Molecular Microbiology (1992) 6(11), 1441-1446

IRE1 encodes a putative protein kinase containing a membrane-spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae Jun-lchi Nikawa^ and Satoshi Yamashita* Department of Biochetvistry, Gunma University School of Medicine, Maebashi371, Japan. Summary A novel gene, IRE1, of Saccharomyces cerevisiae was cloned through genetic complementation of a myoinositol auxotrophic mutant. The predicted amino acid sequence indicated that tREI encodes a protein of 126 983 Da with two highly hydrophobic regions, probably a signal sequence and a membrane-spanning region. The carboxy-terminal region of IRE1 showed close sequence similarity to the catalytic domains of protein kinases. Disruption of the IRE1 locus caused myo-inositol auxotrophy. The IRE1 product is very likely a protein kinase required for myo-inositol synthesis.

Introduction Protein kinases play imporiant regulatory roles in intermediate metabolism, gene transcription, cell division, homione actions, and tumourigenesis {reviewed by Hanks et al,, 1988). Some mammalian protein kinases contain one or more membrane-spanning regions and function as membrane receptors. Binding of specific ligands to these receptors causes activation of their protein kinase activity and, in some cases, stimulation of inositol phospholipid metabolism also. Although 30 or more protein kinase genes have been isolated from the budding yeast Saccharomyces cerevisiae, to date none of them have been shown to have a transmembrane region. Several types of yeast mutant defective in inositol phospholipid metabolism have been isolated. Henry and coworkers (reviewed by Carman and Henry, 1989) isolated a number of mutants defective in myo-inositol (inositol) synthesis by isolating inositoi auxotrophs. They included not only structural gene mutants (inoi). but also regulatory Received 6 January. 1992: revised and accepted 2 March, 1992. "^Present address: Department of Biochemical Engineering and Science, Faculty cf Computer Science and Systems Engineering, Kyushu Institute of Technclogy, lizuka, Fukuoka 820. Japan. 'For correspondence. Tel. (272) 31 7221 ext. 2557; Fax (272) 31 9278. This work is dedicated to the memory of Stiosaku Numa, Kyoto University, Japan.

gene mutants (ino2, ino4, opil). In our laboratory, a mutant defective in phosphatidylinositol synthesis was obtained by selecting an inositol auxotroph requiring a high concentration of inositol for grovi^h (Nikawa and Yamashita, 1982). This mutant was shown to contain an altered phosphatidylinositol synthase with a greatly increased Km for inositol and was used for cloning of the phosphatidylinositol synthase gene, PIS {Nikawa and Yamashita, 1984; Nikawa e/a/., 1987). In the present study, using a different inositol auxotroph, we cloned a novel gene, IRE1, which very likely encodes a protein kinase involved in inositol synthesis. The IRE1 product contained an extended hydrophobic region thought to be membrane-spanning at the centre of Its molecule.

Results and discussion In an attempt to isolate a gene involved in the metabolism of inositol phospholipids, an inositol auxotroph requiring a high concentration of inositol for growth was isolated from mutagenized X2180-1B (aSUC2 mat gal2 CUP1: Yeast Genetic Stock Center, University of California). The mutant {strain 425) was obtained as a high-inositol-requiring mutant which grew well on 100 ^.g m r ' inositol, but poorly on 2 M-g m l " ' inositol. When determined in the presence of a lower concentration of inositol (0.2 |xg mP'), the doubling time of the mutant was 11 h, i.e., much longer than that of the wild type {4.5 h). But in the presence of 100 ^.g m l ' inositol, the doubling time was 4h, which was comparable with that of the wild type {3.5 h). Ieu2 his4 ttp1 markers were introduced into strain 425 and the resulting strain, D437-1B, was transformed with yeast genomic libraries, LL1 and LL3, constructed in a multicopy LEU2 vector, YEpM4. Leu+ Ino"" transformants were selected on minimum medium without inositol. Plasmids were obtained from the transformants and amplified in Escherichia coli HB101, and then their complementation was reexamined. Two plasmids {plRE2 and plRE3) were obtained from library LL1 and one {plRE4) from LL3 (Fig. 1). The inserts of plRE2 and plRE4 showed identical overlapping maps, but their orientations in the vector were opposite. Both plasmids effectively complemented the mutation in D437-1B. The gene carried on these plasmids was designated as IRE1 ([nositol requiring) and studied as

