Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells
Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells
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DOI：
10.1073/pnas.2004606117
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发表时间：
2020-07-14
影响因子：
11.1
通讯作者：
Inaba, Kenji
中科院分区：
文献类型：
--
作者：
Kadokura, Hiroshi;Dazai, Yui;Inaba, Kenji
蛋白质是通过将几个结构单元合并在一个多肽中而进化的。因此，多结构域蛋白质构成了所有蛋白质组的很大一部分。当每个结构域从核糖体或蛋白质易位通道出现时，它们的结构域通常单独地和矢量地折叠到它们的天然结构，从而降低结构域间错误折叠的风险。然而，一些多结构域蛋白在内质网（ER）中通过具有非天然二硫键的中间体非矢量地折叠，这些中间体被认为在合成后缓慢地改组为天然二硫键。然而，它们非矢量折叠的机制仍不清楚。使用二维（2D）凝胶电泳和构象特异性抗体，它使一个正确折叠的结构域，我们在这里表明，改组的非天然二硫键天然的LDL受体（LDLR）的最N-末端区域开始在一个特定的时间在合成过程中。缺失分析鉴定了LDLR上的一个区域，该区域协助上游结构域中的二硫键改组，从而促进其共翻译折叠。因此，质膜结合的多结构域蛋白已经进化出一个序列，促进其上游结构域的非矢量折叠。这些研究结果表明，非矢量折叠的多结构域蛋白质在ER的哺乳动物细胞是更协调和阐述比以前认为的。因此，我们的研究结果改变了我们目前对多结构域蛋白如何在活细胞ER中非矢量折叠的看法。
Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recog-nizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assis-ted with disulfide shuffling in the upstream domain, thereby pro-moting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.