Journal of Biological Chemistry
Volume 292, Issue 50, 15 December 2017, Pages 20570-20582
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Membrane Biology
Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family

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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 α1 subunit of voltage-gated Ca2+ channels (VGCCs). Despite a lack in overall primary sequence similarity, Mid1 resembles in some aspects VGCC α2/δ regulatory subunits and NALCN-associated proteins. Unlike animal α2/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 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 sec71Δ and sec72Δ single mutants defective in the post-translational protein transport into the endoplasmic reticulum (ER), Mid1ΔN23 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 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.

calcium channel
calcium transport
membrane protein
membrane transport
N-linked glycosylation
protein translocation
Saccharomyces cerevisiae
signal peptidase
yeast

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This work was supported by Grant-in-Aid for Scientific Research B 26291026 (to H. I.) from the Japan Society for the Promotion of Science. The authors declare that they have no conflicts of interest with the contents of this article.

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H. Kobayashi and H. Iida, unpublished data.

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Present address: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040.