Cloning and characterization of a p53-related protein kinase expressed in interleukin-2-activated cytotoxic T-cells, epithelial tumor cell lines, and the testes.

A human protein kinase, p53-related protein kinase (PRPK), was cloned from an interleukin-2-activated cytotoxic T-cell subtraction library. PRPK appears to be a homologue of a growth-related yeast serine/threonine protein kinase, YGR262c. However, a complementation assay using YGR262c-disrupted yeast indicated that PRPK is not functionally identical to the yeast enzyme. PRPK expression was observed in interleukin-2-activated cytotoxic T-cells, some human epithelial tumor cell lines, and the testes. The intrinsic transcriptional activity of p53 was up-regulated by a transient transfection of PRPK to COS-7 cells. PRPK was shown to bind to p53 and to phosphorylate p53 at Ser-15. These results indicate that PRPK may play an important role in the cell cycle and cell apoptosis through phosphorylation of p53.

The Saccharomyces cerevisiae Isw2p-Itc1p complex represses INO1 expression and maintains cell morphology.

In the yeast Saccharomyces cerevisiae, IRE1 encodes a bifunctional protein with transmembrane kinase and endoribonuclease activities. HAC1 encodes a transcription factor which has a basic leucine zipper domain. Both gene products play a crucial role in the unfolded protein response. Mutants in which one of these genes is defective also show the inositol-auxotrophic (Ino(-)) phenotype, but the reason for this has not been clear. To investigate the mechanism underlying the Ino(-) phenotype, we screened a multicopy suppressor gene which can suppress the Ino(-) phenotype of the Delta hac1 strain. We obtained a truncated form of the ITC1 gene that has a defect in its 3' region. Although the truncated form of ITC1 clearly suppressed the Ino(-) phenotype of the Delta hac1 strain, the full-length ITC1 had a moderate effect. The gene products of ITC1 and ISW2 are known to constitute a chromatin-remodeling complex (T. Tsukiyama, J. Palmer, C. C. Landel, J. Shiloach, and C. Wu, Genes Dev. 13:686--697, 1999). Surprisingly, the deletion of either ITC1 or ISW2 in the Delta hac1 strain circumvented the inositol requirement and caused derepression of INO1 even under repression conditions, i.e., in inositol-containing medium. These data indicate that the Isw2p-Itc1p complex usually represses INO1 expression and that overexpression of the truncated form of ITC1 functions in a dominant negative manner in INO1 repression. It is conceivable that the repressor function of this complex is regulated by the C-terminal region of Itc1p.

Effect of modifying metabolic network on poly-3-hydroxybutyrate biosynthesis in recombinant Escherichia coli.

The regulatory mechanism for poly-3-hydroxybutyrate (PHB) biosynthesis in recombinant Escherichia coli is markedly different from that of Ralstonia eutropha (formerly, Alcaligenes eutrophus) since the former efficiently synthesizes PHB during growth without any nutrient limitation. To analyze how the central metabolic pathways should be balanced with pathways necessary for cell growth and PHB formation, a stoichiometric model was developed to predict the theoretical maximum PHB production capacity for different metabolic variants. Flux analysis results illustrated the importance of the availability of acetyl-CoA and NADPH for achieving the maximum yield of PHB. In order to examine whether the increased availability of the above substances can enhance PHB synthesis in recombinant E. coli, both genetic and environmental perturbations were attempted. Several E. coli K12 derivatives, namely, HMS174, TA3516 (pta-/ack-), and DF11 (pgi-), were transformed with a plasmid which contains the native phb operon. The fermentation characteristics of these recombinant strains were studied and compared. In this study we examined the effects of intracellular acetyl-CoA accumulation, which may promote PHB synthesis in vivo, by perturbations induced from attenuation of acetate kinase and phosphotransacetylase (TA3516, blocked in the acetate pathway) and by cultivation of E. coli HMS174 on gluconate; it can convert gluconate to acetyl-CoA at a higher rate. The effects of intracellular accumulation of NADPH were investigated by introducing a perturbation induced from attenuation of phosphoglucose isomerase, which redirects the carbon flow to the pentose-phosphate (PP) pathway. Results from the analyses of these perturbations indicate that intracellular buildup of acetyl-CoA may not be able to promote PHB synthesis in vivo. On the other hand, since the biosynthesis of PHB in the pgi- mutant strain can utilize the NADPH overproduced through the PP pathway, the growth of the pgi- mutant on glucose was recovered, indicating that the overproduction of NADPH might be able to enhance PHB synthesis.