1442 J.~i. Nikawa and S. Yamashita H 1

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1 kbp Rg-1- Restriction maps of plRE2 and plR£4. plRE201 and plRE202 are subctones of plRE2. plRE401 and plRE404 are subclones of plRE4. Only the insert is shown for each clone. The ability of each clone to complement the growth phenotype of strain D437-1B is indicated by + or - . Each insert has a multicloning site at each end. The abbreviations used are; H. Hin6m, K. Kpnl, S, Sac\: X, Xho\.

described below. The second gene on ptRE3 was designated as IRE2. but was not studied further because of its rather weak complementation. To localize the IRE1 gene within the inserts, we constructed subclones. plRE201. plRE202, plRE401, and plRE402, and then examined complementation in D4371B. As shown in Fig. 1, the results indicate that the IRE1 gene is located between the Xho\ and second H/ndlll sites on plRE2, This was confirmed by cloning the fragment into a low copy-number vector, YCp50. Sequence determination was carried out by the dideoxy chain termination method (Sanger etai., 1977). Within the 3961 bp sequence shown in Fig. 2, there was a large open reading frame capable of encoding 1115 amino acid residues, with a calculated molecular weight of 126983. The sequence surrounding the ATG codon at positions 356-358 conformed to Kozak's consensus sequence for a translational initiation site, AXXATGXXT {Kozak, 1984). Two putative TATA boxes (Gannon etal., 1977) were found at positions 150-153 and 186-189. There was no intron splice signal TACTAAC (Langford and Gallwitz, 1983). Hydropathy analysis (Kyte and Doolittle, 1982) revealed that the IRE1 product comprised four regions, a putative signal sequence (residues 9-18), an A/-terminal hydrophilic domain, a transmembrane region (residues 527-555). and a C-terminal hydrophiiic domain. The hydropathy profile of the IRE1 product resembled that of the EGF receptor, which also comprises four domains (Ullrich et al., 1984). The N-terminal putative signal sequence was followed by an Arg- and Lys-rich sequence, conforming to the structural features of a signal sequence (Von Heijne, 1983). The extended /V-terminal hydrophilic domain contained over 500 amino acids. There was a short basic amino acid cluster at residues 91-99. At the centre of the molecule, there was an extended (29-amino-acid), hydrophobic region, thought to be a transmembrane region. The

C-terminal region was hydrophilic and contained a basic amino acid cluster of 18 amino acids (residues 642-659) containing five Arg and seven Lys. Interestingly, this extended hydrophilic region shared significant sequence similarity with protein kinases. Comparison of this region with protein kinases is made in Fig. 3. The consensus sequences found in all of the known protein kinases (Hanks et al., 1988) are very well-conserved at similar positions in the IRE1 product as well. The Gly-X-Gly-XX-Gly sequence thought to be part of the ATP-binding region was found at positions 681-686. The Lys residue believed to be involved in the phosphotransfer reaction was found 16 residues downstream. Further downstream, three other protein kinase consensus sequences were observed: Arg-Asp-Leu (residues 795-797), Asp-PheGly (residues 828-830), and Ala-Pro-Glu (residues 857859). Based on the extensive homology with protein kinases and the hydropathy profile, the IRE1 gene was predicted to encode a protein kinase with a receptor-like structure. To study the function of the IRE1 gene, we constructed an irei disruptant and examined its phenotype as follows. A 2.1 kbp Kpnl fragment of IRE1 was replaced with the yeast HIS3 gene. This caused deletion of 73% of the IRE1 coding region. The disrupted ire1 gene was introduced into wild-type diploid DJ11 (a/a Ieu2/leu2 his3/his3 trpi/ trpi). The resulting heterozygous diploid D2013 [IREI/ ire1::HIS3) was allowed to sporulate and then the spores were separated by tetrad dissection. Gene disruption was examined by Southern blot analysis (data not shown). Haploids carrying disrupted ire1 were found to be viable and grew on YPD medium or minimum medium supplemented with 20fxg mi"^ inositol, but grew very poorly on inositoi-free minimum medium (Fig. 4). Thus, the irei disruptant required high concentrations of inositol for growth. Attempts to determine whether the IRE1 locus was allelic to the original mutation in strain 425 were unsuccessful because the cross of the disruptant with strain 425 rarely yielded four viable spores. The present results indicate that the IRE1 gene plays a stimulatory role in the synthesis of inositol, probably through protein phosphorylation. Several possibilities were considered as to the role of IREI phosphorylation in inositol synthesis. First, the 'iREi kinase' may act to phosphorylate one of the enzymes involved in inositol synthesis, causing enzyme activation. IREI disruption would result in dephosphorylation, with concomitant inactivation of the enzyme. The second possibility may be that the 'iREI kinase' stimulates the expression of a structral gene for inositol synthesis through the phosphorylation of a transcription factor or some other component of the transcription machinery. Several examples are known of protein kinases being involved in the control of gene expression in the yeast. The

