Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.
A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.
Sacchetto
R
R
NRC Unit for Muscle Biology and Physiopathology, Department of Experimental Biomedical Sciences, University of Padova, viale Giuseppe Colombo 3, Padua, 35121, Italy.
Damiani
E
E
Turcato
F
F
Nori
A
A
Margreth
A
A
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D004355:Drug Stability; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C489419:HRC protein, Oryctolagus cuniculus; D046911:Macromolecular Substances; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C070048:triadin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium