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• W2155656879 abstract "Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH2-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl β-hydroxylase, a member of the α-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl β-hydroxylase result from alternative splicing of the same gene.The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium 45Ca2+ binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca2+/mol protein with a kDof 217 ± 20 μm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP3 receptor activation.Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl β-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate). Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH2-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl β-hydroxylase, a member of the α-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl β-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium 45Ca2+ binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca2+/mol protein with a kDof 217 ± 20 μm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP3 receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl β-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate). sarcoplasmic reticulum endoplasmic reticulum ryanodine receptor enhanced green fluorescent protein polyacrylamide gel electrophoresis N,N,N′,N′-tetramethylethylenediamine fluorescence in situ hybridization polymerase chain reaction(s) glutathione S-transferase base pair(s) transmembrane antibody(ies) The sarcoplasmic reticulum (SR)1 is an intracellular membrane compartment that controls the intracellular Ca2+concentration thereby playing an important role in the excitation-contraction coupling mechanism (for review see Refs. 1Melzer W. Herrmann-Frank A. Luttgau H.C. Biochim. Biophys. Acta. 1995; 1241: 59-116Crossref PubMed Scopus (481) Google Scholar, 2Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (444) Google Scholar, 3Franzini-Armstrong C. Jorgensen A.O. Annu. Rev. Physiol. 1994; 56: 509-534Crossref PubMed Scopus (343) Google Scholar). The anatomical site of excitation-contraction coupling is the triad, a unique intracellular synapsis that is formed by the association of the following membrane compartments: transverse tubules, which are an invagination of the sarcolemma, and the SR terminal cisternae (3Franzini-Armstrong C. Jorgensen A.O. Annu. Rev. Physiol. 1994; 56: 509-534Crossref PubMed Scopus (343) Google Scholar). The portion of terminal cisternae facing the transverse tubules is referred to as junctional face membrane SR (4Costello B. Chadwick C. Saito A. Chu A. Maurer A. Fleischer S. J. Cell Biol. 1986; 103: 741-753Crossref PubMed Scopus (82) Google Scholar). Ordered arrays of junctional feet (3Franzini-Armstrong C. Jorgensen A.O. Annu. Rev. Physiol. 1994; 56: 509-534Crossref PubMed Scopus (343) Google Scholar, 4Costello B. Chadwick C. Saito A. Chu A. Maurer A. Fleischer S. J. Cell Biol. 1986; 103: 741-753Crossref PubMed Scopus (82) Google Scholar), referable to as ryanodine-sensitive Ca2+release channels (RYR) (5Lai F.A. Erickson H.P. Rousseau E. Liu Q.Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (68) Google Scholar, 6Pessah I.N. Francini A.O. Scales D.J. Waterhouse A.L. Casida J.E. J. Biol. Chem. 1986; 261: 8643-8648Abstract Full Text PDF PubMed Google Scholar, 7Inui M. Saito A. Fleischer S. J. Biol. Chem. 1987; 262: 1740-1747Abstract Full Text PDF PubMed Google Scholar, 8Imagawa T. Smith J.S. Coronado R. Campbell K.P. J. Biol. Chem. 1987; 262: 16636-16643Abstract Full Text PDF PubMed Google Scholar, 9Chu A. Diaz-Munoz M. Hawkes M.J. Brush K. Hamilton S.L. Mol. Pharmacol. 