Putative yeast protein kinase gene IRE1

GGAGCGTAAGCCTCTTCGGGCAATACCTTCGACTATTCCAACAATAAAATTTAAAAAAAG AGATTAATCACATAGTAACAAGAAATAAACGAAAAACATATCATAGGAGATCAATGAGCC AACTTCACATAAACTAACAGTGAAAAGTCTATAACAATATTAATTTTACACAATTAAATC TACACTATAACTGGCACTGTTAATAATCGGTAATCTGCTAAGTAACTTTTTTTTCTCATT CACAAAGCATCGTTTTCCTCTTCCCCACGTCCATTATCACTTTTCTCCATATCACCCTTC ATACACATTAAAAAAACAGCATATCTGAGGAATTAATATTTTAGCACTTTGAAAAATGCG * MR TCTACTTCGAAGAAACATGTTAGTATTGACACTGCTCGTTTGTGTGTTTTCATCCATCAT L L R R N M L _ V L T L L V__C _V F S S I I TTCATGCTCAATCCCATTGTCGTCTCGCACCTCAAGGCGGCAGATAGTGGAAGATGAAGT S C S r P L S S B T S R R Q I V E D E V TGCCTCCACTAAAAAGCTCAATTTCAACTATGGTGTGGATAAAAATATAAACTCGCCCAT A S T K K L N F N y G V D K N I N S P I TCCTGCTCCAAGAACCACTGAAGGTTTACCAAATATGAAACTCAGCTCATATCCAACTCC P A P R T T E G L P N M K L S S Y P T P TAACTTATTGAATACTGCTGATAATCGACGTGCTAACAAAAAAGGACGTAGGGCTGCCAA N L L N T A D N R R A N K K G R R A A N TTCTATAAGTGTACCCTATTTGGAGAATCGTTCCTTGAACGAACTGAGTTTATCAGATAT S I S V P Y L E N R S L N E L S L S D I ACTAATCGCAGCCGACGTTGAGGGTGGACTTCATGCTGTAGATAGAAGAAATGGTCATAT L I A A D V E G G L H A V D R R N G H l CATATGGTCAATCGAACCAGAAAATTTTCAACCTCTGATAGAAATACAAGAACCTTCGAG I W S I E P E N F Q P L I E I Q E P S R GTTAGAAACATATGAAACGTTGATTATAGAACCTTTCGGTGATGGGAACATTTACTACTT L E T Y E T L I l E P F G D G N I Y Y F TAACGCCCATCAAGGGTTACAAAAACTGCCTTTATCCATACGACAACTTGTATCAACTTC N A H Q G L Q K L P L S I R Q L V S T S CCCGCTGCACTTGAAAACAAATATTGTGGTTAATGACTCTGGAAAAATTGTTGAAGATGA P L H L K T N I V V N D S G K I V E D E AAAGGTCTACACTGGATCGATGAGAACTATAATGTATACTATAAACATGTTGAATGGTGA K V Y T G S M R T I M Y T I N H L N G E AATTATATCAGCGTTCGGACCTGGTTCAAAAAACGGGTATTTCGGGAGCCAGAGTGTGGA I I S A F G P G S K N G Y F G S Q S V D TTGCTCACCTGAGGAGAAGATAAAACTTCAGGAATGTGAAAATATGATTGTAATAGGCAA C S P E E K I K L Q E C E N H I V I G K AACTATTTTTGAGCTGGGAATTCACTCTTATGATGGAGCAAGCTACAATGTCACTTACTC T I F E L G I H