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

Abstract

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 theΔ 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.

Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca2+ channel related to the animal VGCC/NALCN family

“…2 A shows that all eight substitution mutants (C587A, C606A, C636A, C642A, C1369A, C1379A, C1727A, and C1738A) showed low Ca 2+ accumulation comparable with the cch1 Δ mutant carrying an empty vector, suggesting that all eight proteins had lost Ca 2+ permeation activity. Based on the previous observation that Ca 2+ accumulation activity is quantitatively correlated with the viability of cch1 mutants ( 16 , 28 ), we also assessed the viability of the transformants after exposure to α-factor, and as expected, the results supported the same conclusions ( Fig. 2 B ).…”

“…Mid1 is a plasma and ER membrane protein with 16 N -glycosylation sites and a C-terminal Cys-rich region composed of 12 Cys residues ( 7 , 16 ) ( Fig. 5 A ).…”

“…Our present study showed that although 12 Cys residues in the Cys-rich domain of Mid1 are highly conserved in fungal Mid1 proteins, only three (Cys-417, Cys-431, and Cys-498) were required for mid1 Δ-complementation activity, and only one (Cys-498) was involved in the interaction with Cch1. Our previous study showed that Cys-417 and Cys-431 are not included in the Frizzled-like motif ( 16 ) ( Fig. 5 A ), and are specifically present in fungal Mid1 proteins and animal Mid1-related proteins, such as the NALCN-associated proteins dmeMid1 (CG33988) in Drosophila melanogaster and NFL-1 in C. elegans , and vertebrate FAM115A and B proteins homologous to NFL-1 ( 17 , 35 ) ( Fig.…”

“…Mid1 is an α 2 /δ-like protein ( 7 , 15 ). Although nonhomologous to the α 2 /δ subunit of animal VGCCs at the amino acid sequence level, Mid1 possesses several structural features of animal α 2 /δ subunits, including an N-terminal signal peptide, a number of N -glycosylation sites, a Cys-rich domain, and extracellular localization ( 7 , 15 , 16 ). The Cys-rich region is of interest because it is highly conserved in Mid1 superfamily proteins in yeasts, fungi, and animals ( 17 ).…”

Cytotoxicity of Oleandrin Is Mediated by Calcium Influx and by Increased Manganese Uptake in Saccharomyces cerevisiae Cells

“…Cch1 is similar to the pore-forming subunit (α1) of the plasma membrane, and voltage-gated Ca 2+ channels (VGCC) from higher eukaryotes, including humans [ 34 ]. Cch1 interacts and partially co-localizes with Mid1p, a stretch-activated cation channel which resembles the VGCC α2/δ regulatory subunits and Na + leak channel non-selective (NALCN)-associated proteins [ 35 ]. Prolonged high [Ca 2+ ] cyt is detrimental to cells, therefore the normal very low [Ca 2+ ] cyt must be restored through the action of Ca 2+ pumps and exchangers [ 33 ].…”