p53 and Tumor Necrosis Factor α Regulate the Expression of a Mitochondrial Chloride Channel Protein
1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês
10.1074/jbc.274.51.36488
ISSN1083-351X
AutoresEster Fernández‐Salas, Manish Sagar, Christina Cheng, Stuart H. Yuspa, Wendy C. Weinberg,
Tópico(s)Cancer-related Molecular Pathways
ResumoA novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53+/+ and p53−/− mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs fromp53−/− mice. mc3s5/mtCLIC mRNA and protein are higher in p53+/+ compared withp53−/− basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor α also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor α. A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53+/+ and p53−/− mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs fromp53−/− mice. mc3s5/mtCLIC mRNA and protein are higher in p53+/+ compared withp53−/− basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor α also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor α. chloride intracellular channel cytochrome oxidase glyceraldehyde-3-phosphate dehydrogenase green fluorescent protein multiplicity of infection open reading frame tumor necrosis factor α base pair(s) kilobase(s) amino acid(s) untranslated region polymerase chain reaction phosphate-buffered saline adenovirus encoding wild-type p53 The multiple functions now ascribed to the p53 tumor suppressor gene include cell cycle control, DNA repair, senescence, differentiation, and apoptosis (reviewed in Refs. 1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6805) Google Scholar, 2Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2301) Google Scholar, 3Selivanova G. Wiman K.G. Adv. Cancer Res. 1995; 66: 143-180Crossref PubMed Google Scholar). p53 exerts part of its biological function by activating or repressing the transcription of several target genes (2Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2301) Google Scholar, 4Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. Nature. 1997; 389: 300-305Crossref PubMed Scopus (2270) Google Scholar, 5Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. Mol. Cell. 1997; 1: 3-11Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar). p53 is particularly important in the skin because it is frequently mutated in preneoplastic and neoplastic human skin lesions (6Kraemer K.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11-14Crossref PubMed Scopus (344) Google Scholar), and loss ofp53 enhances malignant progression of experimentally induced murine skin tumors (7Kemp C.J. Donehower L.A. Bradley A. Balmain A. Cell. 1993; 74: 813-822Abstract Full Text PDF PubMed Scopus (455) Google Scholar). In keratinocytes, p53-dependent transcriptional activity increases in association with terminal differentiation (8Weinberg W.C. Azzoli C.G. Chapman K. Levine A.J. Yuspa S.H. Oncogene. 1995; 10: 2271-2279PubMed Google Scholar). However, the skin of p53-deficient mice appears to be normal in vivo (9Donehower L.A. Harvey M. Slagle B.L. McArthur M.J. Montgomery Jr., C.A. Butel J.S. Bradley A. Nature. 1992; 356: 215-221Crossref PubMed Scopus (4133) Google Scholar), and the expression of differentiation markers by p53−/− keratinocytes in vitro cannot be distinguished from that of wild-type (8Weinberg W.C. Azzoli C.G. Chapman K. Levine A.J. Yuspa S.H. Oncogene. 1995; 10: 2271-2279PubMed Google Scholar). This implies that p53 is dispensable for normal skin development and differentiation. However, deletion of one or both p53alleles enhances the establishment of immortal keratinocyte cell linesin vitro (10Azzoli C.G. Sagar M. Wu A. Lowry D. Hennings H. Morgan D.L. Weinberg W.C. Mol. Carcinog. 