Yasuji Oshima

The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter.

The PHO84 gene specifies Pi-transport in Saccharomyces cerevisiae. A DNA fragment bearing the PHO84 gene was cloned by its ability to complement constitutive synthesis of repressible acid phosphatase of pho84 mutant cells. Its nucleotide sequence predicted a protein of 596 amino acids with a sequence homologous to that of a superfamily of sugar transporters. Hydropathy analysis suggested that the secondary structure of the PHO84 protein consists of two blocks of six transmembrane domains separated by 74 amino acid residues. The cloned PH084 DNA restored the Pi transport activity of pho84 mutant cells. The PHO84 transcription was regulated by Pi like those of the PHO5, PHO8, and PHO81 genes. A PHO84-lacZ fusion gene produced beta-galactosidase activity under the regulation of Pi, and the activity was suggested to be bound to a membrane fraction. Gene disruption of PHO84 was not lethal. By comparison of nucleotide sequences and by tetrad analysis with GAL80 as a standard, the PHO84 locus was mapped at a site beside the TUB3 locus on the left arm of chromosome XIII.

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.

Molecular and functional organization of yeast plasmid pSR1

The nucleotide sequence of a 6251 base-pair plasmid, pSR1, harbored in an osmophilic haploid yeast, Zygosaccharomyces rouxii (formerly Saccharomyces rouxii), was determined. No homology was detected between the sequences of pSR1 and 2-micron DNA of Saccharomyces cerevisiae. pSR1 has a pair of inverted repeats consisting of completely homologous 959 base-pair sequences, which separate two unique sequences 2654 base-pairs and 1679 base-pairs long. Each inverted repeat has an ARS sequence functional in both Z. rouxii and S. cerevisiae hosts. Short direct repeats or dyad symmetries were observed in the inverted repeats similar to those found close to the replication origin of 2-micron DNA. Three open reading frames, P, S and R, each able to encode a protein of molecular weight larger than 10,000, were found. Insertional inactivation of R gave rise to a defect in the intramolecular recombination at the inverted repeats, and that of S reduced the copy number of pSR1 in the S. cerevisiae host. The maintenance stability of the plasmid was also tested in the heterogeneous S. cerevisiae host, but the results of the insertional inactivation of P, S and R were ambiguous. pSR1 and 2-micron DNA were compatible in S. cerevisiae cells, but the protein factors encoded by these plasmids did not complement each other.

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.

Deoxyribonucleic Acid Homology and Taxonomy of the Genus Bacillus

The taxonomic relationships among 56 strains of 16 species of the genus Bacillus were studied by deoxyribonucleic acid (DNA)-DNA hybridization. In general, no significant DNA homology was detected between two strains of different species, except for a group of species consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, and B. pumilus and for another group of species including B. cereus and B. thuringiensis. Species of the former group were related, but they were independent of each other as their DNA homologies were 19% or less. The DNA homology indexes of three strains of B. thuringiensis to B. cereus T, so far tested, showed high DNA homologies (54 to 80%). This fact indicates that B. thuringiensis should be an identical species to B. cereus. The intraspecific DNA homology indexes of 16 strains of B. pumilus were 51% or more to strain IFO 12092 as the standard, and those of 10 strains of B. coagulans were 76 to 113% to strain ATCC 7050 as the standard. Thus, the species identification of B. pumilus and B. coagulans by the conventional taxonomic method was well in accord with the DNA homology data. On the other hand, significant heterogeneities were suggested among the strains of B. circulans and among those of B. sphaericus by the DNA homology data of the three and five strains so far tested, respectively. Although B. lentus was described to be closely related to B. firmus in Bergeys Manual (8th ed., 1974), the interspecific DNA homology index between these two species was 3%. It was concluded that these two species are independent.

Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae

We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand. In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1. GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III. Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate. The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter. Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.

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.