From the Centro de Biología Molecular
"Severo Ochoa," Universidad Autónoma de Madrid,
¶ Departament of Immunology and Oncology, Centro Nacional de
Biotecnología, Consejo Superior de Investigaciones
Científicas, Cantoblanco, 28049 Madrid, Spain, the
Departamento de Biología Cellular, Facultad de Medicina,
Universidad de Murcia, 30071 Murcia, Spain, and the
Departamento de Endocrinología,
Hospital de la Princesa, 28006 Madrid, Spain
Received for publication, October 25, 2000, and in revised form, February 28, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The MAL proteolipid, an integral
protein present in glycolipid- and cholesterol-enriched membrane (GEM)
rafts, is an element of the machinery necessary for apical sorting in
polarized epithelial Madin-Darby canine kidney cells. MAL was the first
member identified of an extended family of proteins that have
significant overall sequence identity. In this study we have used a
newly generated monoclonal antibody to investigate an unedited member
of this family, named BENE, which was found to be expressed in
endothelial-like ECV304 cells and normal human endothelium. Human BENE
was characterized as a proteolipid protein predominantly present in GEM
rafts in ECV304 cells. Coimmunoprecipitation experiments revealed that BENE interacted with caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the
cell surface. After cessation of cholesterol oxidation, a detectable
fraction of the BENE molecules migrated to the plasmalemma accompanying
caveolin-1 and then returned progressively to its steady state
distribution. Together, these features highlight the BENE proteolipid
as being an element of the machinery for raft-mediated trafficking in
endothelial cells.
The compartmentation of cellular membranes in microdomains or
rafts is an emerging concept in cell biology (1). Unlike the bulk of
membranes, which are enriched in phospholipids and packed in a
disordered state, rafts have a high glycosphingolipid and cholesterol
content and appear to be packed in a liquid-ordered structure (2). This
difference makes glycolipid- and cholesterol-enriched membrane
(GEM)1 rafts resistant to
solubilization by nonionic detergents at low temperature (2).
Recruitment of specific proteins into rafts was initially proposed to
explain the segregation and transport of apical proteins during
biosynthetic transport in polarized epithelial cells (3). More
recently, this model has been extended as a general mechanism for
protein recruitment in a variety of cellular processes including
membrane trafficking and signaling (1). Although their characteristic
lipid composition provides the biophysical basis for the specificity of
protein recruitment by compatibility with the raft structure, it is
believed that rafts require protein machinery to be operative in
signaling or transport (1, 3).
Caveolae are raft-containing vesicular invaginations of the plasma
membrane involved in a variety of cellular processes including signaling and clathrin-independent endocytosis (4). Caveolin-1 is a
multifunctional raft-associated protein (5) primarily identified as a
component of the caveolar architecture (6). Caveolin-1 is believed to
be an element of the protein machinery operating in rafts, because: 1)
it is able to direct the organization of rafts in caveolae-like
vesicles (7, 8), and 2) it forms a scaffold onto which many classes of
signaling molecules can assemble to generate preassembled signaling
complexes within caveolae (5). The existence of a family of proteins
similar to caveolin-1 with at least two other proteins, termed
caveolin-2 and caveolin-3, which are resident in GEMs, suggests that
members of the caveolin family are elements of the machinery involved
in raft organization (5). The flotillin/cavatellin family, which so far
groups the raft-associated flotillin-1 and flotillin-2/ESA proteins
(9), whose function is still unknown, might constitute a second family of elements of the raft machinery (5).
Proteolipids are operationally defined as proteins with unusually high
solubility in organic solvents commonly used to extract cell lipids
(10). MAL is an integral membrane proteolipid protein of 17 kDa
expressed in a restricted range of cell types including polarized
epithelial cells (11, 12), oligodendrocytes (13), and T lymphocytes
(14, 15). MAL selectively resides in lipid rafts in all the cell types
in which it is expressed (11-14). An essential role for MAL in apical
sorting has recently been demonstrated by the observation that
depletion of endogenous MAL severely reduces the overall transport of
membrane proteins to the apical surface in polarized epithelial
Madin-Darby canine kidney (MDCK) and Fischer rat thyroid cells
(16-18). This highlights MAL as a component of the machinery acting in
the organization of rafts for apical transport. The presence in the
GenBankTM of cDNAs encoding for proteins with
significant overall sequence identity with MAL was indicative of the
existence of a family of proteins related to MAL, henceforth referred
to as the "MAL family" of proteins (19, 20). The demonstrated role
of MAL as an element of the raft machinery in epithelial cells is
consistent with the early proposal that the MAL family of proteolipid
proteins might be involved in raft organization (19).
The observation that GEMs are resistant to solubilization in nonionic
detergents at low temperatures has been widely exploited for the
biochemical isolation of a membrane fraction that appears to be derived
from cellular rafts (21). So far, no member of the MAL family of
proteolipid proteins has been identified in the GEM fraction of
endothelial cells (22). The BENE gene, a member of the MAL
gene family, was originally cloned during a search for genes present in
the vicinity of the human immunoglobulin Materials--
The mouse hybridoma producing mAb 9E10 (IgG1) to
the human c-Myc epitope EQKLISEED was purchased from the American Type
Culture Collection. Rabbit polyclonal antibodies to the c-Myc tag were from Santa Cruz Biotechnologies (Santa Cruz, CA). Mouse mAb MEM-43 (IgG2a) to CD59 was kindly provided by Dr. V. Horejsi (Institute of
Molecular Genetics, Prague, Czech Republic). The rabbit polyclonal antibody to caveolin-1, and the mouse mAbs to caveolin-1, caveolin-2, and calnexin, were from Transduction Laboratories (Nottingham, United
Kingdom). The anti-human MAL 6D9 mAb has been described previously
(12). Peroxidase-conjugated secondary anti-IgG antibodies were supplied
by Pierce. Fluorescein- and Texas Red-conjugated secondary antibodies were from Southern Biotech (Birmingham, AL). Protein A-gold conjugates were obtained from the Department of Cell
Biology of Utrecht University (Utrecht, The Netherlands). CO was
purchased from Roche Diagnostics (Mannheim, Germany).
Cell Culture Conditions, DNA Constructs, and
Transfections--
Human ECV304 cells (kindly provided by Dr. J. Riese, Centro Nacional de Biotecnología, Madrid) and epithelial
MDCK cells were grown on Petri dishes in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum (Life
Technologies, Inc.), penicillin (50 unit/ml), and streptomycin (50 µg/ml), at 37 °C in an atmosphere of 5% CO2/95% air.