1998; 21: 50-61Crossref PubMed Scopus (20) Google Scholar), suggesting that p53 could participate in keratinocyte mortality in a more subtle way, not involving expression of the major differentiation-related markers. To detect such a contribution of p53 to keratinocyte differentiation or mortality, differential expression of mRNA sequences was examined in keratinocytes isolated from p53 wild-type and null mice and induced to differentiate by Ca2+ in vitro, a condition known to increase p53-dependent transcriptional activity. In this report, we describe the cloning and characterization of a novel p53-regulated gene that we callmc3s5/mtCLIC.1Expression of this gene is up-regulated during keratinocyte maturation and in keratinocytes following treatment with TNF-α. By sequence homology and subcellular localization studies, we found that mc3s5/mtCLIC is a chloride channel protein found in the cytoplasm and mitochondria. Keratinocytes were isolated from newborn Balb/c or p53+/+ and p53−/− mice using established methods (11Dlugosz A.A. Glick A.B. Tennenbaum T. Weinberg W.C. Yuspa S.H. Methods Enzymol. 1995; 254: 3-20Crossref PubMed Scopus (140) Google Scholar). Primary keratinocytes were plated at a density of 3 × 106 cells/60-mm dish, 7 × 106 cells/100-mm dish, and 15 × 106cells/150-mm dish and maintained in Eagle's minimal essential medium without Ca2+ (Bio-Whittaker, Walkersville, MD) containing 8% chelex-treated fetal bovine serum (Gemini, Bio-Products, Inc., Calabasas, CA), 0.05 mm Ca2+, and 20 units ml−1 of penicillin/streptomycin (Life Technologies, Inc.). Differentiation of the keratinocytes was achieved by increasing the extracellular concentration of Ca2+ in the medium to >0.1 mm (11Dlugosz A.A. Glick A.B. Tennenbaum T. Weinberg W.C. Yuspa S.H. Methods Enzymol. 1995; 254: 3-20Crossref PubMed Scopus (140) Google Scholar). Transgenic newborn mice were genotyped as described previously (12Weinberg W.C. Azzoli C.G. Kadiwar N. Yuspa S.H. Cancer Res. 1994; 54: 5584-5592PubMed Google Scholar). The cell line SP1, derived from a chemically induced mouse skin papilloma (13Strickland J.E. Greenhalgh D.A. Koceva-Chyla A. Hennings H. Restrepo C. Balaschak M. Yuspa S.H. Cancer Res. 1988; 48: 165-169PubMed Google Scholar), was cultured in the medium described above. 293 cells were cultured in Dulbecco's modified Eagle's medium (Bio-Whittaker) containing 8% fetal bovine serum (Gemini) and 20 units ml−1 penicillin/streptomycin (Life Technologies, Inc.). The derivation of p53-null keratinocytes was described previously (10Azzoli C.G. Sagar M. Wu A. Lowry D. Hennings H. Morgan D.L. Weinberg W.C. Mol. Carcinog. 1998; 21: 50-61Crossref PubMed Scopus (20) Google Scholar), and these were maintained in medium containing 16% fibroblast conditioned Eagle's minimal essential medium with 8% chelexed serum and 0.05 mm Ca2+. Primary mouse keratinocytes and cell lines were treated with human recombinant TNF-α (R&D Systems, Minneapolis, MN) at final concentrations ranging from 10 to 100 ng/ml for the times detailed. To identify potential p53-regulated genes expressed in differentiating keratinocytes, primary cultures ofp53+/+ and p53−/− keratinocytes were cultured in 0.05 mm Ca2+ medium for 5 days and then switched to 1.3 mm Ca2+ for 12 h. Total RNA was isolated directly from keratinocyte cultures by Trizol extraction (Life Technologies, Inc.). Reverse transcription was done in four independent reactions using four different T12 primers: T12AC, T12AT, T12GA, and T12AG. The cDNA was amplified using 10-mer random primers (Operon Technologies, Alameda, CA). The amplified cDNAs were separated on a 6% polyacrylamide-urea denaturing DNA sequencing gel in Tris-borate buffer at 80 W. Differentially expressed bands were recovered from the gel, reamplified, and cloned into the pCRII TA cloning vector (Invitrogen, Carlsbad, CA). Fragments were sequenced manually using the Sequenase kit (United States Biochemical, Cleveland, OH). GenBankTM comparison computations