The Saccharomyces cerevisiae SCS2 gene product, a homolog of a synaptobrevin-associated protein, is an integral membrane protein of the endoplasmic reticulum and is required for inositol metabolism.

The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 and hac1/ire15 mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39-45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from Aplysia californica (P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580-1583, 1995). In this study we have characterized an SCS2 gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34 degrees C. Genetic studies reveal that the overexpression of the INO1 gene rescues the inositol auxotrophy of the SCS2 disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the SCS2 disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.

PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae.

We developed a novel method for synthesizing marker-disrupted alleles of yeast genes. The first step is PCR amplification of two sequences located upstream and downstream of the reading frame to be disrupted. Due to the addition of non-specific single A overhangs by Taq DNA polymerase, each PCR product can be ligated with a marker DNA which has T residues at its 3' ends. After amplification of individual ligation products through the second PCR, both products are mixed and annealed, and the single strand is converted to a double strand by an extension reaction. The final step is PCR amplification of the fragment composed of a selectable marker and two flanking sequences with the outermost primers. This method is rapid and needs only short oligonucleotides as primers.

Suppression of the Saccharomyces cerevisiae hac1/ire15 mutation by yeast genes and human cDNAs.

We previously reported that the Saccharomyces cerevisiae ire15 mutation results in an inositol-auxotrophic phenotype, and that human cDNAs can suppress the ire15 mutation (Nikawa, J., 1994. A cDNA encoding the human transforming growth factor beta receptor suppresses the growth defect of a yeast mutant. Gene 149, 367 372; Nikawa, J., Nakano, H., Ohi, N., 1996b. Structural and functional conservation of human and yeast HAC1 genes which can suppress the growth defect of the Saccharomyces cerevisiae ire15 mutant. Gene 171, 107-111). Herein, we present evidence that the gene responsible for the ire15 mutation is HAC1, which encodes a transcription factor for KAR2, obtained by isolating a yeast single-copy supressor gene and by performing complementation analysis. Sequencing analysis revealed that the mutant HAC1 gene obtained from the ire15 mutant contained an AAA codon at position 50 instead of the AGA codon observed in the wild-type gene, resulting in the alteration of the aa from Arg to Lys. All human cDNAs and yeast multicopy suppressors, which had been isolated as suppressors for the ire15 mutation, were able to suppress the inositol-auxotrophic phenotype but not the defect in KAR2 induction of the hac1-disrupted strain.

Phosphatidylinositol synthase from yeast.

PI is an important precursor for polyphosphoinositides and some sphingolipids and is also involved in the glycolipid anchoring of plasma membrane proteins. This lipid is synthesized from CDP-diacylglycerol and myo-inositol by PI synthase, an enzyme localized in the outer mitochondrial membranes and microsomes in yeast. PI synthase was highly purified from yeast microsomes after solubilization with Triton X-100. The activity is dependent on Mn2+ or Mg2+ and Triton X-100. The reaction follows a sequential Bi-Bi mechanism with binding to CDP-diacylglycerol before myo-inositol and releasing PI prior to CMP. Unlike most of the yeast phospholipid-synthesizing enzymes, PI synthase is a constitutive enzyme. Its expression is insensitive to the addition of myo-inositol and choline to culture medium or the transition of growth phase. The primary translate deduced from the encoding gene, PIS, comprises 220 amino acid residues with a calculated molecular mass of 23,613. The sequence contains several hydrophobic regions and resembles that of the human enzyme. The sequence also contains the local, conserved region found in enzymes catalyzing the transfer of the phosphoalcohol moiety from CDP-alcohol, such as phosphatidylserine synthase, cholinephosphotransferase and phosphatidylglycerolphosphate synthase. Substitution of amino acid at position 114 from His (CAC) to Gln (CAA) results in a 200-fold increase in Km of the enzyme for myo-inositol, making cells auxotrophic for myo-inositol. Disruption of the PIS locus in the genome is lethal, indicating that PI is essential for the survival and growth of yeast cells.

