Essential Hydrophilic Carboxyl-terminal Regions Including Cysteine Residues of the Yeast Stretch-activated Calcium-permeable Channel Mid1
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m111603200
ISSN1083-351X
AutoresTakashi Maruoka, Yurika Nagasoe, S. Inoue, Yasunori Mori, June Goto, Mitsunobu Ikeda, Hidetoshi Iida,
Tópico(s)Redox biology and oxidative stress
ResumoThe yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca2+-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp441, generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn389 to Thr548. In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe522 (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca2+ uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn389 to Asn521 is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function. The yeast Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca2+-permeable nonselective cation channel composed of 548 amino acid residues. A physiological role of the Mid1 channel is known to maintain the viability of yeast cells exposed to mating pheromone, but its structural basis remains to be clarified. To solve this problem, we identified the mutation sites of mid1 mutant alleles generated by in vivo ethyl methanesulfonate mutagenesis and found that two mid1 alleles have nonsense mutations at the codon for Trp441, generating a truncated Mid1 protein lacking two-thirds of the intracellular carboxyl-terminal region from Asn389 to Thr548. In vitro random mutagenesis with hydroxylamine also showed that the carboxyl-terminal region is essential. To identify the functional portion of the carboxyl-terminal region in detail, we performed a progressive carboxyl-terminal truncation followed by functional analyses and found that the truncated protein produced from the mid1 allele bearing the amber mutation at the codon for Phe522 (F522Am) complemented the mating pheromone-induced death phenotype of the mid1 mutant and increased its Ca2+ uptake activity to a wild-type level, whereas N521Am did not. This result indicates that the carboxyl-terminal domain spanning from Asn389 to Asn521 is required for Mid1 function. Interestingly, this domain is cysteine-rich, and alanine-scanning mutagenesis revealed that seven out of 10 cysteine residues are unexchangeable. These results clearly indicate that the carboxyl-terminal domain including the cysteine residues is important for Mid1 function. The molecular mechanisms by which mechanical signals direct biological responses remain a frontier in the field of signal transduction. Electrophysiological studies have indicated that mechanotransduction can be mediated by ion channels that open or close in response to mechanical stimuli (1.French A.S. Annu. Rev. Physiol. 1992; 54: 135-152Crossref PubMed Scopus (167) Google Scholar, 2.Sackin H. Annu. Rev. Physiol. 1995; 57: 333-353Crossref PubMed Scopus (255) Google Scholar, 3.Garcia-Anoveros J. Corey D.P. Annu. Rev. Neurosci. 1997; 20: 567-594Crossref PubMed Scopus (140) Google Scholar, 4.Sukharev S.I. Blount P. Martinac B. Kung C. Annu. Rev. Physiol. 1997; 59: 633-657Crossref PubMed Scopus (261) Google Scholar, 5.Ghazi A. Berrier C. Ajouz B. Besnard M. Biochimie (Paris). 1998; 80: 357-362Crossref PubMed Scopus (38) Google Scholar). Such channels play essential roles in a wide variety of activities including cell volume control, development, morphogenesis, and neuronal signaling underlying touch, hearing, and balance. However, eukaryotic mechanosensitive ion channels have not been cloned until recently, and thus little is understood of their structures and functions at the molecular level. Recently, genes encoding eukaryotic mechanosensitive channels or their candidates have been found in some species, including MID1 in budding yeast (6.Kanzaki M. Nagasawa M. Kojima I. Sato C. Naruse K. Sokabe M. Iida H. Science. 