were performed at the National Center for Biotechnology Information using the Blastn program, a nucleotide sequence similarity search tool (14Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61086) Google Scholar). A custom cDNA library was prepared by oligo(dT) priming of mRNA from mouse keratinocytes cultured in 0.05 mmCa2+, followed by cloning in the Lambda Zap II vector (Stratagene, La Jolla, CA). Plating and screening of the library was performed as described by the manufacturer. Blotting of the membranes was carried out using 32P-labeled clone mc3s5 excised withEcoRI digestion from the pCR II vector obtained from the differential display. 10–15 of the strongest putative positive clones were cored from the plate and subjected to two more screenings. The Lambda Zap II vector contains the pBlueScript Vector, which contains the cDNA clone. Excision of the vector was done with the ExAssist helper λ phage; supernatants were then transformed into SOLR strain cells that are λ-resistant. Plasmid clones with fragments from 1.7 to 2.5 kb were isolated and sequenced on a Perkin-Elmer ABI Prism 377 DNA sequencer using the dye terminator DNA sequencing kit (PE Applied Biosystems, Foster City, CA) and custom primers (Bioserve Biotechnologies, Laurel, MD). Fragments were assembled using the Sequencher software package (Gene Codes Corp., Ann Arbor, MI). Gapped BLAST homology searches (14Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61086) Google Scholar) in the DNA data bases were performed using the National Center for Biotechnology Information Web site. The 5′ end of the mRNA was cloned using the Marathon-Ready cDNA library (CLONTECH, Palo Alto, CA) from mouse embryo. The nested primer 5′-GCAGCGAACTGTGGAGTGAGACGAC-3′ was used to obtain by polymerase chain reaction a 2-kb fragment containing part of the open reading frame (ORF). Fragments were cloned into the pCR2.1 TA cloning plasmid (Invitrogen). A second round of touchdown PCR using the cDNA library and the nested primer 5′-AAAGCGGATTCTCGGGGGATCG-3′ was performed to obtain the complete ORF sequence and the 5′-untranslated region (UTR). The 762-bp Open Reading Frame was digested from the pCR2.1 and cloned into the pEGFP-N1 and the pEGFP-C1 vectors (CLONTECH), and sequenced to confirm proper ligation in frame with the green fluorescent protein (GFP). This created GFP-mc3s5 (GFP-N-terminal linked) and mc3s5-GFP (GFP-C-terminal linked), which were used for transfection and localization studies. Several clones containing the ORF, the 5′-UTR, and 100 bp of the 3′-UTR, were translated in vitro using the TNT-coupled reticulocyte lysate system (Promega, Madison, WI). Synthesis was performed using [35S]methionine, and samples were analyzed in a 12% acrylamide gel. Bands were detected by autoradiography with X-Omat AR film (Eastman Kodak Co.). The protein encoded by the mc3s5 ORF was translated using MacVector (Oxford Molecular Group). Two peptides were designed to immunize rabbits, an N-terminal peptide (MALSMPLNGLKEEDKEP) and a C-terminal peptide (KEVEIAYSDVAKRLTK). Peptides were synthesized and conjugated to keyhole limpet hemocyanin (Macromolecular Resources, Fort Collins, CO). New Zealand female rabbits (Hazleton Research Products, Denver, PA) were prescreened for antikeratin activity in preimmune serum, and negative animals were immunized with 2 mg of each conjugated peptide in Freund's complete adjuvant. Animals were boosted every 2 weeks with 1 mg of peptide in Freund's incomplete adjuvant until a good titer was achieved, at approximately 10 weeks. By Western blotting on extracts of keratinocytes overexpressing mc3s5-GFP fusion protein, a single band at a molecular mass of 28 kDa for the endogenous protein and a 57 kDa for the fusion protein were identified. Competition with an excess of nonconjugated peptide eliminated