Corbett E F, Oikawa K, Francois P, Tessier D C, Kay C, Bergeron J J, Thomas D Y, Krause K H, Michalak M
Medical Research Council of Canada (MRC) Group in Molecular Biology of Membranes, Protein Engineering Network of Centers of Excellence, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 1999 Mar 5;274(10):6203-11. doi: 10.1074/jbc.274.10.6203.
Casade Blue (CB), a fluorescent dye, was used to investigate the dynamics of interactions between endoplasmic reticulum (ER) lumenal chaperones including calreticulin, protein disulfide isomerase (PDI), and ERp57. PDI and ERp57 were labeled with CB, and subsequently, we show that the fluorescence intensity of the CB-conjugated proteins changes upon exposure to microenvironments of a different polarity. CD analysis of the purified proteins revealed that changes in the fluorescence intensity of CB-ERp57 and CB-PDI correspond to conformational changes in the proteins. Using this technique we demonstrate that PDI interacts with calreticulin at low Ca2+ concentration (below 100 microM), whereas the protein complex dissociates at >400 microM Ca2+. These are the Ca2+ concentrations reminiscent of Ca2+ levels found in empty or full ER Ca2+ stores. The N-domain of calreticulin interacts with PDI, but Ca2+ binding to the C-domain of the protein is responsible for Ca2+ sensitivity of the interaction. ERp57 also interacts with calreticulin through the N-domain of the protein. Initial interaction between these proteins is Ca2+-independent, but it is modulated by Ca2+ binding to the C-domain of calreticulin. We conclude that changes in ER lumenal Ca2+ concentration may be responsible for the regulation of protein-protein interactions. Calreticulin may play a role of Ca2+ "sensor" for ER chaperones via regulation of Ca2+-dependent formation and maintenance of structural and functional complexes between different proteins involved in a variety of steps during protein synthesis, folding, and post-translational modification.
卡萨德蓝（CB）是一种荧光染料，用于研究内质网（ER）腔伴侣蛋白之间的相互作用动态，这些伴侣蛋白包括钙网蛋白、蛋白二硫键异构酶（PDI）和ERp57。PDI和ERp57用CB进行标记，随后，我们发现结合了CB的蛋白质的荧光强度在暴露于不同极性的微环境时会发生变化。对纯化蛋白的圆二色性（CD）分析表明，CB-ERp57和CB-PDI荧光强度的变化与蛋白质的构象变化相对应。利用这项技术，我们证明在低Ca2+浓度（低于100微摩尔）下PDI与钙网蛋白相互作用，而当Ca2+浓度>400微摩尔时该蛋白复合物会解离。这些Ca2+浓度让人联想到在空的或充满Ca2+的内质网Ca2+储存库中发现的Ca2+水平。钙网蛋白的N结构域与PDI相互作用，但蛋白质C结构域与Ca2+的结合决定了这种相互作用的Ca2+敏感性。ERp57也通过该蛋白的N结构域与钙网蛋白相互作用。这些蛋白质之间的初始相互作用不依赖Ca2+，但会受到Ca2+与钙网蛋白C结构域结合的调节。我们得出结论，内质网腔Ca2+浓度的变化可能负责调节蛋白质-蛋白质相互作用。钙网蛋白可能通过调节参与蛋白质合成、折叠和翻译后修饰等多个步骤的不同蛋白质之间Ca2+依赖性结构和功能复合物的形成和维持，发挥内质网伴侣蛋白Ca2+“传感器”的作用。
