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• W2765704865 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. 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. Among Ca2+-permeable ion channels, voltage-gated Ca2+ channels (VGCCs) 3The abbreviations used are: VGCCvoltage-gated Ca2+ channelGPIglycosylphosphatidylinositolERendoplasmic reticulumNALCNNa+ leak channel non-selectiveSRPsignal recognition particleEndo Hendoglycosidase H. have been the most intensively studied from basic to clinical viewpoints because they play crucial roles in synaptic transmission in neurons and contraction in skeletal muscle cells by mediating Ca2+ influx in response to changes in membrane potential in animal cells (1Catterall W.A. Voltage-gated calcium channels.Cold Spring Harb. Perspect. Biol. 2011; 3: a003947Crossref PubMed Scopus (948) Google Scholar). VGCCs are composed of four subunits: the pore-forming α1 subunit and three auxiliary subunits, α2/δ, β, and γ. The α2/δ subunit is composed of four subtypes, each of which is the product of a single gene encoding an ∼140-kDa protein with a C-terminal potential transmembrane segment or glycosylphosphatidylinositol (GPI) anchor attachment site and a number of N-glycosylation sites (2Dolphin A.C. The α2/δ subunits of voltage-gated calcium channels.Biochim. Biophys. Acta. 2013; 1828: 1541-1549Crossref PubMed Scopus (143) Google Scholar). The single polypeptide is post-translationally cleaved into α2 and δ fragments, after which both are bound by disulfide bonding. In the mature form of the α2/δ protein, α2 is exposed entirely to the extracellular space; δ is anchored to the plasma membrane; and both are N-glycosylated, giving this protein a total molecular mass of ∼180 kDa. Although necessary for the modulation of VGCC properties, the α2/δ subunit does not affect single channel conductance, suggesting that it is not required for Ca2+ permeation of the α1 subunit (3Wakamori M. Mikala G. Mori Y. Auxiliary subunits operate as a molecular switch in determining gating behaviour of the unitary N-type Ca2+ channel current in.Xenopus oocytes. J. Physiol. 1999; 517: 659-672Crossref PubMed Scopus (52) Google Scholar, 4Barclay J. Balaguero N. Mione M. Ackerman S.L. Letts V.A. Brodbeck J. Canti C. Meir A. Page K.M. Kusumi K. Perez-Reyes E. Lander E.S. Frankel W.N. Gardiner R.M. Dolphin A.C. Rees M. Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.J. Neurosci. 2001; 21: 6095-6104Crossref PubMed Google Scholar5Brodbeck J. Davies A. Courtney J.M. Meir A. Balaguero N. Canti C. Moss F.J. Page K.M. Pratt W.S. Hunt S.P. Barclay J. Rees M. Dolphin A.C. The ducky mutation in Cacna2d2 results in altered Purkinje cell morphology and is associated with the expression of a truncated α2/δ-2 protein with abnormal function.J. Biol. Chem. 2002; 277: 7684-7693Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). voltage-gated Ca2+ channel glycosylphosphatidylinositol endoplasmic reticulum Na+ leak channel non-selective signal recognition particle endoglycosidase H. The yeast Saccharomyces cerevisiae has a VGCC-related Ca2+ channel composed of essential Cch1 and Mid1 subunits. Cch1 was identified by scanning the yeast sequence database and is 24% identical to the α1 subunit of mammalian L-type VGCCs in overall amino acid sequences and is, thus, called an α1 homolog (6Paidhungat M. Garrett S. A homolog of mammalian, voltage-gated calcium channels mediates yeast pheromone-stimulated Ca2+ uptake and exacerbates the cdc1(Ts) growth defect.Mol. Cell. Biol. 1997; 17: 6339-6347Crossref PubMed Scopus (161) Google Scholar). Mid1 was identified by the screening of mutants showing the mating pheromone-induced death (mid) phenotype because of a defect in Ca2+ influx (7Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating.Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar). Mid1 is composed of 548 amino acid residues and is localized in plasma and endoplasmic reticulum (ER) membranes (7Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating.Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar8Tada T. Ohmori M. Iida H. Molecular dissection of the hydrophobic segments H3 and H4 of the yeast Ca2+ channel component Mid1.J. Biol. Chem. 2003; 278: 9647-9654Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 9Iida K. Tada T. Iida H. Molecular cloning in yeast by in vivo homologous recombination of the yeast putative α1 subunit of the voltage-gated calcium channel.FEBS Lett. 2004; 576: 291-296Crossref PubMed Scopus (28) Google Scholar10Yoshimura H. Tada T. Iida H. Subcellular localization and oligomeric structure of the yeast putative stretch-activated Ca2+ channel component Mid1.Exp. Cell Res. 2004; 293: 185-195Crossref PubMed Scopus (40) Google Scholar). Although Mid1 has no homologous protein in animals or plants with respect to overall amino acid sequences, some features of this protein resemble those of animal α2/δ subunits, such as a potential N-terminal signal peptide, a number of potential N-glycosylation sites, and multiple cysteine residues (see Fig. 1) (7Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating.Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar, 11Martin D.C. Kim H. Mackin N.A. Maldonado-Báez L. Evangelista Jr., C.C. Beaudry V.G. Dudgeon D.D. Naiman D.Q. Erdman S.E. Cunningham K.W. New regulators of a high affinity Ca2+ influx system revealed through a genome-wide screen in yeast.J. Biol. Chem. 2011; 286: 10744-10754Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In addition, many of the fungal Mid1 orthologs have a GPI anchor attachment motif at the C-terminal region, similar to animal α2/δ proteins. Therefore, Mid1 may be called an α2/δ-like protein. In contrast to the α1 subunit of animal VGCCs, Cch1 is completely dependent on Mid1 to mediate Ca2+ influx (6Paidhungat M. Garrett S. A homolog of mammalian, voltage-gated calcium channels mediates yeast pheromone-stimulated Ca2+ uptake and exacerbates the cdc1(Ts) growth defect.Mol. Cell. Biol. 1997; 17: 6339-6347Crossref PubMed Scopus (161) Google Scholar, 12Fischer M. Schnell N. Chattaway J. Davies P. Dixon G. Sanders D. The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating.FEBS Lett. 1997; 419: 259-262Crossref PubMed Scopus (149) Google Scholar). Although homologous to the α1 subunits of animal VGCCs, Cch1 is more homologous to a recently identified subfamily of animal VGCC α1 subunits, the Na+ leak channel non-selective (NALCN), as revealed by a phylogenetic analysis (13Liebeskind B.J. Hillis D.M. Zakon H.H. Phylogeny unites animal sodium leak channels with fungal calcium channels in an ancient, voltage-insensitive clade.Mol. Biol. Evol. 2012; 29: 3613-3616Crossref PubMed Scopus (38) Google Scholar). Functionally, voltage insensitivity is similar between Cch1 and NALCN (14Lu B. Su Y. Das S. Liu J. Xia J. Ren D. The neuronal channel NALCN contributes resting sodium permeability and is required for normal respiratory rhythm.Cell. 2007; 129: 371-383Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 15Hong M.-P. Vu K. Bautos J. Gelli A. Cch1 restores intracellular Ca2+ in fungal cells during endoplasmic reticulum stress.J. Biol. Chem. 2010; 285: 10951-10958Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and this may be because both have fewer positive charges in the voltage-sensing S4 segment than the α1 subunit of VGCCs. In support of the close similarities between Cch1 and NALCN, their auxiliary subunits exhibit similar functional essentialities; as described above, Mid1 is essential for the function of Cch1. Likewise, the auxiliary subunits UNC80, UNC79, NLF-1, and CG33988 of animal NALCN are required for the function of NALCN; however, their localization and overall structure are divergent (16Lu B. Zhang Q. Wang H. Wang Y. Nakayama M. Ren D. Extracellular calcium controls background current and neuronal excitability via an UNC79-UNC80-NALCN cation channel complex.Neuron. 2010; 68: 488-499Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 17Xie L. Gao S. Alcaire S.M. Aoyagi K. Wang Y. Griffin J.K. Stagljar I. Nagamatsu S. Zhen M. NLF-1 delivers a sodium leak channel to regulate neuronal excitability and modulate rhythmic locomotion.Neuron. 2013; 77: 1069-1082Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar18Ghezzi A. Liebeskind B.J. Thompson A. Atkinson N.S. Zakon H.H. Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals.Front. Mol. Neurosci. 