clearly evident (e.g., see Fig. 4). Images were digitally captured and overlaid using the RGB function of Adobe Photoshop. Separate images were adjusted to equivalent intensities. IP Lab Spectrum software was then used to analyze the images for the area occupied by the colocalizing elements as a percentage of the total labeled area. Results are expressed as the mean of five fields ± SEM. Note that this technique allows a comparison of the degree of overlap of different markers but underestimates the actual puncta which are positive for both markers (e.g., cav/p23 showed a 63% colocalization but >90% of puncta were labeled for both markers; see for example Fig. 4).
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Electron Microscopy
Cells were fixed with 8% paraformaldehyde in 100 mM phosphate buffer,
pH 7.35, or with the same fixative containing 0.1% glutaraldehyde for 30 min at RT and then processed for frozen sectioning as described previously (Parton et al., 1997). BSA-gold was internalized as a fluid phase
marker for 10 min or 30 min at 37°C as described previously (Kobayashi
et al., 1998
).
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Results |
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Localization of Expressed Epitope-tagged Cav-1 and Cav-3, and Endogenous Cav-1 in Cultured Cells
We have previously shown that cav-1 is localized to the
Golgi complex and to surface caveolae when expressed in
BHK cells (Dupree et al., 1993). We investigated the use
of a transfection approach to express caveolin mutants to
study caveolin targeting. As caveolin can form homo-oligomers which could potentially influence the distribution
of introduced caveolins, we investigated the use of cav-3 to
study caveolin targeting as BHK cells lack endogenous cav-3. Previous work using GST-fusion proteins of caveolin has shown that cav-1FL or COOH- or NH2-terminal
truncation mutants can oligomerize with full-length cav-1
but not cav-3 (Song et al., 1997
). Also in vivo cav-1 is
sorted away from cav-3 when expressed in differentiating
muscle cells (Parton et al., 1997
).
Epitope-tagged cav-1 and cav-3 were expressed in BHK
cells. As shown in Fig. 1, A and B, cav-1 and cav-3 showed
a similar distribution as judged using antibodies against a
COOH-terminal epitope tag (VSV-G and HA, respectively). The proteins were localized to the cell surface and
to a perinuclear compartment, assumed to represent the
Golgi complex (Dupree et al., 1993; see below). A similar
distribution was observed upon expression of a fusion protein comprising GFP fused to the NH2 terminus of cav-3
(Fig. 1 C). These results suggest that heterologously expressed cav-1 and cav-3 localize to the same compartments
in BHK cells and can be used to study caveolin targeting.
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To further investigate the subcellular localization of caveolin, we examined the distribution of endogenous caveolin using a number of different antibodies and cell lines.
Previous studies differ in their analysis of caveolin distribution; some studies have shown that caveolin is only
present at the cell surface unless cells are subjected to experimental manipulations (Smart et al., 1994), whereas
others have concluded that caveolins exist in Golgi complex-associated and surface pools at steady state (Dupree
et al., 1993
). BHK, Vero, and MDCK cell lines as well as
primary human fibroblasts, which have been extensively
studied by others (Smart et al., 1994
), were labeled with
antibodies to the NH2 terminus of cav-1 (anti-cav1N) and
antibodies to the COOH terminus (anti-cav1C). Similar
labeling patterns were observed in all the cell types studied; surface staining by anti-cav1N (e.g., human fibroblasts, Fig. 1 D) and a striking Golgi-type staining with anti-cav1C antibodies (Fig. 1 E). This suggests that different epitopes are exposed at these two cellular locations.
