The ER-associated protease Ste24 prevents N-terminal signal peptide-independent translocation into the endoplasmic reticulum in Saccharomyces cerevisiae.

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast Saccharomyces cerevisiae, a few proteins devoid of a signal peptide are still translocated into the ER and then N-glycosyl-ated. Using signal peptide-truncated reporter proteins, here we report the detection of significant translocation of N-terminal signal peptide-truncated proteins in a yeast mutant strain (ste24Δ) that lacks the endopeptidase Ste24 at the ER membrane. Furthermore, several ER/cytosolic proteins, including Sec61, Sec66, and Sec72, were identified as being involved in the translocation process. On the basis of screening for 20 soluble proteins that may be N-glycosylated in the ER in the ste24Δ strain, we identified the transcription factor Rme1 as a protein that is partially N-glycosylated despite the lack of a signal peptide. These results clearly indicate that some proteins lacking a signal peptide can be translocated into the ER and that Ste24 typically suppresses this process.

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

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.

Coupling of a voltage-gated Ca2+ channel homologue with a plasma membrane H+ -ATPase in yeast.

Yeast has a homologue of mammalian voltage-gated Ca2+ channels (VGCCs), enabling the efficient uptake of Ca2+ . It comprises two indispensable subunits, Cch1 and Mid1, equivalent to the mammalian pore-forming α1 and auxiliary α2 /δ subunits, respectively. Unlike the physiological roles of Cch1/Mid1 channels, the regulatory mechanisms of the yeast VGCC homologue remain unclear. Therefore, we screened candidate proteins that interact with Mid1 by an unbiased proteomic approach and identified a plasma membrane H+ -ATPase, Pma1, as a candidate. Mid1 coimmunoprecipitated with Pma1, and Mid1-EGFP colocalized with Pma1-mCherry at the plasma membrane. The physiological relevance of their interaction was determined using the temperature-sensitive mutant, pma1-10. At the nonpermissive temperature, the membrane potential was less negative and Ca2+ uptake was lower in pma1-10 than in wild-type cells. Increased extracellular H+ increased the rate of Ca2+ uptake. Therefore, H+ extrusion by Pma1 may be important for Ca2+ influx through Cch1/Mid1. These results suggest that Pma1 interacts physically with Cch1/Mid1 Ca2+ channels to enhance their activity via its H+ -pumping activity.