Iida Kazuko, Teng Jinfeng, Cho Toshihiko, Yoshikawa-Kimura Sato, Iida Hidetoshi
From the Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya, Tokyo 156-8506, Japan and.
the Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei, Tokyo 184-8501, Japan.
J Biol Chem. 2017 Dec 15;292(50):20570-20582. doi: 10.1074/jbc.M117.810283. Epub 2017 Oct 17.
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 α subunit of voltage-gated Ca channels (VGCCs). Despite a lack in overall primary sequence similarity, Mid1 resembles in some aspects VGCC α/δ regulatory subunits and NALCN-associated proteins. Unlike animal α/δ 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, Mid1ΔN23, was -glycosylated and retained Ca influx activity through Cch1. Moreover, an N-terminal truncation analysis revealed that even truncated Mid1 lacking 209 N-terminal amino acid residues was -glycosylated and maintained Ca influx activity. A 219-amino-acid-truncated Mid1 protein lost this activity but was still -glycosylated. In the Δ and Δ single mutants defective in the post-translational protein transport into the endoplasmic reticulum (ER), Mid1ΔN23 could not mediate Ca influx and did not undergo -glycosylation, whereas wild-type Mid1 exhibited normal Ca influx activity and -glycosylation in these mutants. Therefore, the signal peptide-lacking Mid1ΔN23 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.
Mid1由548个氨基酸组成，是Cch1的调节亚基，Cch1是真核生物成孔四结构域阳离子通道家族的成员。Cch1的氨基酸序列和电压不敏感性与非选择性钠泄漏通道（NALCN）的更为相似，而与电压门控钙通道（VGCCs）的α亚基不同。尽管在整体一级序列相似性方面存在不足，但Mid1在某些方面类似于VGCC α/δ调节亚基和NALCN相关蛋白。与动物α/δ亚基不同，Mid1和NALCN相关蛋白对于成孔亚基的功能至关重要。我们在此研究了Mid1的加工和膜转运。发现Mid1具有一个20个氨基酸长的N端信号肽，并且似乎完全定位于细胞外。缺失信号肽的Mid1蛋白Mid1ΔN23进行了O-糖基化，并通过Cch1保留了钙内流活性。此外，N端截短分析表明，即使是缺少209个N端氨基酸残基的截短Mid1也进行了O-糖基化并保持了钙内流活性。一个截短了219个氨基酸的Mid1蛋白失去了这种活性，但仍进行了O-糖基化。在翻译后蛋白质转运到内质网（ER）有缺陷的Δ和Δ单突变体中，Mid1ΔN23不能介导钙内流，也不进行O-糖基化，而野生型Mid1在这些突变体中表现出正常的钙内流活性和O-糖基化。因此，缺少信号肽的Mid1ΔN23蛋白可能仅通过翻译后蛋白质转运进入内质网，而翻译后蛋白质转运通常需要一个N端信号肽。Mid1可能为研究蛋白质转运到内质网的机制提供一个工具。