To investigate this possibility further we studied the exposure of a domain of the molecule which has already
been extensively studied in terms of interacting proteins,
by raising antibodies against the scaffolding domain of the
caveolin family. This domain is conserved between caveolins, thereby acting as a signature motif, and is also conserved in evolution (Tang et al., 1997). Antibodies were
raised in rabbits against a scaffolding domain peptide corresponding to the sequence of cav-3 (see Materials and
Methods) and affinity purified on the corresponding peptide column. By Western blotting the affinity-purified antibody (anti-concav) recognized a doublet of ~21 kD in
BHK membranes (Fig. 2 A), a single band in undifferentiated C2C12 myoblast membranes, and a single <20-kD
band in differentiating C2C12 myotubes (Fig. 2 B). The
signal was completely competed by the specific peptide to
which it was raised (not shown). This suggested that the
antibody (named anti-concav for consensus caveolin) recognized both cav-1 and cav-3, the predominant isoforms
expressed in BHK cells and C2C12 myoblasts, and differentiating C2C12 myotubes, respectively. This was confirmed by blotting of BHK cells overexpressing cav-1 or
cav-3 (Fig. 2 C). By immunofluorescence the antibody was
shown to recognize overexpressed cav-1, cav-2, and cav-3
(Fig. 3, A-C) consistent with the predicted specificity.
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The anti-concav gave a characteristic perinuclear staining on cell lines including BHK, CV-1, and MDCK (Fig. 3,
D-F). Primary human fibroblasts revealed the same pattern of labeling which colocalized with the Golgi marker,
p23 (Fig. 3, G-I). This staining pattern was distinct from
that obtained with antibodies to the NH2 terminus of cav-1,
which as expected gave a surface staining pattern characteristic of caveolae (Fig. 1 D). This result suggested that
the anti-concav antibody was unable to recognize surface caveolin. To test this possibility, we examined the staining
pattern for anti-concav in differentiating C2C12 cells. In
these cells cav-3 gives a characteristic staining pattern representing the surface-connected T-tubule system (Parton
et al., 1997). The cav-3(N) antibody showed the characteristic reticular staining pattern as described previously
(Parton et al., 1997
) but the anti-concav showed no labeling of the T-tubules (results not shown). We then examined whether the strict specificity for intracellular caveolin was retained after overexpression, when interaction with
surface molecules might be expected to be saturated. Even
after high overexpression of cav-3 using the recombinant
SFV expression system, the strict specificity of the concav
antibody for the intracellular caveolin pool was still maintained with no sign of surface staining (compare Fig. 3 J,
anti-cav-3(N) with Fig. 3 K, anti-concav). These experiments, in which antibody concentrations were optimized
to allow only detection of overexpressed caveolin, also
emphasize that the N- and concav antibodies were both
recognizing the same heterologously expressed caveolin.
In summary, the results suggest that the anti-concav and
anti-cavC antibodies specifically recognize Golgi-associated caveolin and that changes in the accessibility or conformation of the epitope on transport of caveolin to the
cell surface may inhibit antibody binding.
Antibodies to the COOH Terminus of Caveolin-1, to Concav, or to Purified Caveolin Localize to the Cis-Golgi Complex
In view of the different models for caveolin cycling, we
sought to pinpoint the domain of the Golgi complex with
which caveolin was associated. For this analysis we used a
monoclonal commercial antibody against purified cav-1
(anti-cav1FL; see Fig. 4 A) together with either a cis-Golgi
marker p23 (Rojo et al., 1997) or a TGN marker (transfected sialotransferase, tST; Rabouille et al., 1995
). We
found that cav-1 immunolabeling colocalized with the
TGN marker tST in control cells as judged by confocal microscopy, consistent with previous studies; (Dupree et al.,
1993
), but the same high degree of colocalization was
found with p23 (not shown). Therefore, we employed the
microtubule-depolymerizing agent, nocodazole, which has
been shown to disrupt the Golgi complex (Kreis, 1990
) and has been used to localize proteins to distinct Golgi
sub-compartments (e.g., Chavrier et al., 1990
; Ullrich et al.,
1996
). Nocodazole treatment caused dispersion of Golgi
markers into discrete puncta (Fig. 4). Double labeling of
nocodazole-treated cells with cis- (p23) and trans-Golgi
(tST) markers showed clear, although incomplete, segregation of the two markers (Fig. 4 B; note that many of the puncta are only labeled for one of the markers). After nocodazole treatment anti-cav1FL showed a relatively low
degree of colocalization with tST (Fig. 4 D) but almost
complete colocalization with p23 (see Fig. 4 C; virtually all
p23 puncta are caveolin-positive). The level of colocalization was also quantitated as the area occupied by the colocalizing elements in the image as a percentage of the total
labeled elements (see Materials and Methods for details). Consistent with the qualitative data, these results showed
a much higher degree of colocalization for caveolin/p23
(63 ± 4%) than for caveolin/tST (46 ± 6%) or p23/tST (41 ± 4%). This suggests caveolin is present in the early Golgi
and is not exclusively present in the TGN.