An incomplete human BENE cDNA clone (a kind gift from Dr. H. G. Zachau, Institute of Physiological Chemistry, Munich, Germany) lacking the 5' end of the coding region was amplified by the polymerase chain reaction with specific oligonucleotide primers that anneal with
the 5' and 3' ends of the BENE coding region contained in the template
plasmid (23). In addition to the annealing sequence, the 5' primer
contained sequences required to reconstitute the entire coding region
of the BENE cDNA in accordance with the additional ATG-containing
5'-upstream BENE sequence found in the EBI/GenBankTM data base
(accession number D83824). To insert the 9E10 c-Myc epitope at the
NH2 terminus of BENE, the reconstituted BENE cDNA was amplified with the same 3' primer and a new 5' end primer with
sequences encoding the 9E10 c-Myc epitope placed between the first and
second codons of the BENE cDNA coding region. After amplification
under standard conditions, the product was cloned into the pCR3.1 DNA
eukaryotic expression vector (Invitrogen, Groningen, The Netherlands)
to generate the pCR/BENE construct.
Transfection of ECV304 cells with pCR/BENE was carried out by
electroporation using the Electro Cell Manipulator 600 equipment (BTX,
San Diego, CA). Selection of stable transfectants was carried out by
treatment with 0.5 mg/ml G418 sulfate (Life Technologies, Inc.) for at
least 4 weeks following transfection. Drug-resistant cells were
selected, screened by immunofluorescence analysis with 9E10 mAb, and
the clones that proved to be positive for tagged BENE expression were
maintained in drug-free medium. After several passages in this medium
>90% of cells within the selected positive clones retained expression
of tagged BENE. The MDCK cell stable transfectants expressing tagged
BENE used for the hybridoma screening were generated following an
identical procedure.
Preparation of Monoclonal Antibodies to Human BENE--
The
peptide EKLLDPRIYYI, corresponding to amino acids 118-128 of the human
BENE molecule, was synthesized in an automated multiple peptide
synthesizer (AMS 422, Abimed, Langerfeld, Germany) using the solid
phase procedure and standard Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry (26). After
coupling to keyhole limpet hemocyanin, the peptide was used to immunize
Wistar rats. Spleen cells from immunized rats were fused to myeloma
cells following standard protocols (27) and plated onto microtiter
plates. The culture supernatants were screened by immunoblot analysis
using BENE-enriched membrane fractions prepared from epithelial MDCK
cells that stably expressed the BENE protein tagged with the 9E10 c-Myc
epitope. The hybridoma clone 5B1, which secretes antibodies to human
BENE, was isolated after several rounds of screening and used to
produce culture supernatants containing 5B1 mAb.
Northern Blot Analysis--
Total RNA from different cell lines
was extracted using the Ultraspec RNA isolation system (Biotecx
Laboratories, Houston, TX). For Northern blot analysis of different
cell lines, ~20 µg of RNA were denatured in 50% formamide and 2.2 M formaldehyde at 65 °C, subjected to electrophoresis in
a 1% agarose/formaldehyde gel, and transferred to Nylon membranes. RNA
samples were hybridized under standard conditions to cDNA fragments
labeled by the random-priming method (28) corresponding to human BENE
(23). As a control of the amounts of RNA present in each lane, blots
were finally hybridized to a 0.6-kilobase pair
HinfI/BamHI DNA fragment from the 3'-untranslated
region of human Detergent Extraction Procedures--
GEMs were prepared
essentially as described by Brown and Rose (21). ECV304 cells grown to
confluence in 100-mm dishes were rinsed with phosphate-buffered saline
and lysed for 20 min in 1 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 at
4 °C. The lysate was scraped from the dishes with a cell lifter, the
dishes were rinsed with 1 ml of the same buffer at 4 °C, and the
lysate was homogenized by passing the sample through a 22-gauge needle.
The lysate was finally brought to 40% sucrose (w/w) in a final volume
of 4 ml and placed at the bottom of an 8-ml 5-30% linear sucrose
gradient. Gradients were centrifuged for 18 h at 39,000 rpm at
4 °C in a Beckman SW41 rotor. Fractions of 1 ml were harvested from
the bottom of the tube, and aliquots were subjected to immunoblot
analysis. Density was determined by measuring the refractive index of
the fractions. In some experiments, centrifugation to equilibrium was
carried out using discontinuous sucrose density gradients consisting of
a 4-ml bottom layer containing the cell lysate in 40% sucrose,
overlaid with 6 ml of 30% sucrose and a 2-ml layer of 5% sucrose at
the top. After centrifugation, the opalescent band containing GEMs,
which migrates in the 5-30% sucrose interphase, was harvested from
the top (fraction I). The 40% sucrose layer containing the cytosolic
proteins and the solubilized proteins was also harvested (fraction S).
Immunoblot and Immunoprecipitation Analyses--
For immunoblot
analysis, samples were subjected to SDS-PAGE in 15% acrylamide gels
under reducing conditions and transferred to Immobilon-P membranes
(Millipore, Bedford, MA). After blocking with 5% nonfat dry milk,
0.05% Tween 20 in phosphate-buffered saline, blots were incubated with
the indicated primary antibody. After several washings, blots were
incubated for 1 h with secondary goat anti-IgG antibodies coupled
to horseradish peroxidase, washed extensively, and developed using an
enhanced chemiluminescence Western blotting kit (ECL, Amersham
Pharmacia Biotech). For immunoprecipitation studies, cells were
incubated for 4 h at 4 °C with a control antibody bound to
protein G-Sepharose, centrifuged and the supernatant immunoprecipitated
by incubation for 4 h at 4 °C with the indicated specific
antibodies bound to protein G-Sepharose. Immunoprecipitates were washed
six times with 1 ml of 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Triton X-100 and analyzed by SDS-PAGE under
reducing conditions. To detect 35S labeling, dried gels
were finally exposed to Fujifilm imaging plates.
Immunofluorescence Analysis--
ECV304 cells grown on
coverslips were fixed in 4% paraformaldehyde for 15 min, rinsed, and
treated with 10 mM glycine for 5 min to quench the aldehyde
groups. The cells were then permeabilized with 0.2% Triton X-100,
rinsed, and incubated with 3% bovine serum albumin in
phosphate-buffered saline for 15 min. For double-label immunofluorescence analysis, cells were incubated for 1 h with mAb
9E10 (IgG1), rinsed several times, and incubated for 1 h with anti-mouse Ig Quantitative Immunoelectron Microscopy--
Stable transfectants
of ECV304 cells expressing tagged BENE were used. The cells were
processed for cryosectioning as described previously (30).
Briefly, the cells were fixed overnight with 4% paraformaldehyde in
0.1 M phosphate buffer, pelleted by centrifugation, embedded in 10% gelatin, and cut into small blocks. The blocks were
infused with 2.3 M sucrose, frozen in liquid nitrogen, and stored until cryoultramicrotomy. Cryosections were immunolabeled as
described by Slot et al. (31). The distribution of BENE was determined with anti-tag mAb 9E10 and that of caveolin-1 with specific
rabbit polyclonal antibodies diluted 1:15 and 1:50, respectively. Nonspecific labeling was measured over the nucleus as described by
Griffiths (32) and was considered not significant (lower than 1 gold
particle per µm2). The substitution of anti-tag 9E10 mAb
by rabbit polyclonal antibodies to the same tag gave a similar
immunolabeling pattern, whereas omission of the primary antibody
abolished the labeling. The quantitative analyses were carried out in
grids double-immunolabeled for tagged BENE and caveolin-1. The sections
with the highest quality were selected and then were scanned
systematically along a fixed track using a magnification of × 20,000. Ultrathin sections were scanned along a fixed track.