1990; 37: 735-741PubMed Google Scholar), bridge the gap of 90–120 Å that separates the membrane of the transverse tubules from the junctional face membrane. The dihydropyridine-sensitive calcium channel of the transverse tubules acts as the voltage sensor for excitation-contraction coupling and plays a crucial role in the regulation of the RYR calcium channel (10Rios E. Pizarro G. Physiol. Rev. 1991; 71: 849-908Crossref PubMed Scopus (492) Google Scholar, 11Lamb G.D. J. Physiol. ( Lond. ). 1986; 376: 85-100Crossref PubMed Scopus (59) Google Scholar, 12Anderson K. Meissner G. J. Gen. Physiol. 1995; 105: 363-383Crossref PubMed Scopus (47) Google Scholar, 13Block B.A. Imagawa T. Campbell K.P. Franzini-Armstrong C. J. Cell Biol. 1988; 107: 2587-2600Crossref PubMed Scopus (589) Google Scholar, 14Tanabe T. Beam K.G. Powell J.A. Numa S. Nature. 1988; 336: 134-139Crossref PubMed Scopus (580) Google Scholar). In addition to the RYR, the junctional face membrane contains several proteins including the histidine-rich calcium binding protein, triadin, calsequestrin, and junctin (15Jones L.R. Zhang L. Sanborn K. Jorgensen A.O. Kelley J. J. Biol. Chem. 1995; 270: 30787-30796Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 16Damiani E. Tobaldin G. Bortoloso E. Margreth A. Cell Calcium. 1997; 22: 129-150Crossref PubMed Scopus (23) Google Scholar, 17Caswell A.H. Brandt N.R. Brunschwig J.P. Purkerson S. Biochemistry. 1991; 30: 7507-7513Crossref PubMed Scopus (134) Google Scholar, 18Kim K.C. Caswell A.H. Talvenheimo J.A. Brandt N.R. Biochemistry. 1990; 29: 9281-9289Crossref PubMed Scopus (106) Google Scholar). During the past decades numerous studies have appeared concerning the biochemical characterization of the protein constituents of the junctional face membrane (19Caswell A.H. Corbett A.M. J. Biol. Chem. 1985; 260: 6892-6898Abstract Full Text PDF PubMed Google Scholar, 20Guo W. Jorgensen A.O. Campbell K.P. J. Biol. Chem. 1994; 269: 28359-28365Abstract Full Text PDF PubMed Google Scholar, 21Guo W. Campbell K.P. J. Biol. Chem. 1995; 270: 9027-9030Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 22Jones L.R. Suzuki Y.J. Wang W. Kobayashi Y.M. Ramesh V. Franzini-Armstrong C. Cleemann L. Morad M. J. Clin. Invest. 1998; 101: 1385-1393Crossref PubMed Scopus (247) Google Scholar, 23Kim D.H. Sreter F.A. Ikemoto N. Biochim. Biophys. Acta. 1988; 945: 246-252Crossref PubMed Scopus (16) Google Scholar, 24Liu G. Pessah I.N. J. Biol. Chem. 1994; 269: 33028-33034Abstract Full Text PDF PubMed Google Scholar, 25Marty I. Robert M. Villaz M. De Jongh K. Lai Y. Catterall W.A. Ronjat M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2270-2274Crossref PubMed Scopus (137) Google Scholar, 26Murray B.E. Ohlendieck K. Biochem. J. 1997; 324: 689-696Crossref PubMed Scopus (77) Google Scholar, 27Saito A. Seiler S. Chu A. Fleischer S. J. Cell Biol. 1984; 99: 875-885Crossref PubMed Scopus (420) Google Scholar, 28Slupsky J.R. Ohnishi M. Carpenter M.R. Reithmeier R.A.F. Biochemistry. 1987; 26: 6539-6544Crossref PubMed Scopus (79) Google Scholar, 29Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). The most abundant polypeptide appears to be calsequestrin, the SR calcium storage protein (30MacLennan D.H. Wong P.T. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 1231-1235Crossref PubMed Scopus (425) Google Scholar), which might also be involved in the regulation of the RYR (31Kawasaki T. Kasai M. Biochem. Biophys. Res. Commun. 1994; 199: 1120-1127Crossref PubMed Scopus (93) Google Scholar, 32Ikemoto N. Ronjat M. Meszaros L.G. Koshita M. Biochemistry. 1989; 28: 6764-6771Crossref PubMed Scopus (187) Google Scholar). Whether this effect is mediated by a direct interaction between the two proteins or via bridging calsequestrin binding proteins such as triadin or junctin is still controversial (17Caswell A.H. Brandt N.R. Brunschwig J.P. Purkerson S. Biochemistry. 