1999; 285: 882-886Crossref PubMed Scopus (178) Google Scholar, 7.Kanzaki M. Nagasawa M. Kojima I. Sato C. Naruse K. Sokabe M. Iida H. Science. 2000; 288: 1347Crossref PubMed Scopus (17) Google Scholar), MEC4 in the nematode (8.Driscoll M. Chalfie M. Nature. 1991; 349: 588-593Crossref PubMed Scopus (458) Google Scholar, 9.Hong K. Mano I. Driscoll M. J. Neurosci. 2000; 20: 2575-2588Crossref PubMed Google Scholar), SIC in the rat (10.Suzuki M. Sato J. Kutsuwada K. Ooki G. Imai M. J. Biol. Chem. 1999; 274: 6330-6335Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and NOMPC in the fly (11.Walker R.G. Willingham A.T. Zuker C.S. Science. 2000; 287: 2229-2234Crossref PubMed Scopus (528) Google Scholar). A MID1 homologue in fission yeast, ehs1+/yam8+, has been reported (12.Carnero E. Ribas J.C. Garcı́a B. Durán A. Sánchez Y. Mol. Gen. Genet. 2000; 264: 173-183Crossref PubMed Scopus (29) Google Scholar, 13.Tasaka Y. Nakagawa Y. Sato C. Mino M. Uozumi N. Murata N. Muto S. Iida H. Biochem. Biophys. Res. Commun. 2000; 269: 265-269Crossref PubMed Scopus (14) Google Scholar). Ca2+ signaling constitutes an important backbone pathway, which is essential for a wide variety of cellular events. In Saccharomyces cerevisiae, Ca2+ has essential roles in the mating process induced by the mating pheromone, α-factor (14.Iida H. Yagawa Y. Anraku Y. J. Biol. Chem. 1990; 265: 13391-13399Abstract Full Text PDF PubMed Google Scholar). This process is divided into early and late stages, and Ca2+ seems to be required for the late stage only (15.Nakajima-Shimada J. Sakaguchi S. Tsuji F.I. Anraku Y. Iida H. Cell Struct. Funct. 2000; 25: 125-131Crossref PubMed Scopus (9) Google Scholar). In the early stage of this process, the mating pheromone binds to its cell surface receptor coupled with a heterotrimeric GTP-binding protein that activates a mitogen-activated protein kinase pathway, and the cells are eventually arrested in the G1 phase of the cell cycle (16.Dohlman H.G. Thorner J.W. Annu. Rev. Biochem. 2001; 70: 703-754Crossref PubMed Scopus (356) Google Scholar). In the late stage, at about 30 min after the binding of the mating pheromone, the cells differentiate into morphologically distinct cells having a mating projection, called shmoos, in which the cell wall and plasma membrane should be rearranged to support the polarized growth. This change accompanies Ca2+ influx necessary for the viability of shmoos (14.Iida H. Yagawa Y. Anraku Y. J. Biol. Chem. 1990; 265: 13391-13399Abstract Full Text PDF PubMed Google Scholar). When incubated in Ca2+-deficient medium, wild-type cells can grow normally in the absence of the mating pheromone but die after differentiation into shmoos if the mating pheromone is added (14.Iida H. Yagawa Y. Anraku Y. J. Biol. Chem. 1990; 265: 13391-13399Abstract Full Text PDF PubMed Google Scholar). Mutants showing the mid (mating pheromone-induced death) phenotype have been isolated, one of which is the mid1 mutant (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar). This mutant dies in response to the mating pheromone even in Ca2+-containing media because of a deficiency of Ca2+ influx and is rescued when high concentrations of CaCl2 are supplemented. Other yeast mutants that show the mid phenotype have been identified. Those include mutants deficient in calmodulin (18.Moser M.J. Geiser J.R. Davis T.N. Mol. Cell. Biol. 1996; 16: 4824-4831Crossref PubMed Scopus (94) Google Scholar), Ca2+/calmodulin-dependent protein kinases (18.Moser M.J. Geiser J.R. Davis T.N. Mol. Cell. Biol. 1996; 16: 4824-4831Crossref PubMed Scopus (94) Google Scholar), calcineurin (18.Moser M.J. Geiser J.R. Davis T.N. Mol. Cell. Biol. 1996; 16: 4824-4831Crossref PubMed Scopus (94) Google Scholar, 19.Withee J.L. Mulholland J. Jeng R. Cyert M.S. Mol. Biol. Cell. 1997; 8: 263-277Crossref PubMed Scopus (73) Google Scholar), and a homologue of the α1 subunit of mammalian, voltage-gated Ca2+ channels (20.Paidhungat M. Garrett S. Mol. Cell. Biol. 1997; 17: 6339-6347Crossref PubMed Scopus (161) Google Scholar, 21.Fischer M. Schnell N. Chattaway J. Davies P. Dixon G. Sanders D. FEBS Lett. 1997; 419: 259-262Crossref PubMed Scopus (149) Google Scholar). The MID1 gene encodes a stretch-activated Ca2+-permeable channel composed of 548 amino acid residues and has four hydrophobic segments named H1–H4 and 16 putative N-linked glycosylation sites (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar). Although the Mid1 polypeptide has no overall sequence similarity to known ion channels, the H2 and H4 segments are partially similar to the transmembrane segments of known ion channels (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar, 22.Jan L.Y. Jan Y.N. Nature. 1990; 345: 672Crossref PubMed Scopus (199) Google Scholar). The region downstream of the H4 segment, which is the carboxyl-terminal region, is hydrophilic, considerably cysteine-rich, and located in the cytoplasm (see Fig. 1). Because little is known about structure-function relationships in eukaryotic stretch-activated channels including the Mid1 channel, we performed mutational analyses on the identification of essential domains necessary for Mid1 function by taking advantage of the ease of molecular genetic approaches in yeast. Here, we report that the carboxyl-terminal region has a positively regulatory domain for Mid1 function. In addition, we have identified seven functionally important cysteine residues located in the carboxyl-terminal region. The yeast strains used in this study are listed in Table I. Rich media and a synthetic medium, SD, were prepared as described by Sherman et al. (23.Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). Because SD medium contains 680.2 μm CaCl2 and 0.8 μm calcium pantothenate, to make the Ca2+-deficient medium SD-Ca, CaCl2 was omitted, and calcium pantothenate was replaced by sodium pantothenate. SD. Ca100 medium was prepared by adding 100 μm CaCl2 to SD-Ca medium. LB and 2× YT media were prepared by the method of Sambrook et al. (24.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).Table IYeast strains used in this studyStrainGenotypeRef.H301–1MAT a mid1–1 his3-Δ1 leu2–3,112 trp1–289 ura3–52 sst1–217.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google ScholarH301–2MAT a mid1–2 his3-Δ1 leu2–3,112 trp1–289 ura3–52 sst1–217.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google ScholarH301–3MAT a mid1–3 his3-Δ1 leu2–3,112 trp1–289 ura3–52 sst1–217.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google ScholarH301–4MAT a mid1–4 his3-Δ1 leu2–3,112 trp1–289 ura3–52 sst1–217.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google ScholarH301–5MAT a mid1–5 his3-Δ1 leu2–3,112 trp1–289 ura3–52 sst1–217.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google ScholarH301–6MAT a mid1–6 his3-Δ1 leu2–3,112 trp1–289 ura3–52 sst1–217.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google ScholarH301–7MAT a mid1–7 his3-Δ1 leu2–3,112 trp1–289 ura3–52 sst1–217.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google ScholarThe strain H301–1 is the same as H301 described in Ref. 17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar. Open table in a new tab The strain H301–1 is the same as H301 described in Ref. 17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar. To isolate mid1 alleles, the gap repair method (25.Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1102) Google Scholar) was employed. The plasmid YCpMID1–21 (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar) containing the wild-type MID1 gene and the selection marker LEU2 was digested with restriction enzymes, HindIII and SnaBI, and digested plasmids without MID1 were introduced into seven mid1 mutants by a lithium acetate method (26.Rose M.D. Broach J.R. Methods Enzymol. 