Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy.
Department of Biochemistry and Molecular Biology, University of British Columbia, V6T 1Z4 Vancouver, Canada.
J Cell Sci. 2022 Jan 15;135(2). doi: 10.1242/jcs.259185. Epub 2022 Jan 25.
Junctin is a transmembrane protein of striated muscles, located at the junctional sarcoplasmic reticulum (SR). It is characterized by a luminal C-terminal tail, through which it functionally interacts with calsequestrin and the ryanodine receptor (RyR). Interaction with calsequestrin was ascribed to the presence of stretches of charged amino acids (aa). However, the regions able to bind calsequestrin have not been defined in detail. We report here that, in non-muscle cells, junctin and calsequestrin assemble in long linear regions within the endoplasmic reticulum, mirroring the formation of calsequestrin polymers. In differentiating myotubes, the two proteins colocalize at triads, where they assemble with other proteins of the junctional SR. By performing GST pull-down assays with distinct regions of the junctin tail, we identified two KEKE motifs that can bind calsequestrin. In addition, stretches of charged aa downstream these motifs were found to also bind calsequestrin and the RyR. Deletion of even one of these regions impaired the ability of junctin to localize at the junctional SR, suggesting that interaction with other proteins at this site represents a key element in junctin targeting.
连接蛋白是一种存在于横纹肌的跨膜蛋白，位于连接肌质网（SR）。它的特征是具有一个腔末端的 C 端尾巴，通过这个尾巴与钙池蛋白和兰尼碱受体（RyR）进行功能交互。与钙池蛋白的相互作用归因于存在伸展的带电荷氨基酸（aa）。然而，能够结合钙池蛋白的区域尚未详细定义。我们在这里报告，在非肌肉细胞中，连接蛋白和钙池蛋白在内质网内的长线性区域组装，反映了钙池蛋白聚合物的形成。在分化的肌管中，这两种蛋白在三联体处共定位，在那里它们与连接 SR 的其他蛋白组装。通过用连接蛋白尾部的不同区域进行 GST 下拉测定，我们鉴定出两个可以结合钙池蛋白的 KEKE 基序。此外，在这些基序下游的带电荷 aa 片段也被发现可以结合钙池蛋白和 RyR。即使删除这些区域中的一个也会损害连接蛋白在连接 SR 定位的能力，这表明与该部位的其他蛋白相互作用是连接蛋白靶向的关键要素。