these bands. Similar dilutions of preimmune serum were also used as a negative control, and no bands were detected. Protein expression was analyzed by Western blot. Cells were washed and then gently scraped in radioimmune precipitation lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.01M Na2HPO4, 2 mm EDTA, 50 mm NaF, 0.2 m Na2VO4, 100 units/ml aprotinin), and 30 μg of protein were separated in 10 or 12% SDS-PAGE gels and transferred onto a nitrocellulose membrane. Antibody against the N terminus of mc3s5 was used at a 1:1000 dilution, and the C-terminal antibody was used at a 1:4000 dilution of rabbit serum. Goat anti-rabbit horseradish peroxidase conjugate (Bio-Rad) was used as a secondary antibody. Monoclonal antibodies directed to murine p53 protein were raised as culture supernatants from the mouse hybridoma cell line PAb122 (15Gurney E.G. Harrison R.O. Fenno J. J. Virol. 1980; 34: 752-763Crossref PubMed Google Scholar). For Western blot, a mixture of antibodies was used including FL-393 (Santa Cruz Biotechnology, Santa Cruz, CA), and PAb122 and PAb240 (NeoMarkers, Fremont, CA). p21/WAF1 polyclonal rabbit antibody was from Oncogene Research Products (Cambridge, MA). β-actin mouse polyclonal antibody was from Roche Molecular Biochemicals. SuperSignal chemiluminescent substrate (Pierce) was used to develop the blot. The antiserum produced against the C-terminal peptide was used for immunofluorescent staining of cultured mouse keratinocytes. Cells on culture plates were fixed for 15 min in MeOH:acetic acid (1:1) and kept at −20 °C until used. Cells were rehydrated with PBS and blocked with 20% normal goat serum for 20 min. The polyclonal monospecific serum against mc3s5 was diluted 1:2000 in 3% bovine serum albumin in PBS and added to the cells. Preimmune serum was used as control at the same dilution, and no staining was detected. Cells were incubated with the primary antibody overnight at 4 °C in a humidified chamber, washed extensively with PBS, and overlaid with goat anti-rabbit antibody conjugated with fluorescein isothiocyanate (Vector Laboratories) at a 1:200 dilution. Secondary antibody was added for 1 h at room temperature. After washing with PBS and dH2O, coverslips were applied with mounting medium containing propidium iodine or 4,6-diamidino-2-phenylindole (Vector Laboratories). Staining was eliminated when the immune serum was competed with an excess of peptide. For the colocalization experiments, cells were fixed in 2% paraformaldehyde, permeabilized in MeOH, and blocked with 100 mm glycine for 30 min. Cells were then incubated for 1 h with the polyclonal serum to mc3s5 at 1:2000 and the mouse monoclonal antibody to cytochrome oxidase (COX) at 5 μg/ml (Molecular Probes, Eugene, OR) in 0.5% bovine serum albumin. After being washed extensively with PBS, cells were incubated for 1 h with goat anti-rabbit antibody conjugated with fluorescein isothiocyanate (Vector Laboratories) at a 1:200 dilution and anti-mouse antibody conjugated with tetramethylrhodamine isothiocyanate (Jackson ImmunoResearch, West Grove, PA) at a 1:300 dilution. After washing, coverslips were applied with mounting medium containing 4,6-diamidino-2-phenylindole (or propidium iodine (Vector Laboratories) in the case of preimmune serum) and examined by confocal microscopy. Transfection of the GFP-mc3s5 fusion vectors into primary Balb/c keratinocytes and the 293 and SP1 cell lines was performed using LipofectAMINE Plus reagent (Life Technologies, Inc.). Briefly, SP1 cells were plated 2 days before transfection at a density of 3 × 105 cells/60-mm dish, and primary cells were plated 2–3 days before transfection at 3 × 106 cells/60-mm dish. Cells were transfected with 2 μg of plasmid DNA per dish in serum-free medium that was replaced after 3 h with complete medium containing 0.05 mmCa2+. Transfected