BENE, a Novel Raft-associated Protein of the MAL Proteolipid Family, Interacts with Caveolin-1 in Human Endothelial-like ECV304 Cells
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m009739200
ISSN1083-351X
AutoresMarı́a del Carmen de Marco, Leonor Kremer, Juan Pablo Albar, José A. Martínez‐Menárguez, J. Ballesta, María Ángeles García-López, Mónica Marazuela, Rosa Puertollano, Miguel A. Alonso,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe 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 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. glycolipid- and cholesterol-enriched membrane cholesterol oxidase Madin-Darby canine kidney monoclonal antibody polyacrylamide gel electrophoresis glycosylphosphatidylinositol The compartmentation of cellular membranes in microdomains or rafts is an emerging concept in cell biology (1Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (7923) Google Scholar). 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 (2Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2029) Google Scholar). This difference makes glycolipid- and cholesterol-enriched membrane (GEM)1 rafts resistant to solubilization by nonionic detergents at low temperature (2Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2029) Google Scholar). 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 (3Simons K. Wandinger-Ness A. Cell. 1990; 62: 207-210Abstract Full Text PDF PubMed Scopus (417) Google Scholar). 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 (1Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (7923) Google Scholar). 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 (1Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (7923) Google Scholar, 3Simons K. Wandinger-Ness A. Cell. 1990; 62: 207-210Abstract Full Text PDF PubMed Scopus (417) Google Scholar). Caveolae are raft-containing vesicular invaginations of the plasma membrane involved in a variety of cellular processes including signaling and clathrin-independent endocytosis (4Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1706) Google Scholar). Caveolin-1 is a multifunctional raft-associated protein (5Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (915) Google Scholar) primarily identified as a component of the caveolar architecture (6Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1828) Google Scholar). 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 (7Fra A.M. Williamson E. Simons K. Parton R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8655-8659Crossref PubMed Scopus (517) Google Scholar, 8Li S. Song K.S. Koh S.S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), and 2) it forms a scaffold onto which many classes of signaling molecules can assemble to generate preassembled signaling complexes within caveolae (5Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (915) Google Scholar). 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 (5Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (915) Google Scholar). The flotillin/cavatellin family, which so far groups the raft-associated flotillin-1 and flotillin-2/ESA proteins (9Volonté D. Galbiati F. Li S. Nishiyama K. Okamoto Lisanti M.P. J. Biol. Chem. 1998; 274: 12702-12709Abstract Full Text Full Text PDF Scopus (185) Google Scholar), whose function is still unknown, might constitute a second family of elements of the raft machinery (5Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (915) Google Scholar). Proteolipids are operationally defined as proteins with unusually high solubility in organic solvents commonly used to extract cell lipids (10Schlesinger M.J. Annu. Rev. Biochem. 1981; 50: 193-206Crossref PubMed Scopus (76) Google Scholar). MAL is an integral membrane proteolipid protein of 17 kDa expressed in a restricted range of cell types including polarized epithelial cells (11Zacchetti D. Peranen J. Murata M. Fiedler K. Simons K. FEBS Lett. 1995; 377: 465-469Crossref PubMed Scopus (94) Google Scholar, 12Martı́n-Belmonte F. Kremer L. Albar J.P. Marazuela M. Alonso M.A. Endocrinology. 1998; 139: 2077-2084Crossref PubMed Scopus (54) Google Scholar), oligodendrocytes (13Kim T. Fiedler K. Madison D.L. Krueger W.H. Pfeiffer S.E. J. Neurosci. Res. 1995; 42: 413-422Crossref PubMed Scopus (114) Google Scholar), and T lymphocytes (14Millán J. Alonso M.A. Eur. J. Immunol. 1998; 28: 3675-3684Crossref PubMed Scopus (64) Google Scholar, 15Alonso M.A. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1997-2001Crossref PubMed Scopus (131) Google Scholar). MAL selectively resides in lipid rafts in all the cell types in which it is expressed (11Zacchetti D. Peranen J. Murata M. Fiedler K. Simons K. FEBS Lett. 1995; 377: 465-469Crossref PubMed Scopus (94) Google Scholar, 12Martı́n-Belmonte F. Kremer L. Albar J.P. Marazuela M. Alonso M.A. Endocrinology. 1998; 139: 2077-2084Crossref PubMed Scopus (54) Google Scholar, 13Kim T. Fiedler K. Madison D.L. Krueger W.H. Pfeiffer S.E. J. Neurosci. Res. 1995; 42: 413-422Crossref PubMed Scopus (114) Google Scholar, 14Millán J. Alonso M.A. Eur. J. Immunol. 1998; 28: 3675-3684Crossref PubMed Scopus (64) Google Scholar). 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 (16Puertollano R. Martı́n-Belmonte F. Millán J. de Marco M.C. Albar J.P. Kremer L. Alonso M.A. J. Cell Biol. 1999; 145: 141-145Crossref PubMed Scopus (147) Google Scholar, 17Cheong K.H. Zacchetti D. Schneeberger E.E. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6241-6248Crossref PubMed Scopus (184) Google Scholar, 18Martı́n-Belmonte F. Puertollano R. Millán J. Alonso M.A. Mol. Biol. Cell. 2000; 11: 2033-2045Crossref PubMed Scopus (91) Google Scholar). 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 (19Pérez P. Puertollano R. Alonso M.A. Biochem. Biophys. Res. Commun. 1997; 232: 618-621Crossref PubMed Scopus (41) Google Scholar, 20Magyar J.P. Ebensperger C. Schaeren-Wiemers N. Suter U. Gene ( Amst. ). 1997; 189: 269-275Crossref PubMed Scopus (44) Google Scholar). 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 (19Pérez P. Puertollano R. Alonso M.A. Biochem. Biophys. Res. Commun. 1997; 232: 618-621Crossref PubMed Scopus (41) Google Scholar). 