Tubular/vesicular elements immunoreactive for BENE were ascribed to one
of the following categories: clathrin-coated membranes (the presence of
a clathrin coat was established on the basis of the typical thickness
and appearance of the coats in cryosections (30, 33), caveolae
(membranes immunoreactive for caveolin-1), and uncoated membranes (the
remaining immunoreactive tubular/ vesicular elements not included in
the former categories). The number of structures found for each
category was expressed as a percentage of the total vesicles counted.
At least 300 vesicles were counted in three independent sessions.
Immunohistochemical Analysis--
Human tonsils were received as
routine specimens obtained from surgery. Samples were fixed for several
hours in 10% neutral buffered formalin and subjected to routine tissue
processing and paraffin embedding. Sections of 5-µm thickness were
prepared from paraffin-embedded tissues and were mounted on
poly-L-lysine-coated glass microslides. Antigen retrieval
was accomplished by subjecting deparaffinized sections to pressure
cooker unmasking for 60 s in 200 mM citrate buffer, pH
6.0. The tissue was then blocked with a 1:20 dilution of normal rabbit
serum in 10 mM Tris-HCl saline buffer, pH 7.6, as described
previously (34). The sections were sequentially incubated with a 1:100
dilution of an ascites stock of anti-BENE 5B1 mAb and
peroxidase-conjugated rabbit anti-rat IgG (Bio-Rad). Each incubation
was followed by three washes with Tris-buffered saline. Then, sections
were developed with Graham-Karnovsky medium containing 0.5 mg/ml of
3,3'-diaminobenzidine tetrahydrochloride (Sigma) and hydrogen peroxide.
Sections were counterstained with Carazzi's hematoxylin, dehydrated,
and mounted by routine methods.
Expression of the BENE Gene in Different Cell
Lines--
A partial BENE cDNA was identified during a search for
genes in the proximity of the human immunoglobulin Generation and Characterization of a Monoclonal Antibody to the
BENE Protein--
The peptide EKLLDPRIYYI, comprising amino acids
118-128 of human BENE, was synthesized (sequence underlined
in Fig. 1), coupled to keyhole limpet hemocyanin, and used to immunize
Wistar rats. The selected peptide is located in the BENE molecule in a
position equivalent to that of the MAL peptide previously used to
generate anti-MAL antibodies (12, 16), which in MAL corresponds to an
extracellular (luminal) loop (36). A hybridoma clone (named 5B1)
producing antibodies to BENE was identified by immunoblot analysis of
membrane fractions enriched in tagged BENE obtained from transfected
MDCK cells. The 5B1 mAb specifically identified a protein band of the
predicted size in COS-7 cells transiently expressing tagged BENE but
not in untransfected cells (Fig.
3A). The BENE peptide used for
the immunizations, at concentrations of 500 ng/ml, was able to totally
neutralize the recognition of BENE by mAb 5B1, whereas other control
peptides did not present any effect (not shown). The observed effect
was specific for the 5B1 mAb, since the same peptide did not influence
the recognition of tagged BENE by anti c-Myc 9E10 mAb (Fig.
3B). As a further test of the specificity of the 5B1 mAb,
Fig. 3C shows that the antibody recognized endogenous BENE
in ECV304 and A498 cells, which were positive for BENE mRNA
expression, but not in the Jurkat T cell line (Fig. 3C) or
HepG-2 cells (not shown), which lack BENE mRNA (Fig. 2). The
endogenous BENE protein migrated with the same electrophoretic mobility
as the endogenous MAL protein from Jurkat cells, as would have been
expected given that the length of the two proteins was calculated to be
equal. In addition, Fig. 3C shows that ECV304 cells lack
detectable expression of MAL, as would be expected given the absence of
MAL transcripts in this cell
line.2 Although ECV304 cells
display some endothelial features (24), the endothelial nature of this
cell line has been questioned recently (37). To examine whether BENE is
expressed in normal endothelia, human tonsil sections were subjected to
immunohistochemical analysis with anti-BENE mAb 5B1. As shown in Fig.
3D, in agreement with the presence of BENE in the
endothelial-like ECV304 cell line, BENE staining was detected in the
endothelial layer lining the blood vessels.
Endogenous BENE Is a Proteolipid Protein Present in GEM
Microdomains in Endothelial ECV304 Cells--
The GEM fraction, which
is resistant to solubilization by nonionic detergent at low
temperatures, was separated from the bulk of cellular membranes, which
are solubilized by the detergent, and from cytosolic proteins by using
an established protocol involving centrifugation to equilibrium on
sucrose density gradients (21). After fractionation from the top of the
gradient, aliquots from each fraction were subsequently separated by
SDS-PAGE and immunoblotted with mAb 5B1. Fig.
4A shows that endogenous BENE
was found selectively in the GEM fraction of ECV304 cells. As controls
we observed that the same fraction contained caveolin-1 and caveolin-2,
two proteins already described in GEMs, but did not contain calnexin, a
transmembrane protein present in the endoplasmic reticulum. To
investigate whether BENE displays lipid-like properties, as is the case
with MAL, the GEM fraction from ECV304 cells was extracted with
n-butyl alcohol, and after phase separation, the
resulting aqueous and organic phases were analyzed by immunoblot with
anti-BENE 5B1 mAb. Fig. 4B shows that BENE partitioned
almost exclusively in the organic phase, indicating that BENE is indeed
a proteolipid protein. BENE was also detected in the proteolipid
fraction obtained from human vein umbilical cells (Fig. 4B),
in agreement with the results of the immunohistochemical analysis (Fig.
3D). To study the ability of BENE to be targeted to GEMs in
greater detail, we prepared ECV304 cell transfectants stably expressing
the BENE protein tagged at the NH2 terminus with the c-Myc
9E10 epitope (ECV304/BENE cells). Fig. 4C shows that
exogenous BENE was correctly incorporated into GEMs in ECV304 cells in
agreement with our previous results obtained by expression of an
incomplete BENE protein in COS-7 cells (19). The relative level of the
exogenous protein was estimated to be approximately one-half of that of
endogenous BENE by immunoblotting with anti-BENE mAb 5B1 of a pool of
the insoluble membrane fractions from ECV304/BENE cells (Fig.
4D).
BENE Is Associated with Caveolin-1 and -2 in ECV304
Cells--
Lipid rafts, such as those containing caveolae or
GPI-anchored proteins, coalesce after detergent extraction, making
impossible the distinction between different types of rafts (2). The
presence of BENE in GEM raft fractions led us to carry out a
comparative immunofluorescence analysis of the distribution of
caveolin-1, the GPI-anchored CD59 molecule, and BENE in ECV304 cells.
