Milstein Michelle L, Houle Timothy D, Cala Steven E
Wayne State University, Department of Physiology, Elliman Building, Room 1107, 421 East Canfield Avenue, Detroit, MI 48201, USA.
Exp Cell Res. 2009 Feb 1;315(3):523-34. doi: 10.1016/j.yexcr.2008.11.006. Epub 2008 Nov 25.
Skeletal muscle calsequestrin (skelCSQ) and cardiac calsequestrin (cardCSQ) are resident proteins of the ER/SR, but mechanisms by which CSQ is retained inside membrane lumens remain speculative. A structural model that predicts linear CSQ polymers has been developed that might explain CSQ concentration and localization inside junctional SR lumens, however little evidence exists for polymer formation in intact cells or for its effects on subcellular localization. We previously showed that cardCSQ is efficiently retained within the ER, but its retention is lost under conditions expected to disrupt its polymerization. In the present study, we found unexpectedly that skelCSQ shows no co-localization with cardCSQ in COS cells or in rat neonatal heart cells, but instead concentrates in a membrane compartment (ERGIC) that is just distal to that of cardCSQ. Consistent with this difference in immunofluorescent localization, the structures of CSQ ((316)Asn-linked) glycans showed two types of pre-Golgi processing. Despite the difference in subcellular distribution of individual wild-type forms of CSQ, however, pairs of different CSQ molecules (for example, different isoforms or different fluorescent fusion proteins) consistently co-localized, suggesting that separate forms of CSQ polymerize in different parts of the same secretory pathway, while different CSQ pairs localize together through heteropolymerization.
骨骼肌肌钙蛋白（skelCSQ）和心肌肌钙蛋白（cardCSQ）是内质网/肌浆网（ER/SR）的驻留蛋白，但肌钙蛋白被保留在膜腔内的机制仍属推测。已建立一个预测线性肌钙蛋白聚合物的结构模型，该模型或许可以解释肌钙蛋白在连接肌浆网腔内的浓度和定位，然而，在完整细胞中聚合物形成的证据或其对亚细胞定位的影响却很少。我们之前表明cardCSQ能有效地保留在内质网中，但其保留在预期会破坏其聚合的条件下会丧失。在本研究中，我们意外地发现，skelCSQ在COS细胞或大鼠新生心脏细胞中与cardCSQ没有共定位，而是集中在一个刚好位于cardCSQ远端的膜区室（内质网-高尔基体中间腔，ERGIC）中。与这种免疫荧光定位的差异一致，肌钙蛋白（（316）天冬酰胺连接）聚糖的结构显示出两种高尔基体前加工类型。然而，尽管单个野生型肌钙蛋白形式在亚细胞分布上存在差异，但不同的肌钙蛋白分子对（例如，不同的同工型或不同的荧光融合蛋白）始终共定位，这表明不同形式的肌钙蛋白在同一分泌途径的不同部分聚合，而不同的肌钙蛋白对通过异源聚合一起定位。
