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

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.