As the anti-BENE 5B1 mAb is only of use for immunoblotting, we used
anti-tag antibodies and ECV304/BENE cells for immunolocalization
studies. Fig. 5 shows that the rafts
containing BENE were mostly intracellular and clearly different from
those of surface caveolae and CD59. Intracellular caveolin-1 and CD59
colocalized with BENE in structures in the Golgi region, as revealed
with antibodies to the Golgi mannosidase II marker. This result
indicated a possible functional and/or biochemical interaction between
caveolin-1 and BENE in internal structures. The amino acid sequence of
BENE contains three motifs that fit with consensus sequences implicated
in interaction with caveolin-1 (Fig. 1) (38). To address the possible
interaction of caveolin-1 with BENE, ECV304 cells were extracted with
1% Triton X-100 and 60 mM octyl glucoside, a procedure
used to solubilize GEMs (21). After centrifugation, the supernatant
containing solubilized GEMs was immunoprecipitated with either control
or anti-caveolin-1 antibodies, and the immunoprecipitates were
subjected to immunoblot analysis with anti-BENE 5B1 mAb. The left
panel in Fig. 6A shows
that endogenous BENE was specifically detected in the caveolin-1
immunoprecipitate, indicating an interaction between the two proteins.
This association was also observed in A498 cells, which express higher
levels of caveolin-1 (Fig. 6A, left panel). The
association of caveolin-1 with BENE was corroborated by analyzing the
association of caveolin-1 with exogenous BENE using ECV304/BENE cells
and anti-tag antibodies (Fig. 6A, middle panel).
As the 5B1 mAb is not of use for immunoprecipitation studies, to carry
out the reciprocal experiment we used anti-tag antibodies to
immunoprecipitate exogenous BENE from extracts obtained from transfected ECV304/BENE cells. Fig. 6B shows that, in
addition to tagged BENE, the anti-tag antibodies immunoprecipitate two protein bands from metabolically labeled ECV304/BENE cells. These are
~22-24 kDa, which corresponds to the size of caveolins. The presence
of caveolin-1 in the BENE immunoprecipitate was confirmed by
immunoblotting with anti-caveolin-1 antibodies (Fig. 6C).
Caveolin-1 is known to interact with caveolin-2 to form heterooligomers
(39), raising the possibility that caveolin-2 was in the same complex. The presence of caveolin-2 in the BENE immunoprecipitates was demonstrated by immunoblotting with anti-caveolin-2 antibodies (Fig.
6C). Fig. 6D shows an example of the efficiency
of the solubilization procedure used to prepare the extracts for the
immunoprecipitation experiments.
Distribution of BENE in ECV304 Cells--
To investigate the
distribution of BENE we carried out immunoelectron microscopy on
ultrathin cryosections with anti-tag antibodies using ECV304 cells
stably expressing tagged BENE. BENE was localized in small
tubular/vesicular elements scattered throughout the cell (Fig.
7). These immunoreactive profiles were
also observed in the Golgi region (Fig.
8a and b).
Occasionally, Golgi cisterna and buds were also labeled (Fig.
8b). No labeling was detected in other cytoplasmic
organelles or the plasma membrane (Fig. 8c). The membranes
containing BENE appeared on an ultrathin section as 50-70 nm vesicular
profiles or as short nonbranching tubules (Fig. 8, c and
d, inset). These membranes were occasionally
covered by a characteristic 18-nm-thick coat (Fig. 7,
inset), which has been unambiguously identified in previous
studies as being made of clathrin (30, 33). To investigate the
relationship between BENE and caveolin-1, we carried out a comparative
analysis of the distribution of these proteins. Membranes immunolabeled
for BENE were screened for the presence of caveolin-1 or a clathrin coat using the procedure described under "Experimental Procedures." The quantitative analysis showed that 8 ± 2% of BENE colocalized with caveolin-1 in the same uncoated membranes (Fig. 8, c
and d), in agreement with our results showing a physical
interaction between these proteins. Quantitative analysis indicated
that most of the remaining BENE molecules are associated with
cytoplasmic tubular/vesicular structures that lack a discernible coat
(88 ± 3% of the total reactive vesicles) and a small fraction is
associated with similar structures displaying a typical clathrin coat
(4 ± 2% of the total labeling). The caveolin-1 associated with
the structures containing BENE represented ~10% of the total
caveolin-1.
Effect of Cholesterol Oxidation on the Distribution of
BENE--
There is a rapid redistribution of surface caveolin-1 to the
Golgi region in response to surface cholesterol oxidation by extracellular CO (25). As both BENE and caveolin-1 reside in insoluble
lipid rafts, we employed confocal immunofluorescence to establish
whether BENE distribution is also sensitive to cholesterol oxidation
using ECV304/BENE cells and anti-tag antibodies. Optical sections were
taken at 0.4-µm intervals along the z axis of the cells at
different times of CO treatment using an optimum pinhole. For
simplicity only two sections are shown illustrating either the
perinuclear region and the plane of the plasma membrane just underneath
the nucleus (a) or the periphery of the cell, also including part of
the perinuclear region (b). Fig. 9 shows
that at steady-state (untreated cells) BENE was mostly found in small discrete structures in the perinuclear region (a) with
little labeling at the cell periphery (b). Although most of
the caveolin-1 labeling was on the cell surface, caveolin-1 was also
found in the perinuclear region as described previously (40). A
significant fraction of the internal caveolin-1 colocalized with BENE,
consistent with our electron microscopic results. In response to
cholesterol oxidation, the distribution of BENE progressively switches
from its steady-state distribution in a large number of small vesicular profiles to become concentrated into a reduced number of structures with a dilated appearance. After 30 min of CO treatment, this redistribution of BENE was already detectable (a), the
typical accumulation of surface caveolin-1 at the leading edge had been lost (a), and caveolin-1 had begun to be internalized
(a and b). After 60 min of CO treatment, most of
the BENE accumulated into dilated structures, and caveolin-1 had been
fully internalized. It is of particular note that, under these
conditions, internalized caveolin-1 was totally concentrated in the
dilated structures stained for BENE.
It has been established that after cessation of cholesterol oxidation,
caveolin-1 returns to the cell surface, probably to replenish the
plasma membrane with fresh cholesterol (25). Fig. 10 shows that 10 min after CO
withdrawal BENE already no longer appeared in the dilated structures
observed in CO-treated cells but rather reacquired a discrete vesicular
pattern. Simultaneously, most of the caveolin-1 originally in the Golgi
area in CO-treated cells returned to the cell surface (a and
b). This movement of caveolin-1 to the plasmalemma was
accompanied by the translocation of a fraction of the BENE to the cell
periphery (see b panels). The translocated BENE molecules
were mostly located underneath surface caveolin-1 (see inset
in the merge profile), although a low level of colocalization between
both proteins was also detected. After 20 min of CO removal the
translocation of BENE was much more pronounced, and BENE was clearly
detected in close proximity to surface caveolin-1. Finally, 1 h
after CO removal BENE and caveolin-1 distributions were both as at
steady state.