1991; 194: 195-230Crossref PubMed Scopus (214) Google Scholar). The transformants were plated onto SD-leucine plates, and then the gap-repaired plasmids were rescued from Leu+transformants by the method of Holm et al. (27.Holm C. Meeks-Wanger D.W. Fangman W.L. Botstein D. Gene (Amst.). 1986; 42: 169-173Crossref PubMed Scopus (224) Google Scholar). The recovered plasmids were propagated in Escherichia coli, purified, and subjected to DNA sequencing (28.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52678) Google Scholar) with a Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Biosciences) and a DNA sequencer, DSQ 2000L (Shimadzu, Kyoto, Japan). The YCpMID1–23 plasmid (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar) containing the wild-type MID1 gene was mutagenized with hydroxylamine (HA) 1The abbreviations used are: HAhydroxylamineMBLmethylene blue liquidGSTglutathione S-transferase with a mutation frequency of 0.9% by the method of Adams et al. (29.Adams A. Gottschling D. Kaiser C. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996Google Scholar). The HA solution contained 0.35 g of HA-HCl and 0.09 g of NaOH in 5 ml of H2O. Ten micrograms of YCpMID1–23 was added to 500 μl of the HA solution sterilized through a filter (Millex-GV, 0.22 μm, Millipore Corp., Bedford, MA), and the mixture was incubated for 20 h at 30 °C. On the following day, 10 μl of 5 mNaCl and 50 μl of 1 mg/ml bovine serum albumin were added to the mixture to stop the reaction, and the plasmid DNA was precipitated by ethanol. The mutagenized and purified plasmids were introduced into the E. coli strain XL1-Blue for amplification. The amplified plasmids were introduced into the mid1-1 mutant, and the viability of the transformants was examined by the methylene blue plate method (see below). The plasmids incapable of complementing the mid1-1 mutation were treated with BamHI and XhoI to cut out the DNA fragment containing the MID1 coding region as well as the 5′- and 3′-flanking regions (150 bp upstream of the initiation codon and 858 bp downstream of the termination codon). The DNA fragments were inserted into the vector portion of unmutagenized YCpMID1–23 treated with BamHI and XhoI and tested again for complementing activity. This procedure eliminates possible mutations in the vector. The plasmids finally identified were subjected to DNA sequence analysis (28.Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52678) Google Scholar). hydroxylamine methylene blue liquid glutathione S-transferase Site-directed mutagenesis of the MID1 gene in the plasmid YCpMID1–23 (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar) was performed with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the method described by the manufacturer. Mutagenic primers (Table II) were synthesized to order by Sawady Technology Co., Ltd. (Tokyo, Japan).Table IIMutagenic primers used in this studyPrimerSequenceCarboxyl-terminal truncation V445Op-F5′-GGGTATGTGCATGATCAATTCCG-3′ V445Op-R5′-CGGAATTGATCATGCACATACCC-3′ C450Op-F5′-CAATTCCGAGATGAACCACGACG-3′ C450Op-R5′-CGTCGTGGTTCATCTCGGAATTG-3′ Y457Am-F5′-CGTCTTCCCAATAGTACATCCACAGAG-3′ Y457Am-R5′-CTCTGTGGATGTACTATTGGGAAGACG-3′ K463Op-F5′-CCACAGAGATTGAAGTCACAACCG-3′ K463Op-R5′-CGGTTGTGACTTCAATCTCTGTGG-3′ Y481Am-F5′-CCTTTGGATGACTAGTACGAAATTCTACC-3′ Y481Am-R5′-GGTAGAATTTCGTACTAGTCATCCAAAGG-3′ V495Op-F5′-GCTATACATTATGACGAAATTGCCC-3′ V495Op-R5′-GGGCAATTTCGTCATAATGTATAGC-3′ F502Am-F5′-GCCCAAGCGATTAGCAATTTTCCTGTCCG-3′ F502Am-R5′-CGGACAGGAAAATTGCTAATCGCTTGGGC-3′ L510Am-F5′-CCTGTCCGAATGATTAGACTACGGAAGATCTTCTTTATC-3′ L510Am-R5′-GATAAAGAAGATCTTCCGTAGTCTAATCATTCGGACAGG-3′ Y520Am-F5′-CGGAAGATCTTCTTTATCAAAGTTAGAATTTCTATATGG-3′ Y520Am-R5′-CCATATAGAAATTCTAACTTTGATAAAGAAGATCTTCCG-3′ N521Am-F5′-CTTTATCAAAGTTACTAGTTCTATATGGACACTGACTACTCAACC-3′ N521Am-R5′-GGTTGAGTAGTCAGTGTCCATATAGAACTAGTAACTTTGATAAAG-3′ F522Am-F5′-CTTTATCAAAGTTACAATTAGTATATGGACACTGACTACTCAACC-3′ F522Am-R5′-GGTTGAGTAGTCAGTGTCCATATACTAATTGTAACTTTGATAAAG-3′ Y523Am-F5′-CTTTATCAAAGTTACAATTTCTAGATGGACACTGACTACTCAACC-3′ Y523Am-R5′-GGTTGAGTAGTCAGTGTCCATCTAGAAATTGTAACTTTGATAAAG-3′ M524Am-F5′-CTTTATCAAAGTTACAATTTCTATTAGGACACTGACTACTCAACC-3′ M524Am-R5′-GGTTGAGTAGTCAGTGTCCTAATAGAAATTGTAACTTTGATAAAG-3′ D525Am-F5′-CTTTATCAAAGTTACAATTTCTATATGTAGACTGACTACTCAACC-3′ D525Am-R5′-GGTTGAGTAGTCAGTCTACATATAGAAATTGTAACTTTGATAAAG-3′ T526Op-F5′-CTATATGGACTGAGACTACTCAACCTG-3′ T526Op-R5′-CAGGTTGAGTAGTCTCAGTCCATATAG-3′ V541Op-F5′-GGTAACTCATCCTTGATGTGAATTCATCCATTGGACGATACG-3′ V541Op-R5′-CGTATCGTCCAATGGATGAATTCACATCAAGGATGAGTTACC-3′Disruption of casein kinase 2 phosphorylation site T511A-F5′-CCTGTCCGAATGATCTTGCGACGGAAGATCTTCTTTATC-3′ T511A-R5′-GATAAAGAAGATCTTCCGTCGCAAGATCATTCGGACAGG-3′ T511V-F5′-CCTGTCCGAATGATCTTGTGACGGAAGATCTTCTTTATC-3′ T511V-R5′-GATAAAGAAGATCTTCCGTCACAAGATCATTCGGACAGG-3′Disruption of sheet-turn-sheet structure Y520A-F5′-CGGAAGATCTTCTTTATCAAAGTGCCAATTTCTATATGG-3′ Y520A-R5′-CCATATAGAAATTGGCACTTTGATAAAGAAGATCTTCCG-3′ Y520G-F5′-CGGAAGATCTTCTTTATCAAAGTGGCAATTTCTATATGG-3′ Y520G-R5′-CCATATAGAAATTGCCACTTTGATAAAGAAGATCTTCCG-3′Cysteine substitution C417A-F5′-GCTCTACAGTTAATATCCGCCGACGCAGACAAAGACGC-3′ C417A-R5′-GCGTCTTTGTCTGCGTCGGCGGATATTAACTGTAGAGC-3′ C431A-F5′-CCGGTCATGACTGCCGACGATTGTGCCG-3′ C431A-R5′-CGGCACAATCGTCGGCAGTCATGACCGG-3′ C434A-F5′-CCGGTCATGACTTGTGACGATGCCGCCGAAGCTTATCG-3′ C434A-R5′-CGATAAGCTTCGGCGGCATCGTCACAAGTCATGACCGG-3′ C443A-F5′-CGTGATTGGGTAGCCGCAGTATCAATTCCG-3′ C443A-R5′-CGGAATTGATACTGCGGCTACCCAATCACG-3′ C450A-F5′-GCAGTATCAATTCCGAGAGCCACCACGACGTCTTTCCC-3′ C450A-R5′-GGGAAGACGTCGTGGTGGCTCTCGGAATTGATACTGC-3′ C487A-F5′-CGAAATTCTACCTGCCATTGACATGTGC-3′ C487A-R5′-GCACATGTCAATGGCAGGTAGAATTTCG-3′ C491A-F5′-CCTTGTATTGACATGGCCTATACATTAGTACG-3′ C491A-R5′-CGTACTAATGTATAGGCCATGTCAATACAAGG-3′ C498A-F5′-GCTATACATTAGTACGAAATGCCCCAAGCG-3′ C498A-R5′-CGCTTGGGGCATTTCGTACTAATGTATAGC-3′ C506A-F5′-GCGATTTTCAATTTTCCGCCCCGAATGATCTTACTACGG-3′ C506A-R5′-CCGTAGTAAGATCATTCGGGGCGGAAAATTGAAAATCGC-3′ C531A-F5′-GGACACTGACTACTCAACCGCCAATTATATAGG-3′ C531A-R5′-CCTATATAATTGGCGGTTGAGTAGTCAGTGTCC-3′Underlines represent substituted nucleotides. Open table in a new tab Underlines represent substituted nucleotides. For screening mutants bearing a nonfunctional mid1 gene after in vitro random mutagenesis with HA, we employed a methylene blue plate (MBP) method (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar). This method is based on the fact that viable cells, but not inviable cells, reduce methylene blue to give the colorless leukomethylene blue (30.Pierce J.S. J. Inst. Brew. 1970; 76: 442-443Crossref Scopus (71) Google Scholar, 31.Rose A.H. Methods Cell Biol. 1975; 12: 1-16Crossref PubMed Scopus (6) Google Scholar). Thus, the viable cells remain white, and inviable cells stain blue. Cells of the mid1 mutant transformed by HA-treated YCpMID1–23 (LEU2) were plated onto SD plates lacking leucine to give about 1000 colonies/plate and incubated for 2–3 days at 30 °C. The Leu+ transformants on the plates received 0.1 ml of 1 mm α-factor and 7 ml of SD medium containing 0.5% agar and 0.01% methylene blue and were incubated for several days. At this stage, the colonies mainly with dead cells become blue but still have viable cells in parts, whereas colonies formed by cells transformed with intact YCpMID1–23 remain white. The blue colonies were selected, suspended in 0.1 ml of SD medium, streaked onto an SD plate, and incubated for 2 days. To avoid selecting cells contaminated from other colonies at the step of pouring 0.5% agar described above, six colonies from each single blue colony were then subjected to quantitative viability assay using the methylene blue