Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family

Kazuko Iida,Jinfeng Teng,Toshihiko Cho,Sato Yoshikawa-Kimura,Hidetoshi Iida

doi:10.1074/jbc.m117.810283

Abstract

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.

Highlights

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 purified protein was subjected to sequencing by Edman degradation, and the first five N-terminal amino acids were identified as LFQDF, which was identical to the Leu21-Phe22Gln23-Asp24-Phe25 sequence deduced from the nucleotide sequence of the MID1 gene
To elucidate how the N-terminal signal peptide– deleted forms of Mid1 are delivered into the endoplasmic reticulum (ER), we focused on yeast mutants lacking Sec proteins, which are present in the ER membrane

Summary

To whom correspondence should be addressed

In contrast to the ␣1 subunit of animal VGCCs, Cch is completely dependent on Mid to mediate Ca2ϩ influx [6, 12]. We show that 20 N-terminal amino acid residues of Mid are a signal peptide This signal peptide functions properly, the signal peptide– deleted forms of Mid are N-glycosylated and become functional for mediating Ca2ϩ influx. One of these forms, Mid1⌬N23, is suggested to be delivered into the ER by the post-translational translocation mechanism in a Sec71- and Sec72-dependent fashion, whereas the complete Mid is delivered by the co-translational translocation mechanism.

Results

Discussion

Experimental procedures