cells were visualized with an inverted fluorescence microscope (Zeiss). Confocal microscopy on live cells was performed 12 h following transfection. MitoTracker staining (Molecular Probes) was performed in live keratinocytes and analyzed by confocal microscopy. Confocal fluorescent images of the overexpressing cells and the colocalization experiments were collected by Susan Garfield at the Confocal Core Facility of the Division of Basic Sciences (NCI, National Institutes of Health) with a Bio-Rad MRC 1024 confocal scan head mounted on a Nikon Optiphot microscope. Excitation was provided by a krypton-argon gas laser. Total RNA was isolated from cultured cells or tissues by Trizol extraction. Multiple tissue Northern blots containing poly(A) + RNA from mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis were purchased from CLONTECH. Poly(A)+ RNA was isolated from cultured cells by digestion of cell lysates with proteinase K and binding to oligo(dT)-cellulose (Invitrogen). RNA was resolved by formaldehyde/agarose gel electrophoresis and blotted as described previously (12Weinberg W.C. Azzoli C.G. Kadiwar N. Yuspa S.H. Cancer Res. 1994; 54: 5584-5592PubMed Google Scholar). A 300-bp cDNA fragment for mc3s5 was amplified from the pCR II vector using M13 amplification primers. The resulting amplified PCR band was isolated by the Gelase (EpiCentre Technologies, Madison, WI) procedure and constituted the 3′probe. A 400-bp probe was selected 2 kb from the 5′ end, generated by PCR from a library clone with custom primers, and gel-purified (Qiagen, Santa Clarita, CA). The TNF-α probe was prepared from mouse keratinocyte total RNA using reverse transcription-PCR and specific primers spanning bases 59–82 and 648–624 of the TNF-α gene. The PCR amplified probes were isolated by the Gelase procedure. cDNA probes were radiolabeled with 32P (Lofstrand, Gaithersburg, MD) and hybridized to the blots as described previously (12Weinberg W.C. Azzoli C.G. Kadiwar N. Yuspa S.H. Cancer Res. 1994; 54: 5584-5592PubMed Google Scholar). Bands were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale CA). Loading was normalized using a probe for gapdh orβ-actin. Adenovirus encoding wild-typep53 (AdWTp53) was a generous gift from Dr. Prem Seth (16Katayose D. Gudas J. Nguyen H. Srivastava S. Cowan K.H. Seth P. Clin. Cancer Res. 1995; 1: 889-897PubMed Google Scholar). β-Galactosidase adenovirus was a generous gift from Dr. Louwei Li. The adenoviruses were propagated in 293 cells and purified by two rounds of CsCl2 density centrifugation (16Katayose D. Gudas J. Nguyen H. Srivastava S. Cowan K.H. Seth P. Clin. Cancer Res. 1995; 1: 889-897PubMed Google Scholar). Adenovirus titers were determined by plaque assay, and viruses were stored at −70 °C. Cells were infected with adenoviruses for 30 min in serum-free medium containing polybrene (2.5 μg/ml) at a multiplicity of infection (MOI) of 10:1, 50:1, and 100:1. Infection of β-galactosidase adenovirus was assessed by β-galactosidase staining of the cells (17Sanes J.R. Rubenstein J.L. Nicolas J.F. EMBO J. 1986; 5: 3133-3142Crossref PubMed Scopus (1023) Google Scholar). Infection of keratinocytes with the AdWTp53 was assessed by immunocytochemistry with rabbit anti-p53 polyclonal serum (BioGenex, San Ramon, CA). Keratinocytes, transfected with the mc3s5-GFP and the GFP-mc3s5 fusion proteins, with GFP vector, and a truncated nonexpressing mc3s5-GFP clone, were harvested 24 h after transfection and subjected to cell fractionation (18Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1247) Google Scholar). Briefly, cells were gently scraped from the dish at 4 °C with sucrose lysis buffer containing dithiothreitol, phenylmethylsulfonyl fluoride, leupeptin, and aprotinin and homogenized with a glass cell homogenizer. Unbroken cells were collected by