Folding of thyroglobulin in the calnexin/calreticulin pathway and its alteration by loss of Ca2+ from the endoplasmic reticulum
Folding of thyroglobulin in the calnexin/calreticulin pathway and its alteration by loss of Ca2+ from the endoplasmic reticulum
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DOI：
10.1042/bj20021257
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发表时间：
2003-03-01
影响因子：
4.1
通讯作者：
Arvan, P
中科院分区：
文献类型：
--
作者：
Di Jeso, B;Ulianich, L;Arvan, P
在内质网（ER）中的初始折叠过程中，新合成的甲状腺球蛋白（Tg）与钙连接蛋白和其他ER分子伴侣相互作用，但其与钙网蛋白的相互作用以前没有研究过。在本研究中，我们研究了内源性Tg与钙网蛋白和其他几种ER分子伴侣的相互作用。我们发现，在FRTL-5和PC-Cl 3细胞中，钙连接蛋白和钙网蛋白与新合成的Tg以碳水化合物依赖性方式相互作用，与Tg链内二硫键的成熟相伴的动力学在很大程度上重叠，在Tg二聚化和从ER退出之前。钙网蛋白共沉淀更多的新合成的Tg比钙连接蛋白;然而，使用两种不同的实验方法，钙连接蛋白和钙网蛋白被发现在与Tg的三元复合物，使其成为第一个内源性蛋白质报告的三元复合物与钙连接蛋白和钙网蛋白在ER的活细胞。由毒胡萝卜素（ER Ca ~（2+）-ATP酶的特异性抑制剂）引起的ER中Ca ~（2+）的消耗导致Tg在该细胞器中的保留。有趣的是，毒胡萝卜素处理诱导Tg过早退出钙连接蛋白/钙网蛋白循环，同时稳定和延长Tg与BiP（免疫球蛋白重链结合蛋白）和GRP 94（葡萄糖调节蛋白94）的相互作用，这两种分子伴侣的结合不依赖于碳水化合物。我们的研究结果表明，钙连接蛋白和钙网蛋白，在三元复合物中与一个大的糖蛋白底物，如Tg，可能从事不同的结构域的折叠，并表明内腔钙强烈影响可输出的糖蛋白的折叠，部分通过调节平衡的底物结合到不同的分子伴侣系统内的ER。
During its initial folding in the endoplasmic reticulum (ER), newly synthesized thyroglobulin (Tg) is known to interact with calnexin and other ER molecular chaperones, but its interaction with calreticulin has not been examined previously. In the present study, we have investigated the interactions of endogenous Tg with calreticulin and with several other ER chaperones. We find that, in FRTL-5 and PC-Cl3 cells, calnexin and calreticulin interact with newly synthesized Tg in a carbohydrate-dependent manner, with largely overlapping kinetics that are concomitant with the maturation of Tg intrachain disulphide bonds, preceding Tg dimerization and exit from the ER. Calreticulin co-precipitates more newly synthesized Tg than does calnexin; however, using two different experimental approaches, calnexin and calreticulin were found in ternary complexes with Tg, making this the first endogenous protein reported in ternary complexes with calnexin and calreticulin in the ER of live cells. Depletion of Ca2+ from the ER elicited by thapsigargin (a specific inhibitor of ER Ca2+-ATPases) results in retention of Tg in this organelle. Interestingly, thapsigargin treatment induces the premature exit of Tg from the calnexin/calreticulin cycle, while stabilizing and prolonging interactions of Tg with BiP (immunoglobulin heavy chain binding protein) and GRP94 (glucose-regulated protein 94), two chaperones whose binding is not carbohydrate-dependent. Our results suggest that calnexin and calreticulin, acting in ternary complexes with a large glycoprotein substrate such as Tg, might be engaged in the folding of distinct domains, and indicate that lumenal Ca2+ strongly influences the folding of exportable glycoproteins, in part by regulating the balance of substrate binding to different molecular chaperone systems within the ER.