The subcellular location was further analyzed by immunoelectron microscopy. All the available antibodies gave
low labeling of the Golgi complex on ultrathin frozen sections of intact cells. However, in frozen sections of BHK
cell fractions enriched for p23 and ERD2 (Rojo et al.,
1997), anti-cavC labeling colocalized with p23 on Golgi
cisternae (Fig. 5). The higher labeling in this preparation
presumably reflects greater accessibility of cytosolic epitopes as described for other Golgi proteins (Griffiths et
al., 1994
). Taken together the results show that under
steady state conditions caveolin is detectable within the
cis-Golgi.
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Mutational Analysis of Caveolin Targeting: Targeting of an NH2-terminal Deletion Mutant
We examined whether a specific region of the caveolin
molecule is responsible for the localization of caveolin to
the Golgi complex. To avoid potential problems of association with endogenous caveolins (Song et al., 1997) we
prepared mutants of cav-3 which are not endogenously expressed in BHK cells. A series of cav-3 deletion mutants
with a COOH-terminal HA tag were prepared (see Fig. 6
A) and expressed transiently in BHK cells. As shown in Fig. 6, C-F, the NH2-terminal deletion mutants showed
overlapping but distinct localization patterns with varying
degrees of surface and intracellular labeling. Most strikingly, Cav3DGV, which lacks the NH2-terminal region up to
the scaffolding domain (residues 54-151), showed no hint
of surface labeling and this mutant was chosen for more
detailed analysis. Cav3DGV localized to the Golgi apparatus and to punctate structures throughout the cell. This
was examined in more detail by immunoelectron microscopy using antibodies to the HA tag (Fig. 7). Consistent
with the confocal microscopic analysis, Cav3DGV was shown
to associate with the Golgi complex (Fig. 7) as well as
intracellular vesicular elements (Roy et al., 1999
). The
Golgi labeling colocalized with ERD2 (KDEL-receptor)
showing that the protein was concentrated in the cis-Golgi complex (Fig. 7 B). In a small proportion of highly
expressing cells labeling was also observed within the
endoplasmic reticulum (not shown).
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Targeting of a COOH-terminal Caveolin Domain
These studies suggest that Golgi targeting/retention information resides in the COOH-terminal portion of the
molecule. To identify the relevant domain we expressed a
construct comprising just the putative COOH-terminal
cytoplasmic domain of cav-3 (residues 108-151). This
domain has previously been assigned as cytoplasmically orientated based on amino acid sequence predictions, on
antibody recognition of the COOH-terminal domain in
permeabilized cells (Dupree et al., 1993), and in vitro import experiments (Monier et al., 1995
). The construct
termed Cav3KSY was expressed in BHK cells with an HA
epitope tag. Cav3KSY was targeted specifically to the perinuclear area of the cell (Fig. 6 F). No surface staining was
apparent in contrast to the full-length protein expressed
under the same conditions. In very highly expressing cells
a cytoplasmic staining in addition to the Golgi complex
was apparent (not shown).
The specific localization of this mutant was confirmed by immunoelectron microscopy (Fig. 8). Expressing cells showed Golgi labeling for the expressed protein. No labeling was associated with the plasma membrane or other organelles. Very high expressing cells showed dispersed cytosolic staining throughout the cell in addition to the Golgi staining but unlike Cav3DGV-expressing cells, Cav3KSY expressing cells never showed ER staining. Together with the immunofluorescence results this suggests that the association of this caveolin fragment with the Golgi complex is a saturable process.
