Purification, primary structure, and immunological characterization of the 26-kDa calsequestrin binding protein (junctin) from cardiac junctional sarcoplasmic reticulum
Purification, primary structure, and immunological characterization of the 26-kDa calsequestrin binding protein (junctin) from cardiac junctional sarcoplasmic reticulum
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DOI：
10.1074/jbc.270.51.30787
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发表时间：
1995-12-22
影响因子：
4.8
通讯作者：
Kelley, J
中科院分区：
文献类型：
--
作者：
Jones, LR;Zhang, L;Kelley, J
以前，我们鉴定了一种表观M（r）= 26，000的蛋白质作为分离自心肌和骨骼肌的连接肌浆网囊泡中的主要钙螯合蛋白结合蛋白（Mitchell，R. D、Simmerman，H. K. B.，和Jones，L. R.（1988）J.Biol.Chem.263，1376-1381）。在这里，我们描述的26 kDa的钙螯合蛋白结合蛋白的纯化和一级结构。从心脏微粒体中纯化了164倍的蛋白质，并通过免疫印迹显示高度富集在交界膜亚组分中。该蛋白在SDS聚丙烯酰胺凝胶电泳上呈紧密的双峰，与I-125-Calsequestrin结合强烈。该蛋白质具有位于细胞质中的短的N-末端区域，并且分子的大部分是高电荷和碱性的，投射到肌浆网腔中。与三醇溶蛋白和β-羟化酶的同源性很高，这表明这三种蛋白质都是一个跨膜内质网蛋白家族的成员。免疫细胞化学标记定位的26 kDa蛋白质的连接肌浆网在心脏和骨骼肌。在这两种组织中表达相同的基因产物。26-kDa蛋白结合钙螯合蛋白的活性及其与钙螯合蛋白和ryanodine受体的共分布强烈表明该蛋白在钙释放复合物的组织和/或功能中起重要作用。由于26-kDa钙螯合蛋白结合蛋白是心肌和骨骼肌中连接肌质网膜的组成部分，我们将其命名为Junctin。
Previously we identified a protein of apparent M(r) = 26,000 as the major calsequestrin binding protein in junctional sarcoplasmic reticulum vesicles isolated from cardiac and skeletal muscle (Mitchell, R. D., Simmerman, H. K. B., and Jones, L. R. (1988) J. Biol. Chem. 263, 1376-1381). Here we describe the purification and primary structure of the 26-kDa calsequestrin binding protein. The protein was purified 164-fold from cardiac microsomes and shown by immunoblotting to be highly enriched in junctional membrane subfractions. It ran as a closely spaced doublet on SDS polyacrylamide gel electrophoresis and bound I-125-calsequestrin intensely, Cloning of the cDNA predicted a protein of 210 amino acids containing a single transmembrane domain. The protein has a short N-terminal region located in the cytoplasm, and the bulk of the molecule, which is highly charged and basic, projects into the sarcoplasmic reticulum lumen. Significant homologies were found with triadin and aspartyl beta-hydroxylase, suggesting that all three proteins are members of a family of single membrane spanning endoplasmic reticulum proteins. Immunocytochemical labeling localized the 26-kDa protein to junctional sarcoplasmic reticulum in cardiac and skeletal muscle. The same gene product was expressed in these two tissues. The calsequestrin binding activity of the 26-kDa protein combined with its codistribution with calsequestrin and ryanodine receptors strongly suggests that the protein plays an important role in the organization and/or function of the Ca2+ release complex, Because the 26-kDa calsequestrin binding protein is an integral component of the junctional sarcoplasmic reticulum membrane in cardiac and skeletal muscle, we have named it Junctin.