2014; 7: 15Crossref PubMed Scopus (20) Google Scholar). The cysteine-rich domains of Mid1, NLF-1, CG33988, and FAM155A and -B are highly conserved and, thus, designated as the Mid1 domain, leading to the hypothesis that the relationship between the Mid1 domain and NALCN has persisted over one billion years of evolution (18Ghezzi A. Liebeskind B.J. Thompson A. Atkinson N.S. Zakon H.H. Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals.Front. Mol. Neurosci. 2014; 7: 15Crossref PubMed Scopus (20) Google Scholar). In this context, it has become more important to characterize the basic molecular nature of Mid1 as a model of essential subunits of the NALCN family. There are two major protein translocation mechanisms that transfer secretory and plasma membrane proteins across the ER membrane (19Zimmermann R. Eyrisch S. Ahmad M. Helms V. Protein translocation across the ER membrane.Biochim. Biophys. Acta. 2011; 1808: 912-924Crossref PubMed Scopus (171) Google Scholar, 20Delic M. Valli M. Graf A.B. Pfeffer M. Mattanovich D. Gasser B. The secretory pathway: exploring yeast diversity.FEMS Microbiol. Rev. 2013; 37: 872-914Crossref PubMed Scopus (137) Google Scholar). One is co-translational translocation, which is enabled by a direct link between translation and translocation. Before the synthesis of these proteins is finished, their signal peptides are recognized and bound by the signal recognition particle (SRP) in the cytoplasm. The SRP mediates the binding of the complex to the SRP receptor at the ER membrane. The signal peptide is then inserted into the translocation channel or translocon composed of the Sec61 protein and others. S. cerevisiae cells with the sec61Δ mutation are inviable (21Deshaies R.J. Schekman R. A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.J. Cell Biol. 1987; 105: 633-645Crossref PubMed Scopus (254) Google Scholar). The other mechanism is post-translational translocation. Whereas this mechanism also requires the translocon and the signal peptides of the proteins, it needs the Sec62/Sec63 complex, composed of Sec62, Sec63, Sec71 (also termed Sec66), and Sec72 (also termed Sec67) in place of SRP. Sec71 and Sec72 are fungi-specific subunits. In S. cerevisiae, the sec62Δ and sec63Δ single mutants are inviable (22Rothblatt J.A. Deshaies R.J. Sanders S.L. Daum G. Schekman R. Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast.J. Cell Biol. 1989; 109: 2641-2652Crossref PubMed Scopus (231) Google Scholar), whereas the sec71Δ and sec72Δ single mutants are viable (23Green N. Fang H. Walter P. Mutations in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae.J. Cell Biol. 1992; 116: 597-604Crossref PubMed Scopus (77) Google Scholar). The viability of the latter two mutants provides a useful tool for testing the dependence of protein translocation on the post-translational mechanism. In the present study, we show that 20 N-terminal amino acid residues of Mid1 are a signal peptide. Although this signal peptide functions properly, the signal peptide–deleted forms of Mid1 are also N-glycosylated and become functional for mediating Ca2+ influx. One of these forms, Mid1ΔN23, is suggested to be delivered into the ER by the post-translational translocation mechanism in a Sec71- and Sec72-dependent fashion, whereas the complete Mid1 is delivered by the co-translational translocation mechanism. We suggest that Mid1 is delivered into the ER by at least two protein translocation mechanisms, and this is dependent on its molecular form. We also demonstrate that most of the 16 potential N-glycosylation sites of Mid1 are N-glycosylated and that the N and C termini are exposed to the extracellular space, suggesting that the entire sequence of Mid1 is present outside the cell. All of the public web software servers utilized, including PrediSi (24Hiller K. Grote A. Scheer M. Münch R. Jahn D. PrediSi: prediction of signal peptides and their cleavage positions.Nucleic Acids Res. 2004; 32: W375-W379Crossref PubMed Scopus (351) Google Scholar), Phobius (25Käll L. Krogh A. Sonnhammer E.L.L. A combined transmembrane topology and signal peptide prediction method.J. Mol. Biol. 2004; 338: 1027-1036Crossref PubMed Scopus (1650) Google Scholar), Signal-BLAST (26Frank K. Sippl M.J. High performance signal peptide prediction based on sequence alignment techniques.Bioinformatics. 