Phosphatidylserine synthase from yeast.

Whereas mammalian cells produce PS by a base exchange reaction from preexisting phospholipids, yeast cells synthesize PS from CDP-diacylglycerol and serine by the PS synthase reaction. Yeast PS synthase was purified to homogeneity and shown to have a molecular mass of 23 kDa. The activity is dependent on either Mg2+ or Mn2+ and Triton X-100. The enzyme specifically transfers the phosphatidyl group from CDP-diacylglycerol or dCDP-diacylglycerol to L-serine, but not to threonine, cysteine and ethanolamine. The PSS/CHO1 gene encoding the enzyme was cloned by the complementation of the choline auxotrophic pss/cho1 mutant. The deduced protein comprises 279 amino acids with a calculated molecular mass of 30,804. The primary translate undergoes proteolytic processing to the enzymatically more active 23-kDa enzyme. The deduced amino acid sequence contains several putative membrane-spanning regions and resembles that of the Bacillus subtilis enzyme, but not those of the E. coli and Haemophilus influenzae enzymes. The sequence also contains the local, conserved region found in enzymes catalyzing the transfer of the phosphoalcohol moiety from CDP-alcohol, such as PI synthase, cholinephosphotransferase and phosphatidylglycerolphosphate synthase. The activity of PS synthase is maximal in the exponential phase, but decreases when cells enter the stationary phase. The enzyme is phosphorylated at a single serine residue by cyclic AMP-dependent protein kinase with a 60-70% decrease in enzymatic activity, but the primary translation product is not phosphorylated. PS synthase is inhibited by CTP, probably due to the chelation of the divalent cations, Mg2+ and Mn2+, and also by sphingoid bases, such as sphinganine and phytosphingosine. Phosphatidate, phosphatidylcholine and phosphatidylinositol are stimulatory, whereas cardiolipin and diacylglycerol are inhibitory. The expression of yeast PS synthase is transcriptionally repressed by myo-inositol and choline in a coordinate manner with other phospholipid-synthesizing enzymes. The upstream regulatory region of the PSS/CHO1 gene responsible for the myo-inositol-choline regulation was identified. An octameric sequence, CATRTGAA (R = A or G), plays an important role in the conferral of the myo-inositol-choline transcriptional regulation.

Cloning of a human cDNA for CTP-phosphoethanolamine cytidylyltransferase by complementation in vivo of a yeast mutant.

CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.

Saccharomyces cerevisiae IRE2/HAC1 is involved in IRE1-mediated KAR2 expression.

The Saccharomyces cerevisiae IRE1 gene, encoding a putative receptor-type protein kinase, is known to be required for inositol prototrophy and for the induction of a chaperon molecule, BiP, encoded by KAR2, under stress conditions such as tunicamycin addition. We have characterized a yeast gene, IRE2, which was isolated as a suppressor gene that complements the inositol auxotrophic phenotype of the ire1 mutation. Sequencing analysis revealed that IRE2 is identical to HAC1, which encodes a transcription factor having a basic-leucine zipper motif. Introduction of IRE2/HAC1 into the ire1 mutant clearly restored the expression of KAR2 upon tunicamycin treatment. ire2/hac1-disrupted yeast cells showed not only the inositol auxotrophic phenotype but also the tunicamycin sensitivity, and failed to induce the expression of KAR2. These results clearly indicate that the IRE2/HAC1 gene product plays a critical role in the induction of KAR2 expression and in the inositol prototrophy mediated by IRE1.