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We then localized the Cav3KSY mutant with respect to
the defined Golgi markers p23, tST, and giantin (Linstedt
and Hauri, 1993) at the light and electron microscopic
levels. The Cav3KSY colocalized with all three markers in
untreated cells (Fig. 9, A-F). However, after nocodazole treatment, colocalization with cis markers was more
complete than with the medial or trans Golgi markers (Fig.
10, A-I). This confirms that the localization of Cav3KSY
closely follows that of endogenous cav-1 and suggests
that the Cav3KSY is not mislocalized to an irrelevant domain. We also examined the distribution of Cav3KSY at
the electron microscopic level with respect to defined
markers. Cav3KSY showed a clear colocalization with the
cis-Golgi markers p23 (Fig. 8, B and C) and with ERD2
(not shown). In extracted cells (e.g., Fig. 8 C) labeling
was clearly shown to be associated with the cytoplasmic
face of the membrane.
|
|
The distribution of Cav3KSY with respect to endogenous caveolin was also examined. Cav3KSY showed colocalization, as judged by confocal immunofluorescence microscopy, with antibodies to the COOH terminus of caveolin (Fig. 9, G and H). In contrast, as expected antibodies to the NH2 terminus of caveolin which only label the surface caveolin, showed no colocalization with Cav3KSY (not shown). A consistent observation in the Cav3KSY-expressing cells was a dramatic decrease in the endogenous Golgi caveolin labeling in a subpopulation (30-40%) of expressing cells (Fig. 9, I and J). This decrease appeared unrelated to expression levels and was observed with different labeling sequences, in combination with nocodazole treatment (Fig. 9, K and L) and with the three different antibodies against caveolin which recognize different domains of the molecule. This suggests that the association of Cav3KSY with the Golgi complex either decreases accessibility of caveolin antibodies to the endogenous caveolin or, more likely, decreases the Golgi-associated pool of caveolin.
Although the oligomerization of caveolin has been
shown to be isotype-specific in vitro (Song et al., 1996), the
possibility remained that the Golgi-association of Cav3KSY
could represent association with endogenous Golgi caveolin. To test this in vivo we made use of the epithelial cell
line, FRT, which lacks caveolin and caveolae (Lipardi et al.,
1998
). Cav3KSY expressed in these cells showed the characteristic staining of the Golgi complex (Fig. 11, A and B)
which colocalized with p23 (not shown), strongly suggesting that the association of Cav3KSY is independent of endogenous caveolin. This was further tested by double
transfection of Cav3KSY and wild-type cav-1 or GFP-tagged cav-3. Cav3KSY colocalized with the expressed proteins in the Golgi complex but even after high overexpression of full-length cav-3, Cav3KSY was not recruited to the
cell surface (not shown). To examine whether removal of
the COOH terminus of Cav-3 caused a loss of Golgi localization, Cav-3
C with an NH2 terminal GFP tag was expressed in BHK cells. The protein showed only a reticular
staining pattern consistent with an ER localization (not
shown). Although these results are consistent with a role
for the COOH terminus in association of caveolin with the
Golgi complex they could indicate a general perturbation
of folding/oligomerization leading to lack of transport from the ER. Nevertheless our results show that the
COOH-terminal cytoplasmic domain of caveolin associates in a saturable fashion with the cis-Golgi complex.
|
Cav3KSY Can Target a Heterologous Cytoplasmic Protein to the Golgi Complex
To investigate whether the COOH terminus of cav-3 could target a heterologous protein to the Golgi complex, a fusion protein comprising Cav3KSY fused to the COOH terminus of GFP was prepared. The GFP-Cav3KSY fusion protein was expressed in BHK cells under a CMV promoter. The fusion protein was specifically targeted to the Golgi complex (Fig. 11 C) where it colocalized with p23 (not shown). In the majority of cells cytoplasmic staining was also apparent consistent with saturation of the targeting machinery (Fig. 11 C). In addition, in higher expressing cells GFP-Cav3KSY labeled the periphery of large vesicular structures in the perinuclear area of the cell (Fig. 11 D). These experiments show that the COOH-terminal domain of cav-3 is sufficient to localize a heterologous cytosolic protein to the Golgi complex.
