Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family
Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family
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DOI：
10.1074/jbc.m117.810283
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发表时间：
2017-12-15
影响因子：
4.8
通讯作者：
Iida, Hidetoshi
中科院分区：
文献类型：
--
作者：
Iida, Kazuko;Teng, Jinfeng;Iida, Hidetoshi
酿酒酵母Mid 1由548个氨基酸和Cch 1的调节亚基组成，Cch 1是真核孔形成的四结构域阳离子通道家族的成员。Cch 1的氨基酸序列和电压不敏感性与Na+漏通道非选择性（NALCN）的氨基酸序列和电压不敏感性比与电压门控Ca 2+通道（VGCC）的（1）亚基更相似。尽管缺乏总体一级序列相似性，但Mid 1在某些方面类似于VGCC（2）/调节亚基和NALCN相关蛋白。与动物（2）/亚基不同，Mid 1和NALCN相关蛋白对孔形成亚基的功能至关重要。我们在此研究了Mid 1的加工和膜转位。Mid 1被发现有一个20个氨基酸长的N-末端信号肽，似乎是完全本地化的细胞外。一个信号肽缺失的Mid 1蛋白，Mid 1 N23，N-糖基化，并保留Ca 2+内流活性，通过Cch 1。此外，N-末端截短分析显示，即使是截短的Mid 1缺乏209个N-末端氨基酸残基的N-糖基化，并保持Ca 2+内流活性。一个219个氨基酸截短的Mid 1蛋白失去了这种活性，但仍然是N-糖基化。在sec 71和sec 72的翻译后蛋白转运到内质网（ER）缺陷的单一突变体，Mid 1 N23不能介导的Ca 2+内流，并没有进行N-糖基化，而野生型Mid 1表现出正常的Ca 2+内流活动和N-糖基化在这些突变体。因此，缺乏信号肽的Mid 1 N23蛋白可以仅通过翻译后蛋白质易位而易位至ER，这通常需要N末端信号肽。Mid 1可能为研究蛋白质易位到ER的机制提供了一个工具。
Saccharomyces cerevisiae Mid1 is composed of 548 amino acids and a regulatory subunit of Cch1, a member of the eukaryotic pore-forming, four-domain cation channel family. The amino acid sequence and voltage insensitivity of Cch1 are more similar to those of Na+ leak channel non-selective (NALCN) than to the (1) subunit of voltage-gated Ca2+ channels (VGCCs). Despite a lack in overall primary sequence similarity, Mid1 resembles in some aspects VGCC (2)/ regulatory subunits and NALCN-associated proteins. Unlike animal (2)/ subunits, Mid1 and NALCN-associated proteins are essential for the function of the pore-forming subunit. We herein investigated the processing and membrane translocation of Mid1. Mid1 was found to have a 20-amino-acid-long N-terminal signal peptide and appeared to be entirely localized extracellularly. A signal peptide-deleted Mid1 protein, Mid1N23, was N-glycosylated and retained Ca2+ influx activity through Cch1. Moreover, an N-terminal truncation analysis revealed that even truncated Mid1 lacking 209 N-terminal amino acid residues was N-glycosylated and maintained Ca2+ influx activity. A 219-amino-acid-truncated Mid1 protein lost this activity but was still N-glycosylated. In the sec71 and sec72 single mutants defective in the post-translational protein transport into the endoplasmic reticulum (ER), Mid1N23 could not mediate Ca2+ influx and did not undergo N-glycosylation, whereas wild-type Mid1 exhibited normal Ca2+ influx activity and N-glycosylation in these mutants. Therefore, the signal peptide-lacking Mid1N23 protein may be translocated to the ER exclusively through the post-translational protein translocation, which typically requires an N-terminal signal peptide. Mid1 may provide a tool for studying mechanisms of protein translocation into the ER.