Molecular cloning of rat phosphatidylinositol synthase cDNA by functional complementation of the yeast Saccharomyces cerevisiae pis mutation.

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol: 3-phosphatidyltransferase, EC 2.7.8.11) catalyzes the formation of phosphatidylinositol and CMP from CDP-diacylglycerol and myo-inositol. We have cloned a phosphatidylinositol synthase cDNA from rat brain by functional complementation of the yeast pis mutation, which is defective in phosphatidylinositol synthase. The deduced protein comprised 213 amino acids with a calculated molecular mass of 23,613 Da. The predicted protein sequence is highly homologous to the previously determined yeast phosphatidylinositol synthase sequence. The cDNA hybridized to a 1.7-kb mRNA that was abundantly expressed in rat brain and kidney.

Structural and functional conservation of human and yeast HCP1 genes which can suppress the growth defect of the Saccharomyces cerevisiae ire15 mutant.

The Saccharomyces cerevisiae ire15 mutant has a defect in the expression of the IN01 gene, showing an inositol auxotrophic phenotype. The growth defect of this mutant is suppressed by human cDNAs such as for the TGF-beta receptor-encoding gene (TGFR) [Nikawa, Gene 149 (1994) 367-372]. Here, we isolated a new human cDNA, HCP1, which suppresses the ire15 mutation by genetic complementation. Sequencing analysis revealed that HCP1 encodes 360 amino acid residues (40,515 Da). The product of HCP1 is highly conserved among species and the yeast homolog was also found to suppress the ire15 mutation. Northern blot analysis revealed that multicopies of the yeast and human HCP1, as well as TGFR, resulted in an increase in the IN01 mRNA level in the yeast mutant. These results clearly indicate that the products of human and yeast HCP1 are structural and functional homologs, and are involved in expression of genes such as of IN01.

Cloning and sequence of the SCS2 gene, which can suppress the defect of INO1 expression in an inositol auxotrophic mutant of Saccharomyces cerevisiae.

Saccharomyces cerevisiae ire15 mutant has a defect in the expression of INO1, showing the inositol auxotrophic phenotype [Nikawa, J. (1994) Gene 149, 367-372]. We have isolated five yeast genes which suppress the ire15 mutation in multiple copies by genetic complementation. Among them, one gene, designated as SCS2, also suppressed the choline-sensitive dominant mutation, CSE1 [Hosaka, K. et al. (1992) J. Biochem. 111, 352-358]. The CSE1 mutation is not allelic to ire15. Sequencing analysis revealed that the SCS2 gene encodes 244 amino acid residues with a calculated molecular mass of 26,866. INO2/SCS1, which is another suppressor gene for CSE1 and is known to be a positive factor for INO1 expression, also suppressed the growth defect of the ire15 mutant. These results clearly indicate that the ire15 and CSE1 mutations genetically interact and the SCS2 and INO2/SCS1 genes are involved in the regulation of INO1 expression.

Isolation and characterization of genes that promote the expression of inositol transporter gene ITR1 in Saccharomyces cerevisiae.

The expression of many genes of Saccharomyces cerevisiae, such as ITR1, is regulated by inositol and choline. In this work, a yeast strain has been constructed in which HIS3 expression is controlled by the ITR1 promoter. Using this strain, three genes were isolated which, when introduced as multicopies, abolish the repression caused by inositol via the ITR1 promoter. Northern blot analysis revealed that two of these three genes, designated as DIE1 and DIE2, clearly increased the expression of ITR1. DIE2 is more effective for ITR1 expression than DIE1. Gene-disruption experiments revealed that DIE1 was essential for the expression of ITR1 but that DIE2 was not. The sequence of the DIE1 gene was shown to be identical to that of INO2 (also called SCS1), which encodes a protein required for the expression of INO1. DIE2 is a new gene and is capable of encoding 525 amino acid residues with a calculated molecular weight of 61,789. Experiments involving lacZ fusion genes showed that multicopy DIE2 resulted in an increase in the expression of both ITR1 and INO1. These results strongly suggest that the DIE1 and DIE2 gene products have an important regulatory function for gene expression of not only ITR1 but also INO1.

Functional analysis of mutations in the PIS gene, which encodes Saccharomyces cerevisiae phosphatidylinositol synthase.

The PIS gene for an enzyme phosphatidylinositol synthase having an increased Km for myo-inositol, was isolated from Saccharomyces cerevisiae. The mutant PIS gene contained a CAA codon at position 114 instead of the CAC codon observed in the wild-type gene, resulting in alteration of the amino acid from His to Gln. Oligonucleotide mediated site-directed mutagenesis of PIS at codon 114 revealed that mutant genes with codons for Ala, Thr and Leu could support yeast cell growth in vivo, but those for Asp, Lys and Tyr could not. All mutant enzymes when expressed in Escherichia coli showed greatly reduced in vitro activity.

The SNF2/SWI2/GAM1/TYE3/RIC1 gene is involved in the coordinate regulation of phospholipid synthesis in Saccharomyces cerevisiae.

Genes involved in the phospholipid synthesis of Saccharomyces cerevisiae, such as PEM1, PEM2, PSS, and INO1, are coordinately repressed by myo-inositol and choline. In order to investigate this regulation, we transformed wild-type yeast with a PEM1 promoter-lacZ fusion and isolated two mutants, named ric1 and ric2 (regulation by myo-inositol and choline), exhibiting decreased PEM1 expression. The lowered PEM1 expression in the mutants was monitored in colonies in terms of their failure fully to develop blue color on 5-bromo-4-chloro-3-indolyl-beta-galactopyranoside-containing agar. ric1 mutant was auxotrophic for myo-inositol, indicating that INO1 expression was also affected, whereas ric2 mutant required myo-inositol only in the presence of choline. The RIC1 gene was isolated by complementation of the Ino- phenotype of ric1 mutant and its identity was confirmed by genetic cross between the original ric1 mutant and a gene disruptant. The RIC1 gene was sequenced and found to be identical with the previously identified gene, SNF2/SWI2/GAM1/TYE3, which is known to encode a general transcription factor required for the expression of various genes including INO1. Analysis using various lacZ fusion constructs containing promoters for genes in phospholipid synthesis revealed that the expression of myo-inositol-choline-regulated genes, PEM1, PEM2, PSS, CKI, and INO1, was markedly decreased in the snf2/swi2/gam1/tye3/ric1 background, but the expression of a constitutive gene, PIS, was not. We conclude that SNF2/SWI2/GAM1/TYE3/RIC1 is a positive regulatory gene required for the expression of not only INO1 gene, but also of myo-inositol-choline-regulated genes in general.

Isolation and characterization of a SCT1 gene which can suppress a choline-transport mutant of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae possesses a choline-transport system encoded by the CTR1 gene. We isolated a yeast gene, SCT1, that can suppress a CTR1 defect when introduced as a multicopy. The SCT1 coding frame is capable of encoding 759 residues with a calculated molecular weight of 85,734. On Northern blot analysis, an RNA species that hybridized with the coding region was detected in the total RNA of the wild-type yeast. The transcription of SCT1 is constitutive. The primary translation product contains three membrane-spanning domains, a PEST-like sequence, and a glutamic acid-rich sequence at the C terminal end. Gene disruption experiments showed that SCT1 is not an essential gene under the standard culture conditions. SCT1 did not suppress a null mutant of ctr1, indicating that a mutant form of choline transporter is necessary for the suppression caused by SCT1.

Cloning and sequence of the SCS3 gene which is required for inositol prototrophy in Saccharomyces cerevisiae.

The SCS3 gene of Saccharomyces cerevisiae was cloned by functional complementation, using a conditional mutant exhibiting myo-inositol auxotrophy in the presence of choline, and sequenced. The sequence contained an open reading frame capable of encoding 380 amino acids with a calculated molecular weight of 42,734. Disruption of the SCS3 locus caused myo-inositol auxotrophy. The gene appeared to be involved in the synthesis of inositol phospholipids from inositol but not in the control of inositol synthesis.