We carried out a preliminary characterization of the regions of the COOH-terminal cytoplasmic domain which were required for Golgi location by expressing COOH-terminal fragments as fusion proteins with GFP. A 32- amino acid segment (Cav3IYS; see Fig. 6 B) fused to GFP showed the characteristic staining pattern of the Golgi complex (Fig. 11 E). In contrast, all cells expressing the Cav3IRT mutant, which lacks the first five amino acids of Cav3IYS (see Fig. 6 B), consistently showed dispersed labeling throughout the cell (Fig. 11 F). The observed differences between the two mutants were independent of expression level. In conclusion we have identified a unique domain of the caveolin molecule which can target a heterologous protein to a specific domain of the Golgi complex.
Caveolin COOH-terminal Constructs Show Tight Binding to Membranes: Putative Role for Palmitoylation in Golgi Association
Finally, we examined the nature of the association of Cav3KSY with the Golgi complex. BHK cells expressing Cav3KSY were harvested, homogenized, and separated by centrifugation into crude cytosol and membrane fractions. Membranes were initially extracted with buffer containing 1% SDS or 1% Triton X-100. Fig. 12 A shows that both these detergents extracted a peptide of ~5 kD recognized by the anti-HA antibody which was not present in mock transfected control cells. Treatment with either 1 M KCl or alkaline 0.1 M Na2CO3 both failed to extract Cav3KSY from the pellet (Fig. 12 B). To confirm the apparent hydrophobicity of Cav3KSY the membranes were treated with Triton X-114 which enables phase separation of amphiphilic from hydrophilic proteins. Fig. 12 C shows that Cav3KSY was detected only in the amphiphilic Triton X-114 phase. We then examined the membrane association of the GFP-Cav3KSY chimera. While expressed WT-GFP was predominantly in the soluble fraction, as predicted for a cytosolic protein, the majority of GFP-Cav3KSY was targeted to the membrane fraction (Fig. 12 D).
|
Cav-1 has been shown to be palmitoylated and so we examined whether this lipid modification might be required
for the Golgi localization of the COOH-terminal caveolin
fragment. A full-length cav-1 construct in which the three
COOH-terminal palmitoylated cysteines have been mutated to alanine (Cav-1Cys-Ala) has already been described and characterized (Dietzen et al., 1995; Monier et al.,
1996
). We used a WT cav-1 construct to generate the cav-1 equivalent of the Cav3KSY (Cav-1KSF) and the Cav-1Cys-Ala cDNA to generate the corresponding Cys-Ala mutant
(Cav-1KSF
p). Each construct incorporated a COOH-terminal HA-tag. The constructs were expressed in BHK
cells and their distribution examined by immunofluorescence. As expected the Cav-1KSF was specifically localized to the Golgi complex consistent with the results
with the cav-3 constructs (Fig. 11 G). In contrast, cells expressing Cav-1KSF
p did not show a characteristic Golgi staining (Fig. 11 H) although both Cav-1KSF and Cav-1KSF
p
were membrane associated as judged biochemically (not
shown). These results suggest a role for palmitoylation
in the specific association of the COOH terminus of caveolin with the Golgi complex.
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Discussion |
---|
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---|
Crucial to understanding the role of caveolins is the determination of the exact cellular distribution and intracellular itinerary of this family of proteins. In the present study we have localized caveolin using different specific antibodies and analyzed the targeting information in the caveolin molecule. In particular, we have shown that in addition to surface caveolae, caveolin is also associated with the cis-Golgi in all cell types studied. However, different epitopes are exposed in these locations. We have then used a mutational approach to examine caveolin cycling and targeting. The NH2 terminus of the protein was shown to be required for caveolae targeting while the COOH terminus contains a Golgi targeting motif which is sufficient to target a heterologous protein to the Golgi.