centrifugation at 300 × g. Nuclei were then pelleted at 750 × g for 10 min, and the mitochondria pellet was collected by further centrifugation at 10,000 × g for 30 min. Membrane and cytoplasmic fractions were separated by ultracentrifugation (Beckman) at 100,000 × g for 1 h. All pellets were washed twice with lysis buffer to avoid contamination from the cytoplasmic fraction. Mitochondria were purified from Fisher rat liver as described by Rickwood et al. (19Rickwood D. Wilson MT Darley-Usmar V.M. Darley-Usmar V. Rickwood D. Wilson M.T. Mitochondria: A Practical Approach. IRL Press Ltd., Oxford, England1987: 1-16Google Scholar). Liver was homogenized, and crude nuclei and mitochondrial extracts were pelleted as described above. The pellets were washed several times with lysis buffer. Nuclei were pelleted at 80,000 × g for 80 min at 4 °C in 2.2 msucrose, 1 mm MgCl2 and collected at the bottom of the tube (20Rickwood D. Messent A. Patel D. Graham J.M. Rickwood D. Subcellular Fractionation. 1st Ed. Oxford University Press, Oxford, England1997: 71-103Google Scholar). Crude mitochondria were resuspended in 2 ml of 0.8m sucrose and loaded over an isopycnic sucrose density gradient from 1 to 2 m. Mitochondria were separated by ultracentrifugation for 2 h at 80,000 × g at 5 °C. Intact mitochondria could be detected as a brown band at about 1.19 g/ml. Twenty fractions of approximately 250 μl were collected from the gradient through a hole in the bottom of the tube and were immediately frozen on liquid nitrogen. The amount of protein in each fraction was determined by Bradford assay (Bio-Rad). Cytochrome oxidase activity (21Trounce I.A. Kim Y.L. Jun A.S. Wallace D.C. Methods Enzymol. 1996; 264: 484-509Crossref PubMed Google Scholar) was assayed on alternate fractions. Cytoplasmic and membrane fractions were obtained as described for keratinocytes. 30 μg of protein from the liver crude extract, purified nuclei, crude mitochondria, cytoplasm, and membrane fractions were assayed for the presence of mc3s5 by Western blot. Initial PCR differential display of differentiatingp53+/+ and p53−/− keratinocytes yielded 10 differentially expressed clones confirmed by Northern blotting. Of these, three presented higher expression in p53+/+ cells and seven presented lower expression. On Blastn searches, one of the clones, named mc3s5, revealed a high homology with a 202-bp fragment induced by TNF-α, M88757, that was obtained through a subtraction library of murine fibroblasts treated with TNF-α (22Gordon H.M. Kucera G. Salvo R. Boss J.M. J. Immunol. 1992; 148: 4021-4027PubMed Google Scholar). mc3s5 mRNA is expressed in both p53+/+ and p53−/− keratinocytes, but basal levels are 5–7 times higher in +/+ cells (Fig. 1 A). In both cases, expression of the mRNA increased approximately 3 times when cells were induced to differentiate in 1.3 mm Ca2+medium for 9 h. The cDNA fragment obtained from the differential display was used to screen a murine keratinocyte cDNA library. Five clones obtained from the screening were sequenced and aligned, revealing a 2.5-kb fragment corresponding to the 3′ end of the mRNA. The apparent size of mc3s5 mRNA by Northern blots is 4.25 kb. To obtain the 5′ sequences we performed 5′ rapid amplification of cDNA ends using the primers described under “Experimental Procedures.” An ORF of 762 bp was localized, encoding a 253 amino acid protein. The nucleotide and deduced amino acid sequences of mc3s5 are shown in Fig. 1 B. The mRNA contains a 5′-UTR sequence of 170 bases and a long 3′-UTR of 3.25 kb (GenBankTM accession number AF102578). The 5′-UTR contains the ribosome binding sequence or Kozak sequence immediately upstream of the AUG (23Kozak M. Cell. 1980; 22: 7-8Abstract Full Text PDF PubMed Scopus (129) Google Scholar). The cDNA contains two possible polyadenylation consensus