1991; 30: 7507-7513Crossref PubMed Scopus (134) Google Scholar, 21Guo W. Campbell K.P. J. Biol. Chem. 1995; 270: 9027-9030Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 29Zhang L. Kelley J. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 33Franzini-Armstrong C. Kenney L.J. Varriano-Marston E. J. Cell Biol. 1987; 105: 49-56Crossref PubMed Scopus (167) Google Scholar). The junctional face membrane is endowed with numerous other less abundant proteins having a molecular mass ranging from 20 kDa up to 120 kDa, which have yet to be identified and characterized at the molecular level (4Costello B. Chadwick C. Saito A. Chu A. Maurer A. Fleischer S. J. Cell Biol. 1986; 103: 741-753Crossref PubMed Scopus (82) Google Scholar). Because of their localization, the proteins that are present on the junctional face membrane are deemed to be involved in the excitation-contraction coupling mechanism, and a defect in the function of these proteins could potentially lead to alterations of the Ca2+ release process and/or intracellular Ca2+ homeostasis. Thus the characterization of all the molecular components of the junctional face membrane is important not only for our understanding of the basic mechanism of Ca2+storage and release in muscle and non-muscle cells but also in view of the enormous effort aimed at identifying novel genes which, upon mutation, may be linked to neuro/muscular diseases (34MacLennan D.H. Phillips M.S. Science. 1992; 256: 789-794Crossref PubMed Scopus (413) Google Scholar, 35Mickelson J.R. Louis C.F. Physiol. Rev. 1996; 76: 537-592Crossref PubMed Scopus (259) Google Scholar). In the present report, we demonstrate the existence of junctate, a novel integral Ca2+ binding protein of sarco(endo)plasmic reticulum membranes. The Ca2+ binding properties of junctate indicate that the protein might have an active role in the Ca2+ storage/release process in ER membranes in a variety of tissues including heart, brain, pancreas, lung, liver, kidney, and skeletal muscle. In addition, we report that an enzyme (aspartyl β-hydroxylase), a structural protein of sarco(endo)plasmic reticulum membranes (junctin), and a calcium binding protein (junctate) belong to a family of single membrane-spanning proteins that result from alternative splicing events of the same gene located in human chromosome 8. Nitrocellulose was from Amersham Pharmacia Biotech; isopropyl-β-d-thiogalactoside, restriction enzymes, the chemiluminescence kit, synthetic primers, DNA-modifying enzymes, the DNA-digoxigenin labeling kit, and anti-digoxigenin peroxidase-conjugated antibodies were from Roche Molecular Biochemicals and New England Biolabs; the λtriplex, λgt10 human skeletal muscle, and λgt10 human cardiac cDNA libraries, pEGFP plasmids, and multiple tissue Northern blots were from CLONTECH; the Bluescript cloning vector was from Stratagene; the pGex plasmid was from Amersham Pharmacia Biotech; heparin-agarose and glutathione were from Sigma; protein molecular weight markers were from Bio-Rad; [45Ca] and [32P]dATP were from PerkinElmer Life Sciences; Lipofectin was from Life Technologies, Inc.; indo-1 was from Molecular Probes; and all other chemicals were reagent grade. Terminal cisternae, longitudinal sarcoplasmic reticulum, transverse tubules, and sarcolemma were obtained from the white skeletal muscle of New Zealand rabbits as described (27Saito A. Seiler S. Chu A. Fleischer S. J. Cell Biol. 1984; 99: 875-885Crossref PubMed Scopus (420) Google Scholar). Protein concentration was according to Lowryet al. (36Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as standard. Vesicles obtained from the various fractions of rabbit skeletal muscle were resuspended at a final concentration of 1 mg/ml in 1% Triton X-100, 200 mm NaCl, 50 mm Tris-HCl, pH 8.5, 1 mm dithiothreitol, 1 mm EDTA containing 1 mg/ml leupeptin, 100 μmphenylmethylsulfonyl fluoride, and benzamidine, and 1 