Caveolin has been shown to play an important role in
signal transduction at the cell surface and many signaling
molecules have been postulated to interact with caveolin
directly. Upon cell transformation caveolin levels decrease
(Koleske et al., 1995) and it has also been shown that caveolin is phosphorylated upon Rous sarcoma virus transformation (Glenney, 1989
; Li et al., 1996b
) or upon stimulation of adipocytes with insulin (Mastick et al., 1995
;
Mastick and Saltiel, 1997
). In view of these functions, primarily assigned to the plasma membrane, it initially appears somewhat surprising that caveolin is cycling between
the cell surface and intracellular compartments. In fact
early studies suggested that caveolin was exclusively a surface protein based on immunofluorescence and pre-embedding immunoelectron microscopy (Rothberg et al., 1992
).
Other studies showed that caveolin is associated with the
Golgi complex but this location was only visualized with
certain antibodies (Dupree et al., 1993
) or with overexpressed protein (Kurzchalia et al., 1992
). This labeling was
assigned to the TGN and exocytic vesicles consistent with a
conventional cycling pathway between the Golgi and the
cell surface. Later work outlined a quite distinct cycling
pathway for the caveolin molecule involving transport of
caveolin to the ER and cis-Golgi. In these studies caveolin
was associated with the plasma membrane in control cells
(human fibroblasts) but redistributed to intracellular compartments only upon treatment with cholesterol oxidase or
nocodazole (Smart et al., 1994
; Conrad et al., 1995
). Subsequent studies have raised the possibility that the primary
function of caveolin in mammalian cells is to regulate cholesterol transport (Fielding and Fielding, 1997
). To understand these processes it is essential that the caveolin distribution and routing is determined and the molecular
machinery defined. This study has started to address these
issues and to provide tools for further analysis.
First, we have shown that in all cell types studied a pool
of caveolin is present in the Golgi complex. However, our
studies demonstrate some of the difficulties associated
with localizing caveolin. Different antibodies clearly recognize different pools of the protein, at least as seen by immunofluorescence. Of particular interest is a new antibody, characterized here, which was raised against the
scaffolding domain of the caveolin molecule. This domain is highly conserved both in evolution (Tang et al.,
1997) and between different mammalian caveolins (Parton, 1996
) and consistent with this we have shown that it
recognizes all three mammalian caveolins. This domain
interacts with a number of signaling molecules (Li et al.,
1996a
) and is also involved in self-association to form
oligomers (Sargiacomo et al., 1995
). Remarkably, despite these protein-protein interactions, the antibody gave a specific signal by immunofluorescence but only recognized
the Golgi form of the protein by this technique. A similar
specificity was seen with antibodies against the COOH terminus. The molecular basis for the selectivity is unclear.
Oligomerization has been shown to occur early in the biosynthetic pathway immediately after cotranslational insertion into the endoplasmic reticulum. We suspect that
higher order complexes of proteins and lipids might restrict
antibody accessibility. In fact, in ultrathin frozen sections,
antibodies against the COOH terminus or against the conserved domain do recognize surface protein suggesting that
epitopes are exposed upon sectioning (results not shown).
This suggests that protein-protein interactions might not
be responsible for blocking accessibility. Whatever the
mechanism, it is apparent that care should be taken in interpreting caveolin localization based on single antibodies.
We were also able to define the domain of the Golgi
with which the caveolin is detectable as the cis-Golgi complex based on immunoelectron microscopic colocalization
with defined markers. This presumably represents a pool
of caveolin which cycles through the entire Golgi and
TGN to the surface as cav-1 has been detected within
Golgi derived exocytic vesicles (Kurzchalia et al., 1992) and is directly implicated in exocytic transport to the apical cell surface of epithelial cells (Scheiffele et al., 1998
).