BENE Is a Proteolipid Protein with Selective Residence in Rafts in
Endothelial-like ECV304 Cells--
Previous work aimed at the
systematic identification of protein components of GEMs from
endothelial cells found this membrane fraction to be highly enriched in
caveolin-1 and to contain GPI-anchored proteins, scavenger receptors
for modified forms of low density lipoprotein (CD36 and RAGE), a large
number of signaling molecules, and cytoskeletal elements (22). Although
the presence of MAL and other proteolipid proteins with an apparent
size in the range of 14-20 kDa has been described in GEMs from other
cell types (11, 12), no proteolipid protein of the MAL family has so far been reported as being present in the GEM fraction of endothelial cells. The BENE protein was assigned to the MAL family on the basis of
its significant amino acid sequence identity (39%) with MAL and the
similar hydrophobicity profiles (19, 20, 23). The generation of a mAb
specific to BENE has allowed the detection of endogenous BENE in normal
human endothelial cells and its identification as a 17-kDa proteolipid
protein with selective residence in the GEM fraction of the
endothelial-like ECV304 cell line. Thus, endogenous BENE is the second
member of the MAL family of proteolipid proteins to be identified in
GEMs so far.
BENE Associates with Caveolin-1 and -2--
The distribution of
the rafts containing BENE was clearly different from the surface rafts
containing caveolin-1 or GPI-anchored proteins. Caveolin-1 is a
multifunctional protein that interacts with a wide variety of proteins
through the so-called "scaffolding domain," a 20-amino acid
sequence proximal to the putative membrane insertion sequence (5). In
MDCK cells, caveolin-1 is present as homooligomers and as
heterooligomer complexes with caveolin-2 (39). The fact that BENE
contains three different regions that fit consensus sequences known to
interact with the scaffolding domain of caveolin-1, and the partial
colocalization of BENE with caveolin-1 in internal rafts, led us to
investigate the possible interaction between these proteins. Using
fully solubilized extracts, we found that BENE associates with both
caveolin-1 and -2 as demonstrated by coimmunoprecitation experiments,
whereas in agreement with our previous results (41), no association of
MAL with caveolin-1 was found in parallel experiments in MDCK cells
under the same stringent conditions of solubilization (results not
shown). Thus, interaction with caveolins appears to be a specific
feature of BENE not shared by all members of the MAL proteolipid
family. At the electron microscopic level, BENE was localized in
tubular/vesicular structures scattered throughout the cytoplasm and in
the Golgi region. Approximately 8% of these structures were also
positive for caveolin-1 as revealed by quantitative analysis. This
indicates that BENE and caveolin-1 might cooperate in raft-mediated
processes in endothelial cells. In addition to the structures
containing caveolin-1, BENE was also identified in uncoated and
clathrin-coated cytoplasmic tubular/vesicular elements lacking
caveolin-1. This indicates that, in addition to cooperate with
caveolin-1, BENE might also be involved in caveolin-1-independent
functions mediated by lipid rafts. The distribution of BENE suggests
that, similarly to MAL, which cycles between the cell surface,
endosomes and the trans-Golgi network (36), BENE also moves
between different intracellular compartments.
BENE, Caveolin-1, and Cholesterol Trafficking--
Caveolin-1
moves from surface caveolae to large intracellular structures in
response to cholesterol oxidation by CO (25). Those structures have
been characterized previously by immunofluorescence and electron
microscopic analyses as a distended Golgi apparatus (25). Upon CO
removal, caveolin leaves the Golgi and returns to the cell surface.
This caveolin-1 cycle appears to be similar to constitutive caveolin-1
cycling, which involves the sequential movement of caveolin-1 from
surface caveolae to the endoplasmic reticulum, the endoplasmic
reticulum-Golgi intermediate compartment, the Golgi, and surface
caveolae (42). A role for caveolin-1 in transport of cholesterol from
the endoplasmic reticulum to the cell surface has been proposed based
on the knowledge: 1) that caveolin-1 binds cholesterol (43, 44), 2)
that ectopic caveolin-1 expression in cells lacking endogenous
caveolin-1 causes a 4-fold increase in the rate of delivery of newly
synthesized cholesterol to the plasma membrane (45), and 3) of the
response of caveolin-1 to cholesterol oxidation (25). The interaction between BENE and caveolin-1 in ECV304 cells and their partial colocalization in the Golgi region led us to examine in parallel the
effect of cholesterol oxidation on the distribution of BENE and
caveolin-1. Our results showed that in response to cholesterol oxidation, BENE was redistributed from its steady-state distribution in
a large number of small discrete vesicles and accumulated in a reduced
number of structures with dilated morphology. The CO-triggered redistribution of caveolin-1 to the Golgi was accompanied by the conversion of caveolin-1 from a cytoplasmically oriented membrane protein at the cell surface to a intraluminal protein present in large
perinuclear vesicles (25). This change in distribution is paralleled by
the exit of caveolin-1 from lipid rafts, as evidenced by its loss of
insolubility, and consistently with its luminal location (25).
Interestingly, the insolubility of BENE did not alter after 1 h of
CO treatment.2 This indicates that BENE was still
embedded in membrane rafts in the dilated structures observed after CO
treatment. Moreover, despite the extensive colocalization of caveolin-1
with BENE observed in CO-treated cells, the level of association of
caveolin-1 with BENE relative to that of steady state, as assayed by
coimmunoprecipitation experiments, did not
increase.2 These results are consistent with the
presence of BENE and caveolin-1 in the membrane and lumen,
respectively, of the dilated structures observed in CO-treated cells.
Finally, the migration of a detectable proportion of the BENE molecules
to the plasmalemma accompanying the return of caveolin-1 to the plasma
membrane, as is observed after cessation of cholesterol oxidation,
suggests that BENE might play a role in cholesterol homeostasis and/or
caveolin-1 transport to the cell surface. Thus, in agreement with our
previous proposal (19) and the demonstrated role of MAL in
raft-dependent apical transport (16-18), BENE and possibly
the other members of the MAL family of proteolipids might constitute
elements of the raft machinery for the specialized membrane trafficking
pathways that exist in the different cell types.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain locus (23). BENE
mRNA is expressed in the prostate, small intestine, colon, heart,
and lung and is undetectable in brain, thymus, liver, and spleen (20).
In this study, using a newly developed anti-BENE monoclonal antibody
(mAb) we have identified endogenous BENE in the GEM fraction of ECV304
cells, a human cell line displaying endothelial-like features (24). We
have detected a physical interaction between BENE and caveolin-1 and
observed a partial colocalization between these two proteins in
vesicular/tubular structures in ECV304 cells. Oxidation of surface
cholesterol by cholesterol oxidase (CO) and cessation of that process
by CO withdrawal indicate that BENE participates in
cholesterol-regulated processes also involving caveolin-1 (25) in the
endothelial-like ECV304 cell line.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin mRNA (29). Final blot washing conditions
were 0.5× SSC/0.1% SDS (1× SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at 65 °C.