S Y D G A S Y N V T Y S TACATGGCAGCAAAATGTTTTAGATGTTCCCCTAGCGCTTCAGAATACATTTTCAAAGGA T W Q Q N V L D V P L A L Q N T F S K D CGGCATGTGCATAGCGCCTTTCCGTGATAAATCATTGCTAGCAAGCGATTTAGATTTTAG G M C I A P F R D K S L L A S D L D F R AATTGCTAGATGGGTTTCTCCGACATTCCCCGGAATTATTGTTGGGCTTTTCGATGTGTT I A R W V S P T F P G I r V G L F D V F TAATGATCTCCGCACCAGTGAAAATATACTGGTACCGCATCCCTTTAATCCTGGTGATCA N D L R T S E N I L V P H P F N P G D H TGAAAGTATATCGAGTAACAAAGTTTACTTGGATCAGACTTCGAACCTCTCCTGGTTTGC E S I S S N K V Y L D Q T S N L S W F A ATTATCTAGTCAGAATTTTCCATCTTTAGTCGAATCAGCTCCCATATCAAGATACGCTTC L S S Q N F P S L V E S A P I S R Y A S CAGTGACCGTTGGAGGGTGTCTTCAATTTTTGAAGATGAGACTTTATTCAAGAACGCAAT S D R W R V S S I F E D E T L F K N A I CATGGGTGTTCATCAGATATATAATAATGAATATGATCACCTTTATGAAAACTATGAAAA H G V H Q I Y N N E Y D H L Y E N Y E K AACGAATAGTTTGGACACTACGCACAAATATCCACCTCTGATGATTGATTCGTCCGTTGA T N S L D T T H K Y P P L M I D S S V D TACAACCGATTTACATCAGAATAACGAGATGAATTCACTAAAGGAATACATGTCACCAGA T T D L H Q N N E M N S L K E Y M S P E AGACCTTGAGGCATATAGAAAAAAGATACACGAGCAAATATCGAGAGAATTAGATGAAAA D L E A Y R K K I H E Q I S R E L D E K GAACCAAAATTCTTTGCTACTGAAGTTTGGAAGTCTAGTATATCGAATTATAGAGACTGG N Q N S L L L K F G S L V Y R I I E T G AGTATTTCTGTTGTTATTTCTCATTTTTTGTGCAATACTACAAAGATTCAAAATTTTGCC

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Fig. 2. Nucleotide sequence of the IREI gene and the deduced amino acid sequence of the product. The largest open reading frame is translated into an amino acid sequence and shown below the nucleotide sequence, using the one-letter code. The dashed underline indicates the putative signal sequence and the solid underline the potential membrane-spanning region. The asterisks denote the in-frame termination codons. This sequence has been submitted to the EMBL/GenBank/DDJB. Nucleotide Sequence Data Libraries and appears under the accession number Z11701.