This implies that caveolin follows an unusual cycling pathway to reach early compartments of the biosynthetic pathway, distinct from that followed by molecules such as furin
and TGN38 (Chapman and Munro, 1994
). In this way the
results are consistent with those showing redistribution of
caveolin in response to cholesterol oxidase or nocodazole
(Smart et al., 1994
; Conrad et al., 1995
). However, our results differ significantly in other ways. Most notably, in all
cell types studied including human fibroblasts as used in
the above studies, caveolin is not only detectable on the
surface but also in the Golgi complex under all conditions tested. The distribution of surface caveolin was unchanged
by nocodazole treatment (not shown). Our subsequent experiments were designed to analyze the Golgi association
of caveolin further and to determine the molecular basis of
this targeting through mutational analysis of the caveolin molecule.
Mutational Analysis of Caveolin Targeting
We chose to analyze caveolin targeting using the muscle-specific caveolin isoform, cav-3, expressed in BHK cells. The first striking observation was that truncation mutants lacking the first 54 amino acids no longer associated with surface caveolae as determined by immunofluorescence and immunoelectron microscopy with (COOH-terminal) epitope-tagged constructs and with the (NH2-terminally) GFP-tagged fusion protein. In contrast, the removal of the first 33 amino acids had no effect on surface localization. This may indicate that residues 33-54 are required for caveolae targeting or retention or at least implies that removal of the first 54 amino acids disrupts this process. Expression of the entire NH2 terminus produced a soluble protein with no detectable association with membranes (results not shown).
We then went on to analyze the domains of the caveolin
molecule required for Golgi association. Removal of the
NH2 terminus had no effect on Golgi localization of the
protein. Therefore, we investigated whether the COOH
terminus contains Golgi targeting information. Surprisingly, the putative COOH-terminal domain associated with
the cis-Golgi even when expressed alone, presumably as a soluble protein. Moreover, this domain was able to target a
heterologous soluble protein, GFP, to the Golgi. It is important to note that GFP was fused to the NH2 terminus of
the construct, in place of the putative intramembrane domain. The domain required for targeting GFP to the Golgi
was narrowed down to a relatively hydrophobic region of
32 amino acids of which the first five amino acids were essential. This region showed no significant homology with
other Golgi associated proteins in amino acid sequence.
This small domain is sufficient for Golgi targeting and although normally part of a membrane protein can apparently function in the context of a soluble protein. This
property may not be so surprising in view of recent reports
that caveolin is not always an integral membrane protein
but can exist in a cytosolic complex with cholesterol and
chaperones (Uittenbogaard et al., 1998). Our morphological studies suggest that association with the Golgi complex
is a saturable process as higher expressing cells showed labeling throughout the cell by both immunofluorescence
(particularly with the GFP-tagged construct) and by immunoelectron microscopy.
What is the molecular basis of the association with the
Golgi complex? Our studies show that the protein does
not associate with endogenous caveolins consistent with in
vitro studies of caveolin oligomer formation (Song et al.,
1997). The caveolin COOH terminus may therefore interact with a specific Golgi component. Our biochemical
studies showed a surprisingly tight association with the
membrane. Detergent treatment, but not high pH sodium carbonate, released the GFP-Cav3KSY fusion protein from
membranes. One possibility is that like cav-1 (Dietzen et al.,
1995
; Monier et al., 1996
), cav-3 is palmitoylated and this
modification is involved in Golgi membrane association. Indeed, we showed that the corresponding domain of cav-1
also associates preferentially with the Golgi complex and
that this specific localization is lost upon modification of
the three COOH-terminal cysteines to alanines. These
three cysteine residues have been shown to be palmitoylated in vivo and to play a role in stabilization of higher order oligomers (Monier et al., 1996
) but not to play a role in
targeting to caveolae (Dietzen et al., 1995
; Monier et al.,
1996
; Parton, R., and T. Kurzchalia, unpublished observation). Cav-1 can be palmitoylated on all three cysteine residues within the COOH terminus (Monier et al., 1996
).
