Nobuo Ogawa

CRYSTAL STRUCTURE OF PHO4 BHLH DOMAIN-DNA COMPLEX : FLANKING BASE RECOGNITION

The crystal structure of a DNA‐binding domain of PHO4 complexed with DNA at 2.8 Å resolution revealed that the domain folds into a basic–helix–loop–helix (bHLH) motif with a long but compact loop that contains a short α‐helical segment. This helical structure positions a tryptophan residue into an aromatic cluster so as to make the loop compact. PHO4 binds to DNA as a homodimer with direct reading of both the core E‐box sequence CACGTG and its 3′‐flanking bases. The 3′‐flanking bases GG are recognized by Arg2 and His5. The residues involved in the E‐box recognition are His5, Glu9 and Arg13, as already reported for bHLH/Zip proteins MAX and USF, and are different from those recognized by bHLH proteins MyoD and E47, although PHO4 is a bHLH protein.

Regulation of phosphatase synthesis in Saccharomyces cerevisiae — a review ☆

Transcription of the genes encoding acid and alkaline phosphatases and the inorganic phosphate (Pi) transporter of Saccharomyces cerevisiae are coordinately repressed and derepressed depending on the Pi concentration in the culture medium. This phosphatase system is particularly suited for the study of regulatory mechanisms, because the acid phosphatase activity of each colony on a plate is easily detected by specific staining methods and there is a 500-fold difference between the repressed and derepressed levels of acid phosphatase activity. With these advantages, considerable amounts of genetic and molecular evidence have been accumulated in the past two decades. This article summarizes our current knowledge on this subject.

A series of protein phosphatase gene disruptants in Saccharomyces cerevisiae

Thirty‐two protein phosphatase (PPase) genes were identified in the genome nucleotide sequence of Saccharomyces cerevisiae. We constructed S. cerevisiae disruptants for each of the PPase genes and examined their growth under various conditions. The disruptants of six putative PPase genes, i.e. of YBR125c, YCR079w, YIL113w, YJR110w, YNR022c and YOR090c, were created for the first time in this study. The glc7, sit4 and cdc14 disruptants were lethal in our strain background. The remaining 29 PPase gene disruptants were viable at 30°C and 37°C, but only one disruptant, yvh1, showed intrinsic cold‐sensitive growth at 13°C. Transcription of the YVH1 gene was induced at 13°C, consistent with an idea that Yvh1p has a specific role for growth at a low temperature. The viable disruptants grew normally on nutrient medium containing sucrose, galactose, maltose or glycerol as carbon sources. The ppz1 disruptant was tolerant to NaCl and LiCl, while the cmp2 disruptant was sensitive to these salts, as reported previously, and none of the other viable PPase disruptants exhibited the salt sensitivity. When the viable disruptants were tested for sensitivity to drugs, i.e. benomyl, caffeine and hydroxyurea, ppz1 and ycr079w disruptants exhibited sensitivity to caffeine. Copyright

Genes in PHT plasmid encoding the initial degradation pathway of phthalate in Pseudomonas putida

The nucleotide sequence of a 7-kbp EcoRI fragment of PHT plasmid in Pseudomonas putida encoding the enzymes in the initial step of phthalate degradation (the Pht+ phenotype) revealed five open reading frames (ORFs), pht1 to pht5, with the same orientation. The protein deduced from pht1 showed 17.4% similarity in amino acid sequence with the glycerol-3-phosphate transporter of Escherichia coli, that of pht2 showed 39.2% similarity with VanB protein (vanillate monooxygenase reductase) and that of pht3 showed 20.8% similarity with VanA (vanillate monooxygenase) of Pseudomonas sp. The proteins encoded by pht4 and pht5 showed no appreciable similarity with any proteins in a database. Eight Pht− mutants with a Tn5 insertion in pht1, and one with a similar insertion in the pht5 ORF were isolated. These pht1− and pht5− mutants, and two other Pht− mutants induced by UV irradiation of a Pht+ strain of Pseudomonas testosteroni, were examined by complementation tests with several segments of the 7-kbp pht fragment and feeding experiments with phthalate, 4,5-dihydro-4,5-dihydroxyphthalate, 4,5-dihydroxyphthalate, and protocatechuate. The results of these experiments and similarity searches suggested that pht2 encodes phthalate oxygenase reductase, pht3 encodes phthalate oxygenase, pht4 encodes 4,5-dihydro-4,5-dihydroxyphthalate dehydrogenase, and pht5 encodes 4,5-dihydroxyphthalate decarboxylase. The Pht1 protein was suggested to function as a positive regulator for expression of the other pht genes or as a phthalate transporter. Three A : T rich regions, which may have promoter function, were found in the upstream regions of pht1, pht3, and pht5. Northern blot analyses suggested that transcriptions of pht1 and pht2 are low level and under relaxed control of phthalate. The transcriptions of pht3, pht4, and pht5 might be induced by phthalate.

Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase regulon in Saccharomyces cerevisiae.

The PHO4 gene encodes a positive regulatory factor involved in regulating transcription of various genes in the phosphatase regulon of Saccharomyces cerevisiae. Besides its own coding region, the 1.8-kilobase PHO4 transcript contains a coding region for a mitochondrial protein which does not appear to be translated. Four functional domains were found in the PHO4 protein, which consists of 312 amino acid (aa) residues as deduced from the open reading frame of PHO4. A gel retardation assay with beta-galactosidase::PHO4 fused protein revealed that the 85-aa C terminus is the domain responsible for binding to the promoter DNA of PHO5, a gene under the control of PHO4. This region has similarities with the amphipathic helix-loop-helix motif of c-myc protein. Determination of the nucleotide sequences of four PHO4c mutant alleles and insertion and deletion analyses of PHO4 DNA indicated that a region from aa 163 to 202 is involved in interaction with a negative regulatory factor PHO80. Complementation of a pho4 null allele with the modified PHO4 DNAs suggested that the N-terminal region (1 to 109 aa), which is rich in acidic aa, is the transcriptional activation domain. The deleterious effects of various PHO4 mutations on the constitutive transcription of PHO5 in PHO4c mutant cells suggested that the region from aa 203 to 227 is involved in oligomerization of the PHO4 protein.

Functional domains of Pho81p, an inhibitor of Pho85p protein kinase, in the transduction pathway of Pi signals in Saccharomyces cerevisiae.

The PHO81 gene is thought to encode an inhibitor of the negative regulators (Pho80p and Pho85p) in the phosphatase (PHO) regulon. Transcription of PHO81 is regulated by Pi signals through the same PHO regulatory system. Elimination of the PHO81 promoter or its substitution by the GAL1 promoter revealed that stimulation of the PHO regulatory system requires both increased transcription of PHO81 and a Pi starvation signal. The predicted Pho81p protein contains 1,179 amino acids (aa) and has six repeats of an ankyrin-like sequence in its central region. The minimum amino acid sequence required for Pho81p function was narrowed down to a 141-aa segment (aa 584 to 724), which contains the fifth and sixth repeats of the ankyrin-like motif. The third to sixth repeats of the ankyrin-like motif of Pho81p have significant similarities to that of p16INK4, which inhibits activity of the human cyclin D-CDK4 kinase complex. Deletion analyses revealed that the N- and C-terminal regions of Pho81p behave as negative and positive regulatory domains, respectively, for the minimal 141-aa region. The negative regulatory activity of the N-terminal domain was antagonized by a C-terminal segment of Pho81p supplied in trans. All four known classes of PHO81c mutations that show repressible acid phosphatase activity in high-Pi medium affect the N-terminal half of Pho81p. An in vitro assay showed that a glutathione S-transferase-Pho81p fusion protein inhibits the Pho85p protein kinase. Association of Pho81p with Pho85p or with the Pho80p-Pho85p complex was demonstrated by the two-hybrid system.

Function of the PHO regulatory genes for repressible acid phosphatase synthesis in Saccharomyces cerevisiae

SummaryExpression of the repressible acid phosphatase (rAPase) gene, PHO5, of Saccharomyces cerevisiae is repressed by a certain level of inorganic phosphate (Pi) in the medium and is derepressed when the Pi concentration is lowered. The Pi signals are conveyed to PHO5 by a regulatory system consisting of proteins coded for by the PHO2, PHO4, PHO80 and PHO81 genes. We have found that the transcription of PHO81 is regulated by Pi through the PHO regulatory system. Increasing the dosage of PHO4 and PHO81 by ligating each gene to YEP13 gives rise to, respectively, considerable and weak synthesis of rAPase by cultivation of the transformants in high-Pi medium; but in low-Pi medium, increased dosage of PHO4 stimulates the rAPase synthesis significantly, whereas PHO81 has no effect. Increased dosage of PHO2 stimulates rAPase synthesis considerably in low-Pi but not in high-Pi. A coordinate increase of PHO80 cancels the dosage effect of PHO4, but not that of PHO81. Coordinate increases of PHO80 and PHO2 give rise to the same phenotype as an increased dosage of PHO80 alone. The level of the PHO4 protein was found to be the limiting factor of the rAPase synthesis and the copy number of the PHO5 gene not to be. These facts accord with the idea that the PHO80 protein transmits the Pi signals to the PHO5 gene via the PHO4 protein, whereas the PHO2 protein does not have a direct function in the signal transmission.