mmpepstatin as anti-proteolytic agents (mixture of anti-proteolytic agents). The vesicles were solubilized for 30 min at room temperature under gentle agitation and centrifuged at 100,000 ×gmax for 30 min at 4 °C. The supernatant was incubated with a heparin-agarose resin (1 ml of resin/mg of protein) previously equilibrated in 50 mm Tris-HCl, pH 8.5, 200 mm NaCl, 1 mm dithiothreitol, and 1 mm EDTA (Buffer A) for 60 min at room temperature under gentle agitation. The column was washed with 10 bed volumes of Buffer A containing 0.1% Triton X-100 and proteins eluted with a step gradient of NaCl (300 mm, 500 mm, 750 mm,and 1 m) in Buffer A. Identification of the proteins contained in the 500 mm NaCl fraction was carried by Western blot analysis and microsequencing. SDS-PAGE was carried out as described by Laemmli (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206658) Google Scholar); proteins were visualized by Coomassie Brilliant Blue staining or by Stains All staining as described previously (38Treves S. De Mattei M. Landfredi M. Villa A. Green N.M. MacLennan D.H. Meldolesi J. Pozzan T. Biochem. J. 1990; 271: 473-480Crossref PubMed Scopus (99) Google Scholar). Western blots were prepared as described previously (38Treves S. De Mattei M. Landfredi M. Villa A. Green N.M. MacLennan D.H. Meldolesi J. Pozzan T. Biochem. J. 1990; 271: 473-480Crossref PubMed Scopus (99) Google Scholar). The fraction eluting from the heparin-agarose column at 500 mm NaCl was concentrated by addition of 3 volumes of cold acetone. The insoluble protein fraction was collected as a pellet after centrifugation at 4000 rpm (3000 × gmax) in an ALC 5400 centrifuge. The pellet was resuspended in 50 mm Tris-HCl, pH 6.8, 2% SDS, and 0.1% bromphenol blue, and the proteins were separated by SDS gel electrophoresis in a 10% slab gel. The proteins were revealed by imidazole/ZnSO4 reverse staining. To concentrate the protein, gel slices containing the 27-kDa protein bands (×8) were transferred to a rod gel in a Pasteur pipette. The glass of the Pasteur pipette tube was treated with 5% Cl2(CH3)2Si/CHCl3(dichloromethylsylane), dried, and the glass tubes were filled with a solution containing 5% acrylamide, 0.13% bisacrylamide, 125 mm Tris-HCl, pH 6.7. Polymerization was initiated by the addition of ammoniumpersulfate and TEMED. The gel slices were loaded on the rod gel in the presence of 5 ml of 50 mm Tris-HCl, pH 6.8, 2% SDS, 0.1% bromphenol blue. Electrophoresis was carried out at 150–200 mV until the bromphenol blue reached the narrow part of the Pasteur pipette. The protein band was visualized by Coomassie Brilliant Blue staining. The slice of gel containing the protein was washed 3 times with 1 ml of MilliQ H2O and then incubated in 140 μl of CNBr/HCO2H (58.1 mg of CNBr/1.162 ml of HCO2H) plus 60 ml of H2O overnight at room temperature under vortex stirring. The next day the supernatant was transferred to another microvial and dried in a speed vac. The peptides derived from the 27-kDa protein were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membrane. The bands were visualized by Coomassie Brilliant Blue staining. The NH2-terminal sequence and the polypeptides derived from CNBr cleavage were subjected to amino acid sequencing several times (carried out by Prof. V. Hoppe, Physiologische Chemie II, Institute für Biowissenschaften der Univesität Würzburg, Würzburg, Germany). A human λtriplex oligo(dT)-primed skeletal muscle library was screened using a 32P-labeled cDNA probe obtained by RT-PCR of human skeletal muscle using forward and reverse primers obtained from the protein sequence of peptides 1 and 3; the sequences were 5′-AGA ATT CAC AGA GGA CAA AGA G-3′ and 5′-AGA ATT CTA ACG CGA TGA CCA T-3′, respectively. Amplification conditions were as follows: 45 annealing at 45 °C, 30 extension