1 chain coupled to Texas Red. The procedure was then repeated with rabbit polyclonal antibodies to caveolin-1, or mAb
to CD59 or mannosidase II followed by fluorescein-conjugated anti-rabbit IgG antibodies pre-absorbed against mouse IgG or the appropriate isotype-specific secondary antibodies conjugated to fluorescein. After extensive washing, the coverslips were mounted on
slides. As indicated, images were obtained using either a Bio-Rad Radiance 2000 Confocal Laser microscope or a conventional fluorescence microscope (Zeiss). Controls to assess the specificity of the labeling
included incubations with control primary antibodies or omission of the
primary antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain
locus (23). This cDNA clone (EBI/GenBankTM data library
accession number U17077) has an open reading frame of 148 amino acids
showing ~39% identity with the MAL protein sequence but lacks an
in-frame ATG triplet that could be used as a translational initiation
codon. During a search of the TIGR Human Gene Index we
identified a partial cDNA clone that both matched the BENE sequence
and contained an additional 5'-upstream sequence
(EBI/GenBankTM data library accession number D83824). This
additional sequence displays a unique ATG codon in-frame with the BENE
open reading frame mentioned above. The sequence surrounding this ATG
(AGCATGG) is consistent with the
A/GXXATGG consensus sequence (where X
stands for any nucleotide) for optimal ATG translational start sites in
eukaryotic cells (35). The reconstituted open reading frame predicts a
protein of 153 amino acids containing an NH2-terminal five-amino acid extension compared with the incomplete sequence deduced
previously (23). The complete sequence of BENE, with the additional
residues underlined, and its alignment with the MAL protein are shown
in Fig. 1. To identify a suitable model cell system for studying BENE, we carried out Northern blot analysis using a wide range of human cell lines. Fig.
2 shows that, in addition to the prostate
carcinoma PC3 cell line from which the BENE cDNA was originally
cloned (23), the 2.7-kilobase BENE mRNA band was present in
renal epithelial A498 cells, in cervix carcinoma HeLa cells and in the
endothelial-like ECV304 cell line. BENE transcripts were undetectable
in the rest of the cell lines examined including T cells (Jurkat and
HPB-ALL cells), epithelial MDCK cells, and hepatic HepG-2 cells. To
identify BENE gene expression in ECV304 cells unambiguously,
the BENE cDNA coding sequence was amplified by reverse
transcriptase-polymerase chain reaction using total RNA from
ECV304 cells, and the product was cloned and sequenced. The amino acid
sequence predicted from this analysis was identical to that shown in
Fig. 1.
View larger version (19K):
[in a new window]
Fig. 1.
Alignment of the human BENE and MAL protein
sequences. The sequence of BENE was reconstituted by adding the
NH2-terminal sequences encoded by a partial expressed
sequence tag cDNA clone (GenBankTM accession number
D83824) (underlined with a dashed line) to the
predicted open reading frame of the previously reported incomplete BENE
cDNA (GenBankTM accession number U17077). The position
of three different sequences in the BENE protein that fit with
consensus sequences
( -X-
-X-X-X-
;
-X-X-X-X-
-X-X-
,
where
stands for an aromatic amino acid and X for any
amino acid) of interaction with the scaffolding domain of caveolin-1
are indicated. The sequence of the peptide used to produce antibodies
to BENE is underlined with a continuous line. The
amino acids in identical positions in the BENE and MAL sequences are
boxed.
View larger version (49K):
[in a new window]
Fig. 2.
Expression of the BENE gene in different cell
lines. Total RNA (~20 µg) from the indicated cell lines was
hybridized to DNA probes specific to BENE or -actin.
View larger version (57K):
[in a new window]
Fig. 3.
Characterization of a novel monoclonal
antibody to human BENE. A, immunoblot analysis
of the anti-BENE 5B1 mAb. The hybridoma clone 5B1 producing mAb to the
human BENE protein was isolated after screening of the hybridoma
culture supernatants. To assay the specificity of mAb 5B1, protein
extracts from untransfected ( ) or from transfected COS-7 cells
transiently expressing BENE tagged with the c-Myc 9E10 epitope
(BENE) were subjected to immunoblot analysis with either mAb
5B1 mAb or with the anti-tag mAb 9E10. As COS-7 cells are negative for
BENE gene expression (not shown), no reaction was observed
with endogenous proteins of COS-7 cells. B, to further study
the specificity of the 5B1 mAb, aliquots of 5B1 culture supernatant
were preincubated for 1 h at 4 °C with the indicated amounts of
the BENE peptide used for the immunizations and used to probe blots of
extracts from COS-7 cells transiently expressing the human BENE protein
tagged with the c-Myc 9E10 epitope. Other unrelated peptides used did
not show any effect on the recognition of BENE by the 5B1 mAb (not
shown). The same blots were then reprobed with anti c-Myc 9E10 mAb
preincubated with the BENE peptide to show that the competition
observed with the 5B1 mAb was specific. Note that similar amounts of
tagged BENE were present in each lane. C, mAb 5B1
detects endogenous BENE in endothelial ECV304 cells. Extracts from
ECV304 cells and Jurkat T cells were subjected to immunoblot analysis
with anti-BENE 5B1 mAb and anti-MAL 6D9 mAb as indicated. D,
tonsil sections were subjected to immunohistochemical analysis with
anti-BENE mAb 5B1 and counterstained with hematoxylin to visualize
nuclei. Reactivity was found in endothelial cells lining the blood
vessels (arrows).
View larger version (38K):
[in a new window]
Fig. 4.
Identification of endogenous BENE in GEM
microdomains in ECV304 cells. A, endothelial ECV304
cells were extracted with 1% Triton X-100 at 4 °C and subjected to
centrifugation to equilibrium in sucrose density gradients. Fractions
of 1 ml were collected from the bottom of the tube. Aliquots from each
fraction were subjected to SDS-PAGE analyzed by immunoblotting with
anti-BENE mAb 5B1, or antibodies to caveolin-1, caveolin-2, used as
raft markers, or to calnexin, an endoplasmic reticulum transmembrane
protein excluded from rafts. Fractions 1-4 are the 40%
sucrose layer and contain the bulk of cellular membranes and cytosolic
proteins, while fractions 5-12 are the 5-30% sucrose
layer and contain GEMs. B, GEMs prepared from ECV304 cells
were extracted with an identical volume of n-butyl
alcohol, shaken vigorously, and centrifuged at low speed. The
aqueous phase was withdrawn, and the organic phase was evaporated. The
dry residue containing the proteins that partitioned in
n-butyl alcohol was resuspended in loading buffer.
Aliquots from the original sample (T) and the aqueous
(H20) and organic (n-but)
phases were analyzed in parallel by immunoblot with anti-BENE mAb 5B1.
The same procedure was used to isolate the equivalent proteolipid
fraction from human vein umbilical cells (HUVEC). This
fraction was analyzed by immunoblotting with anti-BENE 5B1 mAb.
C, ECV304 cells stably expressing BENE tagged at
the NH2 terminus with the c-Myc 9E10 epitope
(ECV304/BENE) were extracted with 1% Triton X-100 at
4 °C and subjected to centrifugation to equilibrium in sucrose
density gradients. Fractions of 1 ml were collected from the bottom of
the tube. Aliquots from the different fractions were analyzed by
immunoblotting with anti c-Myc mAb 9E10. D, 10 µl from a
pool of the insoluble membrane fraction obtained from ECV304/BENE cells
were analyzed by immunoblotting with anti-BENE mAb 5B1. The signal
corresponding to the exogenous (tagged BENE) and endogenous
BENE were quantified in a densitometer.
View larger version (28K):
[in a new window]
Fig. 5.
Immunofluorescence analysis of the
distribution of BENE in ECV304 cells. ECV304 cells stably
expressing tagged BENE were doubly labeled with anti-tag 9E10 mAb to
detect BENE and antibodies to caveolin-1 (Cav-1), CD59, or
mannosidase II (Man II) followed by the appropriate
fluorescent secondary antibodies. Finally, the samples were analyzed in
a conventional immunofluorescence microscope. Bar, 8 µM.
View larger version (36K):
[in a new window]
Fig. 6.
BENE associates with caveolin-1
and -2 in endothelial ECV304 cells. A,
ECV304 (left panel), ECV304/BENE (middle panel),
or A498 cells (right panel) were extracted with 1% Triton
X-100 plus 60 mM octyl glucoside at room temperature and
the extracts subjected to immunoprecipitation with control anti-CD8 mAb
or with anti-caveolin-1 mAb. The immunoprecipitates were then subjected
to immunoblot analysis with anti-BENE mAb 5B1 (left and
right panels) or anti-tag mAb 9E10 (middle
panel). B and C, ECV304/BENE cells were
metabolically labeled for 6 h with
[35S]methionine/cysteine. Cells were then extracted with
1% Triton X-100 plus 60 mM octyl glucoside and
immunoprecipitated with anti-tag mAb 9E10. The immunoprecipitates were
then subjected to either autoradiography (B) or to
immunoblot analysis with antibodies to caveolin-1, caveolin-2, and
anti-tag 9E10 mAb (C). The 17-kDa band corresponding to BENE
and the 20-24-kDa bands corresponding to caveolins are indicated in
B by an arrowhead and an arrow,
respectively. D, control of the solubilization procedure.
Extracts from ECV304/BENE cells prepared with 1% Triton X-100 plus 60 mM octyl glucoside were centrifuged to equilibrium in a
discontinuous sucrose density gradient, and the soluble (S)
and insoluble (I) fractions were collected. Equivalent
aliquots from each fraction were immunoblotted with anti-caveolin-1
(top panel), anti-caveolin-2 (middle panel), and
anti-tag antibodies (bottom panel).
View larger version (144K):
[in a new window]
Fig. 7.
General view of the distribution of BENE in
ECV304 cells as revealed by immunoelectron microscopy analysis.
Ultrathin cryosections of ECV304/BENE cells were immunolabeled for BENE
using anti-tag antibodies and 15-nm protein A-gold. Overview of an
ECV304 cell immunolabeled for BENE. BENE was detected in small
tubulovesicular elements (arrows) scattered throughout the
cell. No labeling was detected at the plasma membrane (pm).
Inset, detail of a tubulovesicular structure immunoreactive
for BENE and displaying a characteristic clathrin coat
(arrows). Bar, 200 nm.
View larger version (177K):
[in a new window]
Fig. 8.
Detailed views of the distribution of BENE in
ECV304 cells. Ultrathin cryosections of ECV304/BENE cells were
immunolabeled for BENE using anti-tag antibodies with 15 nm protein
A-gold (a and b) or double immunolabeled for BENE
and caveolin-1 with 15 and 10 nm protein A-gold, respectively
(c and d). a, BENE is detected in
small tubulovesicular membranes (arrows) associated with the
Golgi complex (G). b, the lateral rims of Golgi
cisterna were occasionally immunoreactive (arrows).
c, specific labeling for BENE is observed in small
tubulovesicular membranes (arrows). Note that the plasma
membrane (pm) is not labeled. The arrowhead
points to a vesicle immunoreactive for BENE and caveolin-1.
d, note the high labeling for caveolin-1 in a cluster of
intracellular caveolae-like vesicles. The arrowhead points
to a vesicle immunoreactive also for BENE. The inset in
d shows a vesicle containing BENE and caveolin-1.
n = nucleus; pm = plasma membrane;
G = Golgi stack. Bars, 200 nm.
View larger version (19K):
[in a new window]
Fig. 9.
Redistribution of BENE and caveolin-1 in
response to cholesterol oxidase treatment in endothelial ECV304
cells. ECV304/BENE cells grown on coverslips were left
untreated or treated with 1 unit/ml CO for the indicated times. Cells
were fixed and processed for confocal immunofluorescence analysis with
anti-tag mAb 9E10 and rabbit polyclonal antibodies to caveolin-1,
followed by appropriate Texas Red-labeled and
fluorescein-labeled secondary antibodies to visualize BENE and
caveolin-1, respectively. Two different optical sections of the same
cells, at 1.2 µm (a) and 2.0 µm (b) from the
plane of the coverslips, are shown. NT, nontreated. Bar, 8 µm.
View larger version (21K):
[in a new window]
Fig. 10.
Reversible movement of BENE and caveolin-1
upon withdrawal of cholesterol oxidase in ECV304 cells.
ECV304/BENE cells grown on coverslips were treated with 1 unit/ml CO
for 60 min and then washed and incubated in fresh medium at
37 °C in the absence of CO. At the indicated times, cells were fixed
and processed for confocal immunofluorescence analysis as in Fig. 9.
Two different optical sections of the same cells, at 0.8 µm
(a) and 1.6 µm (b) from the plane of the
coverslips, are shown. Insets show details of the merge
profiles at higher magnification. Bar, 8 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Carlos Sánchez for his expertise with the use of the confocal microscope.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Comunidad de Madrid (08.3/0025/2000), the Dirección General de Enseñanza Superior (PM99-0092 and PM99-137), and Fondo de Investigación Sanitaria (01/0085-01). The Department of Immunology and Oncology is funded and supported by the Consejo Superior de Investigaciones Científicas and Pharmacia & Upjohn. This work was also supported by an institutional grant from the Fundación Ramón Areces to Centro de Biología Molecular "Severo Ochoa."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a predoctoral fellowship from the Ministerio de Educación y Cultura, Spain.
** Supported by a Marie Curie Return Fellowship from the European Commission.
§§ To whom correspondence should be addressed: Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Tel.: 34-91-397-8037; Fax: 34-91-397-8087; E-mail: maalonso@cbm.uam.es.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M009739200
2 M. del Carmen de Marco, L. Kremer, J. P. Albar, J. A. Martínez-Menárguez, J. Ballesta, M. A. García-López, M. Marazuela, R. Puertollano, and M. A. Alonso, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GEM, glycolipid- and cholesterol-enriched membrane; CO, cholesterol oxidase; MDCK, Madin-Darby canine kidney; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; GPI, glycosylphosphatidylinositol.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Simons, K., and Ikonen, E. (1997) Nature 387, 569-572[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Brown, D. A.,
and London, E.