liquid (MBL) method (14.Iida H. Yagawa Y. Anraku Y. J. Biol. Chem. 1990; 265: 13391-13399Abstract Full Text PDF PubMed Google Scholar). In the MBL method, 0.1 ml of exponentially growing culture in SD. Ca100 medium or that treated with α-factor for 4 or 8 h was mixed with an equal volume of 0.01% methylene blue, 2% sodium citrate solution, and the number of viable white cells and inviable blue cells was counted under a differential interference-contrast microscope (Olympus, Tokyo, Japan). The viability was expressed as the number of viable white cells as a percentage of the total number of cells. Colonies that produced about 30% viable culture 8 h after receiving α-factor were selected for further analyses. Rabbit polyclonal antibodies to the Mid1 protein were raised against the glutathione S-transferase-Mid1 (GST-Mid1) fusion protein. To obtain the GST-Mid1 protein, we constructed pGEX-6P-2-MID1, an E. coli plasmid that expresses it under the control of the tac promoter. Fusion of Mid1 with GST was necessary, because Mid1 was toxic to the cells when expressed alone. pGEX-6P-2 (Amersham Biosciences) was digested with BamHI and ligated with a linker DNA containing the NsiI site (5′-pGATCCATGCATCTGCAGATGCATG-3′, where the NsiI sites are underlined) to create the pGEX-6P-2-N vector. pGEX-6P-2-MID1 was constructed by inserting the 1.7-kb NsiI-NsiI fragment that contains the entire MID1 open reading frame from pBSMID1-N2 into the NsiI site of pGEX-6P-2-N. The E. coli strain JM109 was transformed with pGEX-6P-2-MID1, and the transformant was incubated to the stationary phase in 2× YT medium at 37 °C. The culture was diluted to one-tenth by 2× YT medium and incubated for 1 h. Then the culture received 1 mmisopropyl-1-thio-β-d-galactopyranoside and was incubated for 3 h at 37 °C to induce the expression of GST-Mid1. The cells were harvested and disrupted by sonication. The protein extracts were fractionated by centrifuging at 20,000 × g, and the pellets were washed twice with PBS containing 2% Triton X-100 (Bio-Rad). Because the GST-Mid1 fusion protein was present in the inclusion body, 7 m urea solution made in 50 mmTris-HCl (pH 8.5), 10 mm dithiothreitol, and a protease inhibitor mixture (catalog no. P8849; Sigma) was used to solubilize it. The solubilized materials were mixed with an equal volume of 2× SDS-sample buffer and separated on 7.5% SDS-polyacrylamide gel. After staining with Coomassie Brilliant Blue, the desired gel strip containing the 97-kDa GST-Mid1 protein was collected, and it was electrophoretically purified by a model 422 electroeluter (Bio-Rad). Protein concentration was adjusted to 1 mg/ml or higher by using an ultrafiltration filter, Microcon YM-50 (Millipore Corp.). Preparation of rabbit anti-Mid1 antibodies was ordered from Asahi Techno Glass, Inc. (Chiba, Japan). The antibodies specifically recognized the Mid1 protein, as revealed by immunoblot analysis on yeast whole cell proteins, and were used at a dilution 1:10,000. Immunoblot analysis was performed as described previously (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar). Ca2+ accumulation in the cells was measured according to the method described by Iida et al. (14.Iida H. Yagawa Y. Anraku Y. J. Biol. Chem. 1990; 265: 13391-13399Abstract Full Text PDF PubMed Google Scholar) except that SD. Ca100 medium was used instead of SD or SD-Ca medium. The45Ca2+ overlay experiments on the Mid1 protein were carried out by the method of Maruyama et al. (32.Maruyama K. Mikawa T. Ebashi S. J. Biochem. (Tokyo). 