at 72 °C, and 30 denaturation at 95 °C for a total of 35 cycles. The addition of an EcoRI restriction enzyme sequence was used to facilitate subcloning and sequencing of the amplified cDNA. After overnight hybridization of the filters at 42 °C with the labeled probe, the filters were washed at high stringency at 65 °C in 0.2× SSCP, 0.1% SDS; positive plaques were identified and purified, and the DNA was prepared according to the manufacturer's recommendations. Templates for sequencing were prepared in the Bluescript cloning vector. To obtain the full-length sequence we carried out nested exonuclease III/mung bean nuclease deletions according to a previously described procedure (39Zorzato F. Fujii J. Otsu K. Phillips M. Green N.M. Lai F.A. Meissner G. MacLennan D.H. J. Biol. Chem. 1990; 265: 2244-2256Abstract Full Text PDF PubMed Google Scholar). Random/oligo(dT)-primed λgt10 human skeletal and cardiac muscle libraries using a cDNA 32P-labeled probe were obtained from λtriplex library clone. The primary sequence of the clones was obtained with an automated DNA sequencer using the dideoxy method. Human BAC genomic libraries were screened as described previously (40Korenberg J.R. Chen X.N. Sun Z. Shi Z.Y. Ma S. Vataru E. Yimlamai D. Weissenbach J.S. Shizuya H. Simon M.I. Gerety S.S. Nguyen H. Zemsteva I.S. Hui L. Silva J. Wu X. Birren B.W. Hudson T.J. Genome Res. 1999; 9: 994-1001Crossref PubMed Scopus (48) Google Scholar). To identify BAC clones, colonies were lifted onto nylon membrane for hybridization probe analysis with radioactively labeled cDNA probes encompassing the 3′ end of both human junctin and junctate. FISH analysis was performed by Genomics Inc. (St. Louis, MO). Extra long PCR products were obtained using the GeneAmp XL PCR kit (Applied Biosystems, Foster City, CA) from 1 μg of BAC or genomic DNA with either 28 or 37 amplification cycles using the PCR primers reported in Table II. PCRs were carried out according to the manufacturer's instructions; the products were purified with Microcon-100 (Millipore) and sequenced with the ABI PRISM Big Dye terminator cycle sequencing ready reaction kit using the ABI PRISM 377 DNA sequencer (PE Applied Biosystems, Foster City, CA).Figure 7Structure of the 5′ end of the human locus for aspartyl β-hydroxylase, junctin, and junctate. The top of the panel shows some of the PCRs performed using BAC 1, BAC 2, and genomic DNA as template. Three plasmid subclones of BAC 1 are also shown, covering the first and second exons of the locus. B, BglII;E, EcoRI; P, PstI.Arabic numbers over black boxes indicate exons. Intervening sequences are indicated in Roman numbers. Hatched bars represent part of the two BAC clones analyzed. A schematic representation of the assembly of aspartyl β-hydroxylase, junctin, and junctate exon splicing is reported at the bottom of thepanel. The cytoplasmic, TM, positively charged, and calcium-binding domains are indicated. The locations of AUG, stop codons, and poly(A) signals are shown. The base pair length of the exons is as follows: exon 1, 180; exon 2, 161; exon 3, 150; exon 4, 45; exon 5, 69; exon 6, 42; exon 7, 1139 to the first poly(A) signal; exon 8, 93; exon 9, 75; exon 10, 129; exon 11, 90; exon 12, 48; exon 13, 33; exon 14, 42; exon 15, 57; exon 16, 45; and exon 17, 1647 to the second poly(A) signal (numbering of exons is temporary, because the mapping of the cluster is not complete).