While palmitoylation alone is unable to mediate specific
association with the Golgi, the lipid modification may contribute to the tight association with the Golgi membrane in
combination with protein interactions. It is interesting to
note that a number of other Golgi proteins are palmitoylated such as the glutamic acid decarboxylase isoform, GAD65 (Solimena et al., 1994
; Dirkx et al., 1995
), and
eNOS (Sessa et al., 1995
; Liu et al., 1997
) but palmitoylation
is not required for the Golgi association. Interestingly,
eNOS cycles between surface caveolae and the Golgi complex and shows a functional interaction with caveolin
(Michel et al., 1997
; Feron et al., 1998
). Palmitoylation has
been implicated in the lateral segregation of membrane associated proteins into DIGs (see Introduction) but, unlike
the full-length caveolins, Cav3KSY is detergent soluble (results not shown). As detergent insolubility is acquired in the
late Golgi/TGN (Scheiffele et al., 1998
) this is consistent
with the proposed cis-Golgi location.
As well as protein interactions it is possible that the
Golgi association of Cav3KSY relies on specific lipid interactions. Recently it was demonstrated that the cytoplasmic
oxysterol-binding protein, which associates with the Golgi
complex in response to oxysterols, is targeted specifically
to this compartment through interactions with a phosphatidylinositol polyphosphate plus some other Golgi determinant (Levine and Munro, 1998). The COOH-terminal Golgi targeted fragment of caveolin is relatively
hydrophobic and secondary structure predictions show a
possible helical conformation but it appears unlikely that
this domain can insert into the Golgi membrane. Previous
studies have shown that a fusion protein containing the
COOH terminus of cav-1 does not associate with membranes after synthesis in vitro (Monier et al., 1995
). This
domain of cav-1 is also accessible in vivo as COOH-terminal antibodies specifically recognize the Golgi caveolin in
non-detergent permeabilized cells (Dupree et al., 1993
). It has also been shown to interact in vitro with signaling molecules such as n-NOS and c-src (Venema et al., 1997
).
Whatever the molecular mechanism, our experiments show
a striking specificity of this domain for the Golgi complex
and should provide powerful tools for further analysis of
the molecular basis of Golgi localization. In addition, the
KSY and DGV mutants described here have functional effects on caveolae-mediated events including infection by
Simian Virus 40 and Ras signaling (Roy et al., 1999
). Interestingly, the present study showed that expression of the
COOH-terminal mutant caused a striking loss of detectable caveolin in the Golgi region but only in some cells in
the population. This effect was not correlated with expression levels and so the reason for the apparent cell-cell variation is unclear. One possibility is a difference in caveolin
cycling in cells at different stages of the cell cycle.
The data presented here provide important new insights into the localization and targeting of this important class of proteins which should be taken into account in future models of caveolin function. Mutational dissection of the caveolin molecule appears to be a particularly powerful approach to gain insights into the function and dynamics of caveolin proteins and more detailed mutational studies should provide further clues into the complex and dynamic machinery comprising the caveolae membrane system.
![]() |
Footnotes |
---|
Address correspondence to Dr. R.G. Parton, Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: 61-7-3365-6468. Fax: 61-7-3365-4422. E-mail: r.parton{at}mailbox.uq.oz.au
Received for publication 4 January 1999 and in revised form 26 April 1999.
Dr. Espen Stang's present address is Institute of Pathology, The National
Hospital, Oslo, Norway.
We wish to thank David James and Jean Gruenberg for numerous discussions throughout the course of this work and for critical reading of the manuscript. We would also like to thank Tommy Nilsson for cDNA constructs and epitope-tag antibodies; Jean Gruenberg and Manuel Rojo for providing membrane fractions and antibodies; and Elina Ikonen for providing the recombinant cav-2-Semliki Forest Virus construct. Special thanks are due to Colin Macqueen for his help with the light microscopy.
This work was supported by grants from the National Health and Medical Research Council of Australia. The Centre for Molecular and Cellular Biology is a Special Research Centre of the Australian Research Council.
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Abbreviations used in this paper |
---|
cav-1, -2, -3, caveolin-1, -2, -3; GFP, green fluorescent protein; SFV, Semliki Forest Virus; WT, wild-type.
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