2008; 24: 2172-2176Crossref PubMed Scopus (99) Google Scholar), and SignalP 4.1 (27Petersen T.N. Brunak S. von Heijne G. Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions.Nat. Methods. 2011; 8: 785-786Crossref PubMed Scopus (7094) Google Scholar), predicted that the Mid1 protein has a signal peptide with 20 amino acid residues at the N terminus. To confirm this prediction experimentally, we attempted to elucidate the N-terminal amino acid sequence of the mature Mid1 protein. We purified FLAG-tagged Mid1 from yeast cells using differential centrifugation and immunoprecipitation with an anti-FLAG antibody, followed by SDS-PAGE and Western blotting (Fig. 2). 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-Phe22-Gln23-Asp24-Phe25 sequence deduced from the nucleotide sequence of the MID1 gene. Therefore, we concluded that the Mid1 protein has a signal peptide composed of 20 N-terminal amino acid residues (Fig. 2). To establish whether the N-terminal signal peptide is important for Mid1 to become active through proper processing and modifications, we constructed a truncated form of this protein lacking 23 N-terminal amino acid residues (ΔN23), expressed it from the MID1 promoter in the mid1 mutant, and assayed its ability to rescue the mid1 phenotypes, such as weak Ca2+ uptake activity and the loss of viability in the presence of the mating pheromone α-factor. Fig. 3 (A and B) shows that the ΔN23 protein rescued both phenotypes; however, this rescuing activity was slightly weaker than that of the wild-type Mid1 protein. These results indicate that the signal peptide is not essential for Mid1 to acquire activity but is required for full activity. These results also suggest that the ΔN23 protein is targeted properly to the plasma membrane to cooperate with Cch1. The above results with the ΔN23 protein prompted us to investigate how much of the N-terminal region may be truncated while maintaining the activity of Mid1. We constructed a series of N-terminally truncated forms of Mid1, such as ΔN32, ΔN51, ΔN68, ΔN112, ΔN209, and ΔN219, the numbers of which represent the deleted number of amino acid residues from the N terminus. We then expressed them from the MID1 promoter or a transcriptionally strong TDH3 promoter (28Nacken V. Achstetter T. Degryse E. Probing the limits of expression levels by varying promoter strength and plasmid copy number in Saccharomyces cerevisiae.Gene. 1996; 175: 253-260Crossref PubMed Scopus (51) Google Scholar) in the mid1 mutant and measured their activities, similar to ΔN23. As shown in Fig. 3, all of the truncated Mid1 proteins except for ΔN219 retained Ca2+ influx activities and viability maintenance; however, their activities were one-half to one-third wild-type levels when these constructs were expressed from the promoter of MID1 (see white bars). The ΔN209 protein retained weak Ca2+ accumulation activity and viability (mid1 versus ΔN209 in white bars, p < 0.03). However, when these constructs were expressed from the TDH3 promoter, even the ΔN209 protein exhibited activity that was more than one-half that of wild-type levels, whereas the ΔN219 protein did not (see dark gray bars). These results indicate that 209 N-terminal amino acid residues are non-essential for the function of Mid1. We also found that HA-tagged deletion mutant proteins (ΔN32-HA and ΔN51-HA), which were expressed from the MID1 promoter, had slightly stronger activities than the untagged counterparts (Fig. 3), suggesting that the HA tag does not impair the activity of the truncated forms of Mid1. Intact Mid1 is an N-glycosylated protein (7Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating.Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar). Therefore, functionally active, N-terminally truncated forms of Mid1, such as ΔN23, ΔN32, ΔN51, ΔN68, ΔN112, and ΔN209, were expected to be N-glycosylated. To examine this hypothesis, we performed a Western blot analysis of the wild-type and ΔN112 proteins before and after the endoglycosidase H (Endo H) digestion of whole-cell extracts. Fig. 4A shows that the higher-molecular weight population of the two proteins was Endo H-sensitive and, thus, was N-glycosylated. Similar to ΔN112, all of the other truncated proteins were N-glycosylated; however, the amounts of the proteins were reduced to approximately one-half to one-third those of wild-type Mid1 (Fig. 4, A and B). Furthermore, even the non-active form of Mid1, ΔN219, was N-glycosylated. These results indicate that the truncation of at least 219 amino acid residues from the N terminus did not affect N-glycosylation and also imply that yeast cells possess a mechanism by which proteins that have lost the N-terminal signal peptide may still be incorporated into the lumen of the ER and then N-glycosylated. Because the above results suggest that the region spanning from Pro210 to Met219 is essential for the function of Mid1 (Fig. 3, A and B), we deleted this region and examined the activity of the deletion mutant (designated Δ210–219). The Δ210–219 protein completely lost its activity (see the first and second bars from the right ends in Fig. 3, A and B). We then compared the amino acid sequence of this region of the S. cerevisiae Mid1 protein with those of 96 orthologs and found that Asp216 and Asp218 were highly conserved during evolution (Fig. 5A). Therefore, we performed an amino acid replacement analysis for the two acidic residues. Asp216 and Asp218 were replaced by the small, non-polar amino acid, Ala, or the polar, uncharged amino acid, Asn, the side-chain volume of which was similar to that of Asp, resulting in the D216A, D216N, D218A, and D218N mutant proteins. Fig. 5 (B and C) shows that D216A and D216N exhibited Ca2+ influx activities and viability maintenance similar to those of the wild-type Mid1, suggesting that the acidity and side-chain volume of Asp216 are not important for the activity of Mid1. On the other hand, D218N lost both activities, whereas D218A did not. Furthermore, D218Q, but not D218E, lost both activities. In terms of the physicochemical properties of amino acids, the hydrophilicities of Asp, Asn, Glu, and Gln are known to be similar (29Kyte J. Doolittle R.F. A simple method for displaying the hydropathic character of a protein.J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17166) Google Scholar), whereas Ala possesses a small side chain. Therefore, we speculated that an amino acid residue at the position of Asp218 may be acidic or small. Both factors may be important for Mid1. We also found that the D216A/D218A double mutant lost both activities (Fig. 5, B and C), suggesting that at least one Asp residue is essential for the function of Mid1 in this region. To elucidate how the N-terminal signal peptide–deleted forms of Mid1 are delivered into the ER, we focused on yeast mutants lacking Sec proteins, which are present in the ER membrane. Secretory and plasma membrane proteins are translocated into the ER through two major protein translocation mechanisms. One is co-translational and the other post-translational (19Zimmermann R. Eyrisch S. Ahmad M. Helms V. Protein translocation across the ER membrane.Biochim. Biophys. Acta. 2011; 1808: 912-924Crossref PubMed Scopus (171) Google Scholar, 20Delic M. Valli M. Graf A.B. Pfeffer M. Mattanovich D. Gasser B. The secretory pathway: exploring yeast diversity.FEMS Microbiol. Rev. 2013; 37: 872-914Crossref PubMed Scopus (137) Google Scholar). These co-translational and post-translational protein translocation mechanisms both utilize the common translocation channel composed of the Sec61 protein. However, the former depends on the SRP for the targeting of the ribosome-nascent polypeptide complex to the Sec61 complex, whereas the latter depends on the Sec62/63/71/72 complex for the targeting of fully translated proteins to the Sec61 complex in S. cerevisiae. In the present study, we employed the sec71Δ and sec72Δ mutations to investigate the mechanisms by which the N-terminal signal peptide–deleted forms of Mid1 were delivered into the ER and became functional. We also employed the Mid1ΔN23 protein as a model of the N-terminal signal peptide–deleted forms of Mid1. We did not employ the sec61Δ, sec62Δ, or sec63Δ mutation because these are lethal mutations (21Deshaies R.J. Schekman R. A yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum.J. Cell Biol. 1987; 105: 633-645Crossref PubMed Scopus (254) Google Scholar, 22Rothblatt J.A. Deshaies R.J. Sanders S.L. Daum G. Schekman R. Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast.J. Cell Biol. 1989; 109: 2641-2652Crossref PubMed Scopus (231) Google Scholar) and also because Sec61 and Sec63 are involved in co-translational and post-translational protein translocation (19Zimmermann R. Eyrisch S. Ahmad M. Helms V. Protein translocation across the ER membrane.Biochim. Biophys. Acta. 2011; 1808: 912-924Crossref PubMed Scopus (171) Google Scholar, 20Delic M. Valli M. Graf A.B. Pfeffer M. Mattanovich D. Gasser B. The secretory pathway: exploring yeast diversity.FEMS Microbiol. Rev. 2013; 37: 872-914Crossref PubMed Scopus (137) Google Scholar, 30Brodsky J.L. Goeckeler J. Schekman R. Bip and Sec63p are required for both co- and posttranslational protein ttranslocation into the yeast endoplasmic reticulum.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 9643-9646Crossref PubMed Scopus (200) Google Scholar). Mid1ΔN23 and a control protein, wild-type Mid1, were expressed individually from the MID1 promoter in either mid1Δ, mid1Δ sec71Δ, or mid1Δ sec72Δ mutants, and the Ca2+ accumulation activity and viability of each mutant expressing one of the two proteins were examined after cells were exposed to α-factor. Fig. 6A shows that in the mid1Δ mutant, Mid1ΔN23 exhibited approximately half the activity of wild-type Mid1, suggesting that yeast cells possess a mechanism that properly translocates N-terminal signal peptide–deleted proteins. This was also implied from the results shown in Fig. 3A (see the bar of MID1 and ΔN23). In the sec71Δ background, Mid1ΔN23-expressing cells lost Ca2+ accumulation activity to an almost negative control level (compare mid1Δ sec71Δ/MID1ΔN23 with mid1Δ/vector), whereas wild-type Mid1-expressing cells did not lose this activity (Fig. 6A), indicating that Mid1ΔN23 depends almost completely on Sec71 to become functional. In the sec72Δ background, Mid1ΔN23-expressing cells lost Ca2+ accumulation activity completely (Fig. 6A). In addition, the activity of wild-type Mid1-expressing cells was decreased to approximately two-thirds that of wild-type Mid1-expressing cells in the mid1Δ mutant (compare mid1Δ sec72Δ/MID1 with mid1Δ/MID1). These results suggest that Mid1ΔN23 is completely dependent on Sec72 to become functional. The results obtained with the sec71Δ and sec72Δ mutations suggest that Mid1ΔN23 is translocated into the ER post-translationally. The difference in the activity of wild-type Mid1 between the sec71Δ and sec72Δ backgrounds was not unexpected because individual proteins with the N-terminal signal peptide are known to be differentially sensitive to the mutations. For example, the Sec63-invertase fusion protein (an ER membrane protein) was translocated more efficiently into the ER in the sec72Δ mutant than in the sec71Δ mutant, whereas pro-α-factor (a secretory protein) was translocated more efficiently into the ER in the sec71Δ mutant than in the sec72Δ mutant (23Green N. Fang H. Walter P. Mutations in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae.J. Cell Biol. 1992; 116: 597-604Crossref PubMed Scopus (77) Google Scholar). Viability assays led to essentially the same conclusion (Fig. 6B). In the mid1Δ mutant, cells expressing Mid1ΔN23 slightly lost viability 8 h after exposure to α-factor. In the sec71Δ and sec72Δ backgrounds, cells expressing Mid1ΔN23 lost viability completely, whereas those expressing wild-type Mid1 did not. To clarify whether the absence of Ca2+ accumulation activity and lack of viability of Mid1ΔN23-expressing cells in the sec71Δ and sec72Δ backgrounds is due to a failure in the translocation of the Mid1ΔN23 protein across the ER membrane, we performed a Western blot analysis of the wild-type Mid1 and Mid1ΔN23 proteins tagged with the epitope FLAG between the N-terminal 51st" @default.
• W2765704865 created "2017-11-10" @default.
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• W2765704865 creator A5057737749 @default.
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• W2765704865 date "2017-12-01" @default.
• W2765704865 modified "2023-09-26" @default.
• W2765704865 title "Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family" @default.
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