1444 J.-l. Nikawa and S. Yamashita

V F L L L F L I F C A I L Q R F K I L P GCCACTATATGTATTATTATCCAAAATTGGATTTATGCCTGAAAAGGAAATCCCCATAGT P L Y V L L S K I G F M P E K E I P I V TGAGTCGAAATCGCTAAATTGTCCCTCTTCATCGGAAAATGTAACCAAGCCATTCGATAT E S K S L N C P S S S E N V T K P F D M GAAATCAGGGAAGCAAGTTGTTTTTGAAGGTGCTGTGAACGATGGAAGTCTAAAATCTGA K S G K Q V V F E G A V N D G S L K S E AAAAGATAAGCATGATGCTGATGAAGATGATGAAAAATCACTAGATTTAACCACAGAAAA K D K H D A D E D D E K S L D L T T E K GAAGAAGAGGAAAAGAGGTTCGAGAGGAGGCAAAAAGGGCCGAAAATCACGCATTGCAAA K K R K R G S R G G K K G R K S R I A N TATACCAAACTTTGAGCAATCTTTAAAAAATTTGGTAGTATCCGAAAAAATTTTAGGTTA I P N F E Q S L K N L V V S E K I L G Y CGGTTCATCAGGAACAGTAGTTTTTCAGGGAAGTTTTCAAGGAAGACCTGTTGCGGTAAA G S S G T V V F Q G S F Q G B P V A V K GAGAATGTTAATTGATTTTTGTGACATAGCTTTAATGGAAATAAAACTTTTGACTGAAAG R M L I D F C D I A L H E I K L L T E S CGATGATCATCCTAACGTCATACGATACTACTGTTCAGAAACAACAGACAGATTTTTGTA D D H P N V I R Y Y C S E T T D R F L Y TATTGCTTTAGAGCTCTGCAATTTGAACCTTCAAGATTTGGTGGAGTCTAAGAATGTATC I A L E L C N L N L Q D L V E S K N V S AGATGAAAACCTGAAATTACAGAAAGAGTATAATCCAATTTCGTTATTGAGACAAATAGC D E N L K L Q K E Y N P I S L L R Q T A GTCCGGGGTAGCACATTTACATTCTTTAAAGATTATCCATCGAGATTTAAAGCCTCAAAA S G V A H L H S L K I I H R D L K P Q N TATTCTCGTTTCTACTTCGAGTAGGTTTACTGCCGATCAGCAAACAGGAGCAGAAAATCT I L V S T S S R F T A D Q Q T G A E N L TCGAATTTTGATATCAGACTTTGGTCTTTGCAAAAAACTAGACTCTGGTCAGTCTTCATT R I L I S D F G L C K K L D S G Q S S F TAGAACAAATTTGAATAACCCTTCTGGCACAAGTGGTTGGAGGGCCCCAGAGCTGCTTGA R T N L N N P S G T S G W R A P E L L E AGAATCAAACAATTTGCAGTGCCAAGTCGAAACGGAACACTCTTCTAGTAGGCATACAGT E S N N L Q C Q V E T E H S S S R H T V AGTTTCATCTGATTCTTTTTATGATCCGTTCACCAAGAGGAGGCTAACAAGATCTATTGA V S S D S F Y D P F T K R R L T R S I D TATTTTTTCTATGGGATGTGTATTCTATTATATCCTATCCAAAGGGAAGCATCCATTTGG I F S M G C V F Y Y I L S K G K H P F G AGATAAATATTCACGTGAAAGCAATATCATAAGAGGAATATTCAGTCTTGATGAAATGAA D K Y S R E S N I I R G I F S L D E M K ATGTCTACATGATAGATCCTTAATTGCAGAAGCTACAGATCTGATCTCCCAAATGATTGA C L H D R S L I A E A T D L I S Q M I D TCACGATCCGTTAAAAAGACCTACTGCTATGAAAGTTCTAAGGCATCCGTTGTTTTGGCC H D P L K R P T A M K V L R H P L F W P AAAGTCGAAAAAATTGGAGTTCCTTTTAAAAGTTAGTGATAGGCTTGAAATTGAAAACAG K S K K L E F L L K V S D R L E I E N R AGACCCTCCAAGTGCCCTGTTAATGAAATTTGACGCCGGTTCTGACTTTGTAATACCCAG D P P S A L L M K F D A G S D F V I P S TGGAGATTGGACTGTCAAGTTTGATAAAACATTCATGGACAACCTTGAAAGGTACAGAAA G D W T V K F D K T F M D N L E R Y R K ATACCATTCATCAAAGTTAATGGATCTATTAAGAGCACTTAGGAATAAATATCATCATTT Y H S S K L H D L L R A L R N K Y H H F TATGGATTTACCTGAAGATATAGCAGAACTAATGGGGCCGGTACCCGATGGATTTTACGA M D L P E D I A E L M G P V P D G F Y D TTACTTCACCAAGCGTTTTCCAAACCTATTAATAGGTGTTTATATGATTGTCAAGGAAAA Y F T K R F P N L L I G V Y M I V K E N TTTAAGTGACGATCAAATTTTACGTGAATTTTTGTATTCATAACATGTTCATGCCCCTCT L S D D O I L R E F L Y S * GCTTCTTCTTGGTTGATTATTGCATTAATGTTACTTTGATCATTAGTAGTAACCATCCAT AATTTTTACGCCCTTGCATAAATGAAAAAAAGTTGTACTTCTTCATGAACCTATACTACT TTTCTCATCCCTACATAAACTACCCGACAGACATTTAATTTTATAAATACATATCTCATA AACATCGAACATCGACATACTACTCCAGTTATTCAACACTGACTTTTTTGGTGGAAAGCT T Fig. 2. (Gont.).

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Putative yeast protein kinase gene IREI IREl

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1445

Fig. 3. Sequence alignment of the IRE1 product with protein kinases. The kinase catalytic domain of the IREI product (residues 676-660) is aligned with those of cAPK (bovine cardiac cAMP-dependent protein kinase catalytic subunit, a-form. residues 46-208) (Shoji etal.. 1981), cdc2 [SctiizosacctTaromyces pombe cdc2' kinase. residues 8-179) (Hindiey and Phear, 1984), src (chicken c-src kinase. residues 270-432) (Takeya and Hanafusa, 1983), EGFR (human epidermal growth factor receptor, residues 715-8884) (Ullrich ef a/,. 1984). The amina acids tdentical to those of the IREI product are shadowed. The dashes indicate spaces introduced for maximum alignment of the sequences.

EGFR IREl cAPK Cdc2 src EGFR

SRFTADQQTGAEHLRILZSDFQLCXKLDSGQSSFKTiiLNNPSIJiXSQfRAPE GXIQ VTDiraFAKRVKGRTW TLCOStPEYLAJ-S GNLK LAflFQLARSFGVPLR i-HYTHEI viLWYRAI'i LVCK VABFCiLARLIEDNEYTAli QGAKFPIKWTAf i QHVK ITnFQI.AKLI.GAEEKETHA-~EGGKVPI KHMRli

Fig. 4. Inositol requirement of the irei disruptant. Diploid strain D2013 (iRE1/ir0i::HIS3) was induced to sporulate and then the growth ptienotypes of the segregants were determined on minimum medium without inositol (A) or with 20p.g ml ' inositol (B). Each horizontal row represents the spore progeny from the same ascus, Tiie progenies which did not grow in the absence of inosrtol were all His'.

SNF1 and GCN2 protein kinases are positive regulators of the expression of SUC2 (Celenza and Carlson, 1986) and GCN4 (Roussou et al., 1988), respectively. The PHO85 protein kinase is a negative regulator of the expression of the phosphatase system (Toh-e etai., 1988). Moreover, it was recently reported that mutations in certain subunits of RNA polymerase II caused inositol auxotrophy (Arndt et ai, 1989; Nonet and Young, 1989). Therefore, the possibility that IREI phosphorylates RNA polymerase II subunits could not be excluded. Because of its receptor-like structure, it is tempting to speculate that the 'IRE1 kinase' might be involved in the signal transduction in the yeast.