1984; 95: 511-519Crossref PubMed Scopus (628) Google Scholar) with minor modifications. The two-hybrid assay was used to examine the possible protein-protein interactions as described previously (33.Bartel P.L. Chen C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar,34.James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Interactions were tested between (a) the carboxyl terminus of Mid1 fused to the Gal4 DNA-binding domain and that fused to the Gal4 activation domain, (b) the carboxyl terminus of Mid1 fused to the Gal4 DNA-binding domain and a central region of Mid1 (designated H2-H4) fused to the Gal4 activation domain, and (c) the carboxyl terminus of Mid1 fused to the Gal4 activation domain and the H2-H4 region fused to the Gal4 DNA-binding domain. The following plasmids were used: pGBT9 carrying the Gal4 DNA-binding domain (33.Bartel P.L. Chen C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar); pGAD424 carrying the Gal4 activation domain (33.Bartel P.L. Chen C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford1993: 153-179Google Scholar); pGBT-Mid1C carrying the carboxyl terminus of Mid1 (codons 397–548) fused to the Gal4 DNA-binding domain; pGAD-Mid1C carrying the carboxyl terminus of Mid1 (codons 398–548) fused to the Gal4 activation domain; pGBT-Mid1 H2-H4 carrying a central region of Mid1 (codons 132–374 starting after the H2 domain and terminating in the H4 domain and thus designated H2–H4) fused to the Gal4 DNA-binding domain; pGAD-Mid1 H2–H4 carrying the H2–H4 region fused to the Gal4 activation domain; and a positive control plasmid, pYN870, carrying the full-length Gal4 (35.Yao Y. Yamamoto K. Nishi Y. Nogi Y. Muramatsu M. J. Biol. Chem. 1996; 271: 32881-32885Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Combinations of control and fusion plasmids were transformed into the PJ69-4A strain (34.James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Transformants were streaked onto agar plates containing synthetic medium without histidine or adenine. The transformants were also examined for β-galactosidase activity by the filter method (36.Breeden L. Nasmyth K. Cold Spring Harbor Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (470) Google Scholar) and the quantitative liquid method (37.Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). To identify the amino acids required for the function of Mid1, we isolated and analyzed the seven EMS-induced mid1 alleles that we reported previously (17.Iida H. Nakamura H. Ono T. Okumura M.S. Anraku Y. Mol. Cell. Biol. 1994; 14: 8259-8271Crossref PubMed Scopus (194) Google Scholar). The mid1 alleles were recovered by the gap repair method as described under "Experimental Procedures," and the gap-repaired mid1 alleles that did not complement the mid phenotype (see above) of the corresponding mid1 mutants were selected and then subjected to DNA sequencing and computational polypeptide analysis. As shown in Fig. 2, the mid1-1 allele product had two mutations, Y254H (TAC to CAC) and W441Op (TGG to TGA). Analysis of the single mutations generated by site-directed mutagenesis showed that the Y254H mutation was a silent mutation and that the W441Op mutation was responsible for the mid phenotype associated with the mid1-1 mutation (data not shown). The mid1-2 and mid1-5 allele products had a common mutation, Y254H (TAC to CAC), indicating that the two alleles have no loss-of-function mutation in the coding region. The mid1-3 allele product had G104D (GGC to GAC) and Y254H (TAC to CAC) mutations. Again, analysis of the single mutations generated by site-directed mutagenesis showed that only the G104D mutation was responsible for the mid phenotype associated with the mid1-3 mutation (data not shown). It is of interest that Gly104 is located in the hydrophobic segment H2. The mid1-4 allele product had Y254H (TAC to CAC) and W441Am (TGG to TAG) mutations. Note that this allele product is the same as the mid1-1 allele product with different stop codons. The mid1-6 allele had a deletion of a nucleotide in the codon for Met109, resulting in a frameshift mutation producing
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