Ca2+ regulation of interactions between endoplasmic reticulum chaperones
Ca2+ regulation of interactions between endoplasmic reticulum chaperones
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DOI：
10.1074/jbc.274.10.6203
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发表时间：
1999-03-05
影响因子：
4.8
通讯作者：
Michalak, M
中科院分区：
文献类型：
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作者：
Corbett, EF;Oikawa, K;Michalak, M
用荧光染料Casade Blue(CB)研究了内质网(ER)腔伴侣蛋白钙网蛋白、蛋白质二硫键异构酶(PDI)和ERp57之间的相互作用动力学。PDI和ERp57被CB标记，随后，我们发现CB结合的蛋白质在不同极性的微环境中的荧光强度发生了变化。CD分析表明，CB-ERp57和CB-PDI荧光强度的变化与蛋白质构象的变化相对应。利用这一技术，我们证明了在低的钙离子浓度下(低于100mM)，PDI与钙网硬蛋白相互作用，而在钙离子浓度为400mU时，蛋白质复合体解离。这些钙离子浓度使人想起在空的或满的内质网钙离子储存库中发现的钙离子水平。钙网蛋白的N-结构域与PDI相互作用，但与蛋白质C-结构域的钙结合是导致相互作用的钙敏感性的原因。ERp57还通过蛋白的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是不依赖于钙离子的，但它受钙离子与钙网蛋白C结构域结合的调节。我们的结论是，内质网腔内钙离子浓度的变化可能参与了蛋白质-蛋白质相互作用的调节。在蛋白质合成、折叠和翻译后修饰的过程中，不同蛋白质之间的结构和功能复合体的形成和维持可能通过调节钙离子依赖的形成和维持，钙网蛋白可能扮演着内质网伴侣的钙“传感器”的角色。
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 mu M), whereas the protein complex dissociates at >400 mu M 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.