A series of double disruptants for protein phosphatase genes in Saccharomyces cerevisiae and their phenotypic analysis.

Thirty‐two protein phosphatase (PPase) genes were identified in Saccharomyces cerevisiae based on the nucleotide sequences of the entire genome. In an effort to understand the role of PPases and their functional redundancy in the cellular physiology of one of the reference eukaryotic organisms, a series of single and double PPase gene disruptants were constructed in the W303 strain background. Two single disruptants for the CDC14 and GLC7 genes were lethal. Double disruptants for 30 non‐essential PPase genes were constructed in all possible 435 combinations. No double disruptant showed synthetic lethality. Several phenotypes of the viable 30 single and 435 double disruptants were examined; temperature‐sensitive growth, utilization of carbon sources and sensitivity to cations and drugs. Four double disruptants exhibited synthetic phenotypes in addition to eight single ones: the pph21 pph22 double disruptant showed slow growth on complete medium, as did the sit4 and yvh1 single ones. In addition to the ptc1, ynr022c and ycr079w single disruptants, the ppz1 ppz2 double disruptant showed temperature‐sensitive slow growth. The msg5 ptp2 double disruptant, like the ynr022c single one, did not grow on complete medium containing 0.3 M CaCl2. The double msg5 ptc2 disruptant failed to grow on medium containing 1.0 M NaCl and, like the ynr022c single deletion, also could not grow on medium containing 0.3 M CaCl2. The synthetic phenotypes in the two latter cases where each of the PPases is categorized in a different phosphatase family led us to discuss the novel mechanism involved in the functional redundancy of the PPases. Copyright

A putative membrane protein, Pho88p, involved in inorganic phosphate transport in Saccharomyces cerevisiae.

Transcription of a regulatory gene,PHO81, in the phosphatase regulon ofSaccharomyces cerevisiae is repressed by inorganic phosphate (Pi) in the medium via that same regulatory system. The activity of Pho81p, the product ofPHO81, is also inhibited by a high concentration of Pi in the medium. Increased dosage ofPHO86, a gene encoding a putative membrane protein associated with a Pi transporter complex, activates the Pi-inhibited Pho81p produced under the control of theGAL1 promoter. A new gene,PHO88/YBR106w, has now been identified as a multicopy suppressor of the rAPase− phenotype of the cells caused by thePi inhibition of Pho81p. Thepho86 disruptant expressed rAPase activity in high-Pi medium, while thepho88 disruptant did not. The Δpho86 Δpho88 double disruption resulted in enhanced synthesis of rAPase under the high-Pi condition and conferred arsenate resistance on the cells than those in single disruptants of these genes. Its hydropathy profile and the results of an analysis of its cellular localization suggested that Pho88p is a membrane protein similar to Pho86p. Both disruption and high dosage ofPHO88 orPHO86 resulted in reduced Pi uptake. These findings suggest that Pho88p is also involved in Pi transport and modulates Pho81p function together with Pho86p.

A putative new membrane protein, Pho86p, in the inorganic phosphate uptake system of Saccharomyces cerevisiae.

The PHO84 gene in Saccharomyces cerevisiae encodes the P(i) transporter Pho84p. The other three genes, GTR1, PHO86 and PHO87, are also suggested to be involved in the P(i) uptake system. We cloned and sequenced PHO86 and found that it encodes a 34-kDa protein consisting of 311 amino acid residues with two strongly hydrophobic segments in its N-terminal half. Western blotting analysis of cell extracts revealed that Pho86p, tagged with c-Myc, was fractionated into a water-insoluble fraction. Disruption of PHO86 did not affect cell viability even in combination with the pho84 and/or pho87 disruptions. The triple disruptants showed high levels of constitutive rAPase synthesis and arsenate resistance similar to the pho84 mutant, but showed slower cell growth than the pho84 mutant. PHO86 has two putative binding sites for the transcriptional activator, Pho4p, at nucleotide positions -191 and -497 relative to the ATG start codon, and showed substantial levels of transcription under high-P(i) conditions and more enhanced levels in low-P(i) medium.