Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane.
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 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-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 beta-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 (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (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 InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).
Treves
S
S
Departments of Anaesthesia and Research, Hebelstrasse 20, Kantonsspital, 4031 Basel, Switzerland.
Feriotto
G
G
Moccagatta
L
L
Gambari
R
R
Zorzato
F
F
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry; D002845:Chromatography; D002874:Chromosome Mapping; D002898:Chromosomes, Human, Pair 8; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D015723:Gene Library; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007425:Intracellular Membranes / Q000378:metabolism; D007438:Introns; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011189:Potassium Chloride / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D012685:Sepharose / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D011189:Potassium Chloride; D012685:Sepharose; C498842:Junctin protein, Oryctolagus cuniculus; D006899:Mixed Function Oxygenases; C058291:aspartic acid 2-oxoglutarate-dependent dioxygenase; C419389:ASPH protein, human; D005982:Glutathione Transferase; D002118:Calcium