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IISynthetic oligonucleotides used for long PCR experimentse1F5′-CAAGAGCAGCGGCAACAG-3′i1R5′-GTACCAACATTTGATGGTCCAG-3′e2F5′-ACCTGCCAGCAGTACTTTTG-3′e3F5′-CAAAGCATGGAGGACACAAGAATG-3′e3R5′-TTCCTGAGAGTCCGCCTTTC-3′e4F5′-TAACTTATCAGAGGTGCTTCA-3′e4R5′-TGAAGCACCTCTGATAAGTTA-3′e5F5′-GATGGAGATTTTGATGTGGATG-3′e5R5′-AATAAAACTTTGGCATCATCCACATCAAAATCTCC-3′e6F5′-GGGGTAGCCAAGAGAAAAACTAAG-3′e6R5′-TTTCTCTTGGCTACCCCACTGG-3′e7F5′-AAAGGGGAAGAAAGAGGATGTCC-3′e7R5′-CAAACTGCAATCTGGAACATGACC-3′e8F5′-ATCTACTTCAGAGCCAGCAGTCCC-3′e8R5′-CCTCCACAGGAACCTGCTCCTC-3′e9F5′-CAGTCCCTTCTCCATGAAATGG-3′e9R5′-TTCTGCGTGTACCATTTCATGG-3′e10F5′-TGCAACAAGAAGATGGACCCAC-3′e10R5′-TCAGGTTCCAGGGTCTCAAATC-3′e11F5′-CGTGGAAGAGACAGTTTCACAAG-3′e11R5′-GTCTTGTGAAACTGTCTCTTCCAC-3′e13F5′-GTAACATACCAAGTCTATGAGGAAC-3′e13R5′-TGCTTGTTCCTCATAGACTTGG-3′i13F5′-TCTCTGCCCTTAAAGCATG-3′e14R5′-TACCTGTGATTTCTATCCCTTC-3′e15F5′-TGCTCCCCCTGAGGATAATC-3′e15R5′-GGATTATCCTCAGGGGGAG-3′e16F5′-GCATTTTTCCTGTGGAAGAAC-3′e16R5′-TGGTGGTACTTCCTGCTG-3′e17F15′-TCAAAAAGACTGCCCCTACC-3′e17R15′-AGCGTATGGTTAGGCTGGTC-3′e17R25′-GGACATGCAGGAATGTAACATGAC-3′e17R35′-TCAGGTAAGACTAAAAGCTGTCTC-3′e17F25′-CTGAATTTATGTGTGTTTTTCCGAC-3′e17F35′-CCTGCATGGGGTACTCGATG-3′e17R45′-ACACATCGAGTACCCCATGCAG-3′e18R5′-GCTTTTTGTTCTGGATCATCTG-3′e = exon; i = intron; F = forward; R = reverse. Open table in a new tab e = exon; i = intron; F = forward; R = reverse. 2 μg of poly(A)+ RNA from eight different human tissues (CLONTECH) and 20 μg of total human genomic DNA digested with EcoRI and PvuII and blotted onto nylon membranes were hybridized with the appropriate cDNA probes as described previously (38Treves S. De Mattei M. Landfredi M. Villa A. Green N.M. MacLennan D.H. Meldolesi J. Pozzan T. Biochem. J. 1990; 271: 473-480Crossref PubMed Scopus (99) Google Scholar). Hybridization was performed twice with two distinct membranes. Blots were washed under high stringency (0.2× SSCP, 0.1%SDS at 65 °C for 60 min), and autoradiography was performed for 1 week (Northern blot) or 3 weeks (Southern blots) at −80 °C with an intensifying screen. Northern blots were also probed with digoxigenin-labeled β-actin probe and washed under high stringency, and the tissue distribution of β-actin was revealed using an anti-digoxigenin peroxidase-conjugated (1:10,000) antibody followed by chemiluminescence. A GST fusion protein containing the COOH-terminal domain of junctate was constructed by fusing the 610-bp PCR-amplified cDNA (amino acid residues 98–298) in frame into the multiple cloning site of pGex5 × 3. The forward and reverse primers were, respectively, 5′-TCT CGA GGGGCA GTC TTT TTG AA-3′ and 5′-AGA ATT CTA CTT CAG AGC CAG CA-3′. Amplification conditions were as described for cDNA cloning except that the annealing temperature was 60 °C; the addition of theXhoI/EcoRI restriction enzyme sequence was used to facilitate cloning and sequencing of the amplified sequence. The GST fusion protein was purified from the bacteria using glutathione-Sepharose 4B as described by the manufacturer. Calcium overlays on Western blots were carried out as described (41Maruyama K. Mikawa T. Ebashi S. J. Biochem. ( Tokyo ). 1984; 95: 511-519Crossref PubMed Scopus (628) Google Scholar). Calcium equilibrium binding was carried out by the use of a continuos flow microdialysis chamber as described previously (38Treves S. De Mattei M. Landfredi M. Villa A. Green N.M. MacLennan D.H. Meldolesi J. Pozzan T. Biochem. J. 1990; 271: 473-480Crossref PubMed Scopus (99) Google Scholar). To monitor the intracellular distribution of junctate, we cloned it into the pEGFPC1 mammalian expression vector and monitored expression of the recombinant protein after transfection into COS-7 cells. Two NH2-terminal green fluorescent protein-tagged constructs were made; one encompassed the whole coding sequence of junctate (nucleotides 1–979; EGFP-junctate), whereas the other encompassed the putative hydrophobic transmembrane domain between nucleotides 137 and 254 (EGFP-TM-junctate). PCR amplification conditions were as described for cDNA cloning except that the annealing temperature was 58 °C. TheEcoRI/BamHI restriction enzyme sequence was used to facilitate cloning and sequencing of the amplified sequence into the MCS of the pEGFPC1 plasmid. Forward and reverse primers for the EGFP-junctate construct were 5′-AGA ATT CAC AAA TGG CTG AAG-3′ and 5′-GAA GCT TTT AGG ATC CTG GTG-3′, respectively; forward and reverse primers for the EGFP-TM-junctate construct were 5′-GGA ATT CCA CCA TGA GGA AAG GCG GAC TCT CA-3′, and 5′-GGG ATC CCT TTG CTT TGG CTA GA, respectively. COS-7 cells grown on glass coverslips were transfected using Lipofectin as described previously (42Censier K. Urwyler A. Zorzato F. Treves S. J. Clin. Invest. 