Summary
The Ca2+ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the Junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m) [125I]iodophenyl diazirine and then isolated and proteolysed with calpain II, the [125I] was traced from the 565 kDa parent to M r, 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa frament was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin
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Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein dye binding. Anal. Biochem 72:248–254
Brandt, N.R., Caswell, A.H., Brunschwig, J.-P. 1980. ATP-energized Ca2+ pump in isolated transverse tubules of skeletal muscle. J. Biol. Chem 225:6290–6298
Brandt, N.R., Caswell, A.H., Wen, S.-R., Talvenheimo, J.A. 1990. Molecular interactions of the junctional foot protein and dihydropyridine receptor in skeletal muscle triads. J. Membrane Biol 113:237–251
Brunner, J., Franzusoff, A.T., Luscher, B., Zugliani, C., Semenzo, G. 1985. Membrane protein topology: Amino acid residues in a putative transmembrane of helix of bacteriorho-dopsin labeled with the hydrophobic carbene-generating reagent 3-(trifluoromethyl)-3-m-([125I] iodophenyl) diazirine. Biochemistry 24:5422–5430
Caswell, A.H., Brandt, N.R., Brunschwig, J.-P., Purkerson, S. 1991. Localization and partial characterization of the oligomeric disulfide-linked molecular weight 95000 protein (Triadin) which binds the ryanodine and dihydropyridine receptors in skeletal muscle triadic vesicles. Biochemistry 30:7507–7513
Caswell, A.H., Lau, Y.H., Brunschwig, J.-P. 1976. Ouabain-binding vesicles from skeletal muscle. Arch. Biochem. Biophys 176:417–430
Clarke, D.M., Loo, T.W., Inesi, G., MacLennan, D.H. 1989. Localization of high affinity Ca2+ binding sites within the predicted transmembrane domain of the sacroplasmic reticulum Ca2+ ATPase. Nature 339:476–478
Dayhoff, M.O., Burker, W.C., Hunt, L.T. 1983. Establishing homologies in protein sequences. Methods Enzymol 91:524–545
Dayton, W.R., Reville, W.J., Goll, D.E., Stromer, M.A. 1976. A Ca2+-activated protease possibly involved in myofibriilar protein turnover. Partial characterization of the purified enzyme. Biochemistry 15:2159–2167
Dombradi, W.K., Silberman, S.R., Lee, E.Y.C., Caswell, A.H., Brandt, N.R. 1984. The association of phosphorylase kinase with rabbit muscle T-tubules. Arch. Biochem. Biophys 230:615–630
Finer-Moore, J., Stroud, R. 1984. Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc. Natl. Acad. Sci. USA 81:155–159
Gopalakrishna, R., Barsky, S.H. 1986. Hydrophobic association of calpains with subcellular organelles compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues. J. Biol. Chem 261:13936–13942
Kasuga, N., Umazume, Y. 1990. Deterioration introduced by physiological concentration of calcium ions in skinned muscle fibers. J. Muscle Res. Cell Motil 11:41–47
Kawamoto, R.M., Brunschwig, J.-P., Kim, K.C., Caswell, A.H. 1986. Isolation, characterization, and location of the spanning protein from skeletal muscle triads. J. Cell. Biol 103:1405–1414
Kim, K.C., Caswell, A.H., Brunschwig, J.-P., Brandt, N.R. 1990. Identification of new subpopulation of triad junctions isolated from skeletal muscle. Morphological correlations with intact muscle. J. Membrane Biol 113:221–235
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685
Lai, F.A., Meissner, G. 1989. The muscle ryanodine receptor and its instrinsic Ca2+ channel activity. J. Bioenerg. Biomembr 21:227–246
Lai, F.A., Misra, M., Xu, L., Smith, H.A., Meissner, G. 1989. The ryanodine receptor-Ca2+ release channel complex of skeletal muscle sarcoplasmic reticulum: Evidence for a cooperative coupled, negatively charged homotetramer. J. Biol. Chem 264:16776–16785
Marks, A.R., Fleischer, S., Tempst, P. 1990. Surface topography analysis of the ryanodine receptor/junctional channel complex based on proteolysis sensitivity mapping. J. Biol. Chem 265:13143–13149
McLachlen, A.D. 1976. The 14-fold periodicity in α-tropomyosin and the interaction with actin. J. Mol. Biol 103:271–298
Mellgren, R.L., Lane, R.D. 1988. Myocardial calpain 2 is inhibited by monoclonal antibodies specific for small, noncatalytic subunit. Biochim. Biophys. Acta 954:154–160
Mellgren, R.L., Repetti, A., Muck, T.C., Easly, J. 1982. Rabbit skeletal muscle calcium-dependent protease requiring millimolar Ca2+. J. Biol. Chem 257:7203–7209
Meyer, W.L., Fischer S.H., Krebs, E.G. 1964. Activation of skeletal muscle phosphorylase b kinase by Ca2+. Biochemistry 3:1033–1039
Otsu, K., Willard, H.F., Khanna, V.K., Zorzato, F., Green, N.H., MacLennan, D.H. 1990. Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sacroplasmic reticulum. J. Biol. Chem 265:13472–13483
Penner, R., Neher, E., Takeshima, H., Nishimura, S., Numa, S. 1989. Functional expression of the calcium release channel from skeletal muscle ryanodine receptor DNA. FEBS Lett 259:217–221
Rardon, D.P., Cefali, D.C., Mitchell, R.D., Seiler, S.M., Hathaway, D.R., Jones, L.R. 1990. Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Circ. Res 67:84–96
Rogers, S., Wells, R., Rechsteiner, M. 1986. Amino acid sequences common to rapidly degraded proteins: The pest hypothesis. Science 234:364–368
Seiler, S., Wegener, A.D., Whang, D.D., Hathaway, D.R., Jones, L.R. 1983. High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum bind calmodulin, are phosphorylated and are degraded by Ca2+ activated protease. J. Biol. Chem 257:8550–8557
Solaro, R.S., Pang, D.C., Briggs, F.N. 1971. The purification of cardiac myofibrils with Triton X-100. Biochim. Biophys. Acta 245:259–262
Takeshima, H., Nishimura, S., Matsumoto, R., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., Numa, S. 1989. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339:439–445
Thieleczek, R., Mayr, G.W., Brandt, N.R. 1989. Inositol polyphosphate-mediated repartitioning of aldolase in skeletal muscle triads and myofibrils. J. Biol. Chem 264:7349–7356
Walker, J.E., Saraste, M., Runwick, M.J., Gay, N.J., 1982. Distinctly related sequences in the α and β subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBOJ 1:945–951
Wang, K.K.W., Villalobo, A., Roufogalis, B.D. 1989. Calmodulin-binding proteins as calpain substrates. Biochem. J 262:693–706
Yoshimura, N., Murachi, T., Heath, R., Kag, J., Jasani, B., Newman, G.R. 1986. Immunogold electro-microscopic localisation of calpain I in skeleton muscle of rats. Cell Tissue Res 244:265–270
Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N.M., Lai, F.A., Meissner, G., MacLennan, D.H. 1990. Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sacroplasmic reticulum. J. Biol. Chem 265:2244–2256
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Brandt, N.R., Caswell, A.H., Brandt, T. et al. Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction. J. Membarin Biol 127, 35–47 (1992). https://doi.org/10.1007/BF00232756
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DOI: https://doi.org/10.1007/BF00232756