(2000)
J. Biol. Chem.
275,
17221-17224 |
3. | Simons, K., and Wandinger-Ness, A. (1990) Cell 62, 207-210[Medline] [Order article via Infotrieve] |
4. | Anderson, R. G. W. (1998) Annu. Rev. Biochem. 67, 199-225[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Smart, E. J.,
Graf, G. A.,
McNiven, M. A.,
Sessa, W. C.,
Engelman, J. A.,
Scherer, P. E.,
Okamoto, T.,
and Lisanti, M. P.
(1999)
Mol. Cell. Biol.
19,
7289-7304 |
6. | Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y., Glenney, J. R., and Anderson, R. G. W. (1992) Cell 68, 673-682[Medline] [Order article via Infotrieve] |
7. | Fra, A. M., Williamson, E., Simons, K., and Parton, R. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8655-8659[Abstract] |
8. |
Li, S.,
Song, K. S.,
Koh, S. S.,
Kikuchi, A.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
28647-28654 |
9. |
Volonté, D.,
Galbiati, F.,
Li, S.,
Nishiyama, K.,
Okamoto,
and Lisanti, M. P.
(1998)
J. Biol. Chem.
274,
12702-12709 |
10. | Schlesinger, M. J. (1981) Annu. Rev. Biochem. 50, 193-206[CrossRef][Medline] [Order article via Infotrieve] |
11. | Zacchetti, D., Peranen, J., Murata, M., Fiedler, K., and Simons, K. (1995) FEBS Lett. 377, 465-469[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Martín-Belmonte, F.,
Kremer, L.,
Albar, J. P.,
Marazuela, M.,
and Alonso, M. A.
(1998)
Endocrinology
139,
2077-2084 |
13. | Kim, T., Fiedler, K., Madison, D. L., Krueger, W. H., and Pfeiffer, S. E. (1995) J. Neurosci. Res. 42, 413-422[Medline] [Order article via Infotrieve] |
14. | Millán, J., and Alonso, M. A. (1998) Eur. J. Immunol. 28, 3675-3684[CrossRef][Medline] [Order article via Infotrieve] |
15. | Alonso, M. A., and Weissman, S. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1997-2001[Abstract] |
16. |
Puertollano, R.,
Martín-Belmonte, F.,
Millán, J.,
de Marco, M. C.,
Albar, J. P.,
Kremer, L.,
and Alonso, M. A.
(1999)
J. Cell Biol.
145,
141-145 |
17. |
Cheong, K. H.,
Zacchetti, D.,
Schneeberger, E. E.,
and Simons, K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6241-6248 |
18. |
Martín-Belmonte, F.,
Puertollano, R.,
Millán, J.,
and Alonso, M. A.
(2000)
Mol. Biol. Cell
11,
2033-2045 |
19. | Pérez, P., Puertollano, R., and Alonso, M. A. (1997) Biochem. Biophys. Res. Commun. 232, 618-621[CrossRef][Medline] [Order article via Infotrieve] |
20. | Magyar, J. P., Ebensperger, C., Schaeren-Wiemers, N., and Suter, U. (1997) Gene (Amst.) 189, 269-275[CrossRef][Medline] [Order article via Infotrieve] |
21. | Brown, D. A., and Rose, J. K. (1992) Cell 68, 533-544[Medline] [Order article via Infotrieve] |
22. | Lisanti, M. P., Scherer, P. E., Vidugiriene, J., Tank, Z., Hermanowski-Vosatka, A., Tu, Y-H., and Sargiacomo, M. (1994) J. Cell Biol. 126, 111-126[Abstract] |
23. | Lautner-Rieske, A., Thiebe, R., and Zachau, H. G. (1995) Gene (Amst.) 159, 199-202[CrossRef][Medline] [Order article via Infotrieve] |
24. | Hughes, S. E. (1996) Exp. Cell Res. 225, 171-185[CrossRef][Medline] [Order article via Infotrieve] |
25. | Smart, E. J., Ying, Y.-S., Conrad, P. A., and Anderson, R. G. W. (1994) J. Cell Biol. 127, 1185-1197[Abstract] |
26. | Gausepohl, H., Boulin, C., Kraft, M., and Frank, R. W. (1992) Peptide Res. 5, 315-320 |
27. | Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
28. | Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 8-13 |
29. | Ponte, P., Gunning, P., Blau, H., and Kedes, L. (1983) Mol. Cell. Biol. 3, 1783-1791[Medline] [Order article via Infotrieve] |
30. | Martínez-Menárguez, J. A., Geuze, H. J., Slot, J. W., and Klumperman, J. (1999) Cell 98, 81-90[Medline] [Order article via Infotrieve] |
31. | Slot, J. W., Geuze, H. J., Gigengack, S., Lienhard, G. E., and James, J. E. (1991) J. Cell Biol. 113, 123-135[Abstract] |
32. | Griffiths, G. (1993) Fine Structure Immunocytochemistry , Springer-Verlag, Berlin |
33. | Martínez-Menárguez, J. A., Geuze, H. J., and Ballesta, J. (1996) Eur. J. Cell Biol. 71, 137-143[Medline] [Order article via Infotrieve] |
34. | Marazuela, M., Sánchez-Madrid, F., Acevedo, A., Larrañaga, E., and Landázuri, M. O. (1995) Clin. Exp. Immunol. 102, 328-334[Medline] [Order article via Infotrieve] |
35. | Kozak, M. (1996) Mamm. Genome 7, 563-574[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Puertollano, R.,
and Alonso, M. A.
(1999)
Mol. Biol. Cell
10,
3435-3447 |
37. | Brown, J., Reading, S. J., Jones, S., Fitchett, C. J., Howl, J., Martin, A., Longland, C. L., Michelangeli, F., Dubrova, Y. E., and Brown, C. A. (2000) Lab. Invest. 80, 37-45[Medline] [Order article via Infotrieve] |
38. |
Couet, J.,
Li, S.,
Okamoto, T.,
Ikezu, T.,
and Lisanti, M. P.
(1997)
J. Biol. Chem.
272,
6525-6533 |
39. |
Scheiffele, P.,
Verkade, P.,
Fra, A. M.,
Simons, K.,
and Ikonen, E.
(1998)
J. Cell Biol.
140,
795-806 |
40. | Dupree, P., Parton, R. G., Raposo, G., Kurzchalia, T. V., and Simons, K. (1993) EMBO J. 12, 1597-1605[Abstract] |
41. | Millán, J., Puertollano, R., Fan, L., and Alonso, M. A. (1997) Biochem. Biophys. Res. Commun. 233, 707-712[CrossRef][Medline] [Order article via Infotrieve] |
42. | Conrad, P. A., Smart, E. J., Anderson, R. G. W., and Bloom, G. S. (1995) J. Cell Biol. 131, 1421-1433[Abstract] |
43. | Murata, M., Peranen, J., Schreiner, R., Wieland, F., Kurzchalia, T. V., and Simons, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10339-10343[Abstract] |
44. |
Li, S.,
Song, K. S.,
and Lisanti, M. P.
(1996)
J. Biol. Chem.
271,
568-573 |
45. |
Smart, E. J.,
Ying, Y.-S.,
Donzell, W. C.,
and Anderson, R. G. W.
(1996)
J. Biol. Chem.
271,
29427-29435 |