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 (21Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2584) Google Scholar). So far, no member of the MAL family of proteolipid proteins has been identified in the GEM fraction of endothelial cells (22Lisanti M.P. Scherer P.E. Vidugiriene J. Tank Z. Hermanowski-Vosatka A. Tu Y-H. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Crossref PubMed Scopus (804) Google Scholar). 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 κ chain locus (23Lautner-Rieske A. Thiebe R. Zachau H.G. Gene ( Amst. ). 1995; 159: 199-202Crossref PubMed Scopus (11) Google Scholar). BENE mRNA is expressed in the prostate, small intestine, colon, heart, and lung and is undetectable in brain, thymus, liver, and spleen (20Magyar J.P. Ebensperger C. Schaeren-Wiemers N. Suter U. Gene ( Amst. ). 1997; 189: 269-275Crossref PubMed Scopus (44) Google Scholar). 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 (24Hughes S.E. Exp. Cell Res. 1996; 225: 171-185Crossref PubMed Scopus (115) Google Scholar). 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 (25Smart E.J. Ying Y.-S. Conrad P.A. Anderson R.G.W. J. Cell Biol. 1994; 127: 1185-1197Crossref PubMed Scopus (376) Google Scholar) in the endothelial-like ECV304 cell line. 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 (12Martı́n-Belmonte F. Kremer L. Albar J.P. Marazuela M. Alonso M.A. Endocrinology. 1998; 139: 2077-2084Crossref PubMed Scopus (54) Google Scholar). 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). Human ECV304 cells (kindly provided by Dr. J. Riese, Centro Nacional de Biotecnologı́a, 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 (23Lautner-Rieske A. Thiebe R. Zachau H.G. Gene ( Amst. ). 1995; 159: 199-202Crossref PubMed Scopus (11) Google Scholar). 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. 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 (26Gausepohl H. Boulin C. Kraft M. Frank R.W. Peptide Res. 1992; 5: 315-320PubMed Google Scholar). 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 (27Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) 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. 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 2.2m 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 (28Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 8-13Crossref Scopus (16561) Google Scholar) corresponding to human BENE (23Lautner-Rieske A. Thiebe R. Zachau H.G. Gene ( Amst. ). 1995; 159: 199-202Crossref PubMed Scopus (11) Google Scholar). As a control of the amounts of RNA present in each lane, blots were finally hybridized to a 0.6-kilobase pairHinfI/BamHI DNA fragment from the 3′-untranslated region of human β-actin mRNA (29Ponte P. Gunning P. Blau H. Kedes L. Mol. Cell. Biol. 1983; 3: 1783-1791Crossref PubMed Google Scholar). Final blot washing conditions were 0.5× SSC/0.1% SDS (1× SSC = 0.15 m NaCl, 0.015m sodium citrate, pH 7.0) at 65 °C. GEMs were prepared essentially as described by Brown and Rose (21Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2584) Google Scholar). 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). 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, 0.15m 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. 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 Igγ1 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. Stable transfectants of ECV304 cells expressing tagged BENE were used. The cells were processed for cryosectioning as described previously (30Martı́nez-Menárguez J.A. Geuze H.J. Slot J.W. Klumperman J. Cell. 1999; 98: 81-90Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). 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. (31Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James J.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (703) Google Scholar). 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 (32Griffiths G. Fine Structure Immunocytochemistry. Springer-Verlag, Berlin1993Crossref Google Scholar) 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 (30Martı́nez-Menárguez J.A. Geuze H.J. Slot J.W. Klumperman J. Cell. 1999; 98: 81-90Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 33Martı́nez-Menárguez J.A. Geuze H.J. Ballesta J. Eur. J. Cell Biol. 1996; 71: 137-143PubMed Google Scholar), 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. 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 (34Marazuela M. Sánchez-Madrid F. Acevedo A. Larrañaga E. Landázuri M.O. Clin. Exp. Immunol. 1995; 102: 328-334Crossref PubMed Scopus (29) Google Scholar). 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. A partial BENE cDNA was identified during a search for genes in the proximity of the human immunoglobulin κ chain locus (23Lautner-Rieske A. Thiebe R. Zachau H.G. Gene ( Amst. ). 1995; 159: 199-202Crossref PubMed Scopus (11) Google Scholar). 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/GXX ATGG consensus sequence (where Xstands for any nucleotide) for optimal ATG translational start sites in eukaryotic cells (35Kozak M. Mamm. Genome. 1996; 7: 563-574Crossref PubMed Scopus (755) Google Scholar). 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 (23Lautner-Rieske A. Thiebe R. Zachau H.G. Gene ( Amst. ). 1995; 159: 199-202Crossref PubMed Scopus (11) Google Scholar). 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 (23Lautner-Rieske A. Thiebe R. Zachau H.G. Gene ( Amst. ). 1995; 159: 199-202Crossref PubMed Scopus (11) Google Scholar), 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.Figure 2Expression 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 β-actin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The peptide EKLLDPRIYYI, comprising amino acids 118–128 of human BENE, was synthesized (sequence underlinedin 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 (12Martı́n-Belmonte F. Kremer L. Albar J.P. Marazuela M. Alonso M.A. Endocrinology. 1998; 139: 2077-2084Crossref PubMed Scopus (54) Google Scholar, 16Puertollano R. Martı́n-Belmonte F. Millán J. de Marco M.C. Albar J.P. Kremer L. Alonso M.A. J. Cell Biol. 1999; 145: 141-145Crossref PubMed Scopus (147) Google Scholar), which in MAL corresponds to an extracellular (luminal) loop (36Puertollano R. Alons
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