1998; 101: 1233-1242Crossref PubMed Scopus (91) Google Scholar). 24 or 48 h after transfection cells were washed 3 times with phosphate-buffered saline, fixed with 3.7% formaldehyde in phosphate-buffered saline for 20 min, and examined under fluorescent light (excitation, 480 nm; emission, 510 nm) using a Nikon Diaphot 300 inverted microscope equipped with a PlanApo × 100/1.40 objective. The free cytoplasmic Ca2+ concentration of COS-7 cells was determined using the fluorescent Ca2+ indicator indo-1 using a Nikon Diaphot 300 inverted fluorescent microscope equipped with a × 60 Plan-Apo oil immersion objective attached to two photomultipliers (P100; Nikon Inc.). The cell to be measured was identified by its green fluorescence and then ratio fluorescence measurements (410/480 nm) were obtained as described previously (42Censier K. Urwyler A. Zorzato F. Treves S. J. Clin. Invest. 1998; 101: 1233-1242Crossref PubMed Scopus (91) Google Scholar). Excitation of indo-1 was achieved using a 100-watt mercury lamp attenuated by neutral density filters to avoid dramatic photobleaching. Stimulation of individual cells was obtained by an 8-way 100-μm diameter quartz micromanifold computer-controlled microperfusor (ALA Scientific). The total amount of calcium released and statistical analysis were performed using the Origin computer program (Microcal Software, Inc., Northampton, MA). Kidneys from 3.0–3.5-kg male New Zealand rabbit were homogenized (10% w/v) in a buffer containing 10 mm HEPES, pH 7.2, 150 mm KCl plus a mixture of anti-proteolytic agents (see “Heparin-Agarose Chromatography”). The homogenate was centrifuged at 3,000 ×gmax for 10 min, and the resulting supernatant was centrifuged at 10,000 × gmax for 15 min. The 10,000 × gmax supernatant was then filtered through 10 layers of cheesecloth and centrifuged at 150,000 × gmax for 60 min. The pellet was resuspended in a solution containing 10 mm HEPES, pH 7.2, 0.6 m KCl plus anti-proteolytic agents and was centrifuged at 150,000 × gmax for 60 min. The KCl-washed membranes were resuspended in a solution containing 10 mm HEPES, pH 7.2, 150 mm KCl at a final concentration of 1–2 mg/ml. The microsomal suspension was then incubated for 30 min at 4 °C in the presence of 100 mmNa2CO3, pH 11. The membrane fraction pellet was obtained by centrifugation at 150,000 ×gmax for 60 min and washed with 0.6m KCl. Biochemical analyses of kidney microsomes were carried out with three different preparations with microsomes isolated from two different animals. Preparation of rabbit heart microsomes was essentially as described by Pessah et al. (43Pessah I.N. Durie E.L. Schiedt M.J. Zimanyi I. Mol. Pharmacol. 1990; 37: 503-514PubMed Google Scholar), except that the total microsomal fraction was washed with 0.6 m KCl before being layered onto the sucrose gradient to remove cytoskeletal proteins. Polyclonal Abs were raised by immunizing mice with the proteins present in the fraction elu" @default.
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• W2155656879 title "Molecular Cloning, Expression, Functional Characterization, Chromosomal Localization, and Gene Structure of Junctate, a Novel Integral Calcium Binding Protein of Sarco(endo)plasmic Reticulum Membrane" @default.
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