signals separated by 1.7 kb. A probe 5′ to the first polyadenylation signal (5′ probe) revealed two transcripts in proliferating keratinocytes of 2.5 and 4.25 kb (Fig. 1 C), the latter being more abundant. The presence of both mRNAs was confirmed in all mouse tissues tested (data not shown). mc3s5 cDNA was similar to several reported sequences in the nonredundant GenBankTM/EMBL/DDBJ/Protein Data Bank data base. In addition to the TNF-α-inducible sequence reported (96% homology), mc3s5 shares 85% homology over a stretch of 195 bp in the ORF of the bovine chloride channel p64 cDNA that extends over 6 kb (GenBankTM accession number L16547). It also shares 80% homology over 130 bp of the ORF of NCC27(U93205), a human chloride channel with a cDNA of 1.7 kb, and 83% homology with exons 2 and 3 of another human chloride channelCLIC2 (AJ000218). The most similarities are with rat intracellular chloride ion channel p64H1 (AF104119) with 93% identity over 762 bp, and with two human channels: H1chloride channel (AF097330) with 88% homology over 767 bp, and intracellular chloride channel p64H1 (AF109196) with 87% homology over 791 bp. The protein encoded by the ORF also shared high similarity with the three chloride channel proteins present in the data base, bovine p64 (24Landry D. Sullivan S. Nicolaides M. Redhead C. Edelman A. Field M. al-Awqati Q. Edwards J. J. Biol. Chem. 1993; 268: 14948-14955Abstract Full Text PDF PubMed Google Scholar), human NCC27 or CLIC1 (25Valenzuela S.M. Martin D.K. Por S.B. Robbins J.M. Warton K. Bootcov M.R. Schofield P.R. Campbell T.J. Breit S.N. J. Biol. Chem. 1997; 272: 12575-12582Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 26Tulk B.M. Edwards J.C. Am. J. Physiol. 1998; 274: F1140-F1149PubMed Google Scholar), and CLIC2 (27Heiss N.S. Poustka A. Genomics. 1997; 45: 224-228Crossref PubMed Scopus (67) Google Scholar). All three proteins are members of a new family of CLICs (Fig. 2 A). The homology between mc3s5 and p64 is 73% at the C-terminal region, although p64 extends 184 more amino acids at its N terminus with a total length of 437 amino acids. NCC27/CLIC1, the human nuclear chloride channel, is 244 amino acids long, and it shares a 66% homology with mc3s5. CLIC2 was derived from a genomic clone on Xq28. The putative protein of 243 aa shares 65% homology with mc3s5. The newest member of this emerging family, CLIC3 (AAD16450), was recently identified through a yeast two-hybrid screen for proteins that interact with extracellular signal-regulated kinase 7 kinase (28Qian Z. Okuhara D. Abe M.K. Rosner M.R. J. Biol. Chem. 1999; 274: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), a kinase involved in regulation of cell growth. Furthermore, mc3s5 shares 98% homology with the 253 residues of p64H1 (AF104119), a chloride channel protein present on endoplasmic reticulum in rat brain and shown to have outwardly rectifying anion channel activity (29Duncan R.R. Westwood P.K. Boyd A. Ashley R.H. J. Biol. Chem. 1997; 272: 23880-23886Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). mc3s5 differs from p64H1 in only 3 aa in positions 65 (Asp for His), 162 (Asp for Gly), and 174 (Phe for Ser), but these are nonconservative substitutions. Recently, the human homologue of p64H1 (also called CLIC4) was described by two independent groups (30Chuang J.Z. Milner T.A. Zhu M. Sung C.H. J. Neurosci. 1999; 19: 2919-2928Crossref PubMed Google Scholar, 31Edwards J.C. Am. J. Physiol. 1999; 276: F398-F408PubMed Google Scholar). mc3s5 differs in 7 aa when compared with human p64H1 (30Chuang J.Z. Milner T.A. Zhu M. Sung C.H. J. Neurosci. 1999; 19: 2919-2928Crossref PubMed Google Scholar) and in 3 aa when compared with CLIC4 (31Edwards J.C. Am. J. Physiol. 1999; 276: F398-F408PubMed Google Scholar), suggesting t
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