References Amdt, K.R., Styles, C.A., and Fink, G.R. {1989} A suppressor of a HIS4 transcriptional defect encodes a protein with homology to

the catalytic subunit of protein phosphatases. Cell 56: 527537. Carman, G.M., and Henry, S.A. (1989) Phospholipid biosynthesis in yeast. Annu Rev Biochem 58: 635-669. Celenza, J . L , and Carlson, M. (1986) A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science 233:1175-1180. Gannon. F.. O'Hara, K., Perrin. F., LePennec, L., Benoist, C , Cochet, M,, Breathnach, R., Royal, A., Garapin, A., Cami, B.. and Chambon, P. (1977) Organization and sequences at the 5' end of a cloned complete ovalbumin gene. Nature 278: 428-434. Hanks, S.K., Quinn, A.M., and Hunter, T. (1988) The protein kinase family: conserved features and deduced phytogeny of the catalytic domains. Science 241: 42-52. Hindiey. J., and Phear. G. (1984) Sequence of the cell division gene cdc2 from Schizosacctiaromyces pombe: patterns of splicing and homology to protein kinases. Gene 31: 129-134. Kozak. M. (1984) Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. NucI Acids Res 12; 857-872. Kyte, J., and Ooolittte. R.F. (1982) A simple method for displaying the hydropathic character of a protein. J Mol B/b/157:105-132. Langford. C.J.. and Gallwitz, D. (1983) Evidence for an introncontained sequence required for the splicing of yeast RNA polymerase II transcripts- Ce//33: 519-527. Nikawa, J., and Yamashita, S. (1982) Yeast mutant defective in synthesis of phosphatidylinositol: isolation and characterization of a CDP-diacylglycerol-inositol 3-phosphatidyltransferase Km mutant. Eur J Biochem 125: 445-45:. Nikawa, J., and Yamashita, S. (1984) Molecular cloning of the gene encoding CDPdiacylglycerol-inositol 3-phosphatidyltransferase in Saccharomyces cerevisiae. EurJ Biochem 143: 251-256. Nikawa. J.. Kodaki, T., and Yamashita, S. (1987) Primary structure and disruption of the phosphatidytinositol synthase gene of Saccharomyces cerevisiae. J Blol Chem 262: 4876-4881. Nonet, M.L., and Young, R.A. (1989) Intragenic and extragenic suppressors of mutations in heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II. Genetics 123: 715-724.

1446 J.-L Nikawa and S. Yamashita Roussou, I.. Thireos, B., and Hauge, B.M. (1988) Transcriptionaltransiational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase. Mol Cell Biol Q: 2132-2139. Sanger. F., Nicklen, S.. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc NatI Acad Sci USA 74: 5463-5467. Shoji, S., Parmelee, D.C, Wade, R.D., Kumar, S,. Ericsson, LH., Walsh, K,A., Neurath, H., Long, G.L, Demaille, J,G., Fischer, E.H., and Titani, K. (1981) Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMPdependent protein kinase. Proc NatI Acad Sci USA 78: 848851. Takeya, T,, and Hanafusa. H. (1983) Structure and sequence of the cellular gene homoiogues to the RSV src gene and the

mechanism for generating the transforming virus. Cell 32; 881-890, Toh-e, A., Tanaka, K., Uesono, Y., and Wickner, R.B. (1988) PHO85, a negative regulator of the PHO system, is a homolog of the protein kinase gene, CDC28. of Saccharomyces cerevisiase. Mol Gen Genet2iA: 162-164. Ullrich, A.. Coussens, L, Hayflick, J.S., Dull. J.T., Gray, A.. Tam, A.W., Lee, J., Yarden, Y., Libermann, T.A., Schlessinger. J., Downward, J,, Mayes, E.L.V., Whittle, N,, Waterfield, M.D., and Seeburg, P,H. (1984). Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermal carcinoma cells. Nature 309: 418-425, Von Heijne, G. (1983) Signal-sequence cleavage sites. Eur J :17-21.

IRE1 encodes a putative protein kinase containing a membrane-spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae.

A novel gene, IRE1, of Saccharomyces cerevisiae was cloned through genetic complementation of a myoinositol auxotrophic mutant. The predicted amino ac...
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