Identification and characterization of thiamin repressible acid phosphatase in yeast

We have identified a genetic locus, pho4, in Schizosaccharomyces pombe which encodes a minor expressed cell surface acid phosphatase that is repressed by low concentrations (0.5 microM) of thiamin. The enzyme was purified from a strain that overproduces the enzyme. It is an Asn-linked glycoprotein. Removal of the carbohydrates by endoglycosidase H does not abolish enzymatic activity. The molecular mass of deglycosylated and unglycosylated enzyme that accumulates in membranes when cells are grown in the presence of tunicamycin is 56 kDa as determined by sodium dodecyl sulfate-gel electrophoresis. Thiamin regulation, at least in part, operates by reducing the level of pho4-mRNA. Pho4 is not genetically linked to the phosphate repressible acid phosphatase gene pho1. Phosphate and thiamin repressible acid phosphatase differ in their substrate specificity. Their protein moieties are immunologically related. Pho4 and pho1 are the only genes in S. pombe that express cell surface acid phosphatases being enzymatically active with nitrophenyl phosphate as substrate. S. pombe is not unique in having a thiamin repressible acid phosphatase. In Saccharomyces cerevisiae this enzyme is encoded by PHO3.     

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M _r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression. Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998     

Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae.

A kinetic study of Pi transport with 32Pi revealed that Saccharomyces cerevisiae has two systems of Pi transport, one with a low Km value (8.2 microM) for external Pi and the other with a high Km value (770 microM). The low-Km system was derepressed by Pi starvation, and the activity was expressed under the control of a genetic system which regulates the repressible acid and alkaline phosphatases. The function of the PHO2 gene, which is essential for the derepression of repressible acid phosphatase but not for the derepression of repressible alkaline phosphatase, was also indispensable for the derepression of the low-Km system.     

Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PHO pathway regulates expression of phosphate-responsive genes such as PHO5, which encodes repressible acid phosphatase (rAPase). In this pathway, Pho81p functions as an inhibitor of the cyclin-cyclin-dependent kinase (CDK) complex Pho80p-Pho85p. However, the mechanism regulating the inhibitory activity of Pho81p is poorly understood. Through use of the yeast two-hybrid system, we identified the UbL-UbA protein Ddi1p as a Pho81p-binding protein. Further, Pho81p levels were found to be low under high-phosphate condition and high during phosphate starvation, indicating that Pho81p is regulated by phosphate concentration. However, our results revealed that Ddi1p and its associated protein Rad23p are not involved in the decrease in Pho81p level under high-phosphate condition. Significantly, the Δddi1Δrad23 strain exhibited a remarkable increase in rAPase activity at an intermediate-phosphate concentration of 0.4 mM, suggesting that Ddi1p and Rad23p play a cooperative role as negative regulators in the PHO pathway.     

Genetic Analysis of Pleiotropic Effects of pho85 Mutations in Yeast Saccharomyces cerevisiae

The cyclin-dependent phosphoprotein kinase Pho85p is involved in the regulation of metabolism and cell cycle in the yeast Saccharomyces cerevisiae. It is known that mutations in the PHO85gene lead to constitutive synthesis of Pho5p acidic phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, sensitivity to high temperature, and other phenotypic effects. A lack of growth at 37°C and on a medium with alcohol as the carbon source was shown to be associated with the rapid accumulation of nuclear ts and mitochondrial [rho ^– ] mutations occurring in the background of gene PHO85 inactivation. Thus, Pho85p seems to play an important role in the maintenance of yeast genome stability.     

Structure and function of the PHO82-pho4 locus controlling the synthesis of repressible acid phosphatase of Saccharomyces cerevisiae.

pho4 mutants of Saccharomyces cerevisiae, although rare among phosphatase-negative mutants isolated from wild-type strains, were isolated efficiently from pho80, pho85, or pho80 pho85 strains. The distribution of these pho4 mutants over the pho4 locus was determined by analyzing random spores of two- and three-factor crosses. The pho4-4 mutation confers temperature-sensitive synthesis of repressible acid phosphatase. An intragenic suppressor for the pho4-12 allele results in the temperature-sensitive synthesis of repressible acid phosphatase. Recombination between these sites occurs at 1.0 to 3.0%, the highest for any pair of sites within the pho4 locus. All these results strongly indicate that the information of the pho4 locus is translated into a protein. The PHO82 site was mapped inside the pho4 locus by random spore analysis. The order met10-pho4-1PHO82-1-pho4-9 on the right arm of chromosome VI was confirmed by tetrad analysis. Doubly heterozygous diploids, pho3 PHO82c PHO4+/pho3 pho82+ pho4, produce variable amounts of repressible acid phosphatase under repressive conditions depending on the combination of PHO82c and pho4 alleles. This phenomenon may reflect the constitutive production of the pho82+-pho4 product in the repressed condition, which interferes with the function of the PHO82c-PHO4+ product. The earlier model for the function of the PHO82-pho4 cluster, in which the PHO82 site acts as an operator of the pho4 gene, has been revised to a model in which the PHO82 site codes for the part of the pho4 protein that has affinity for the regulatory protein encoded by the pho80 and pho85 genes.     

Molecular analysis of the PHO81 gene of Saccharomyces cerevisiae.

The PHO81 gene product is a positive regulatory factor required for the synthesis of the phosphate repressible acid phosphatase (encoded by the PHO5 gene) in Saccharomyces cerevisiae. Genetic analysis has suggested that PHO81 may be the signal acceptor molecule; however, the biochemical function of the PHO81 gene product is not known. We have cloned the PHO81 gene and sequenced the promoter. A PHO81-LacZ fusion was shown to be a valid reporter since its expression is regulated by the level of inorganic phosphate and is controlled by the same regulatory factors that regulate PHO5 expression. To elucidate the mechanism by which PHO81 functions, we have isolated and cloned dominant mutations in the PHO81 gene which confer constitutive synthesis of acid phosphatase. We have demonstrated that overexpression of the negative regulatory factor, PHO80, but not the negative regulatory factor PHO85, partially blocks the constitutive acid phosphatase synthesis in a strain containing a dominant constitutive allele of PHO81. This suggests that PHO81 may function by interacting with PHO80 or that these molecules compete for the same target.     

Overexpression of the <Emphasis Type="Italic">PHO84</Emphasis> gene causes heavy metal accumulation and induces Ire1p-dependent unfolded protein response in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells

Pho84p, the protein responsible for the high-affinity uptake and transport of inorganic phosphate across the plasma membrane, is also involved in the low-affinity uptake of heavy metals in the Saccharomyces cerevisiae cells. In the present study, the effect of PHO84 overexpression upon the heavy metal accumulation by yeast cells was investigated. As PHO84 overexpression triggered the Ire1p-dependent unfolded protein response, abundant plasma membrane Pho84p could be achieved only in ire1Δ cells. Under environmental surplus, PHO84 overexpression augmented the metal accumulation by the wild type, accumulation that was exacerbated by the IRE1 deletion. The pmr1Δ cells, lacking the gene that encodes the P-type ATPase ion pump that transports Ca²⁺ and Mn²⁺ into the Golgi, hyperaccumulated Mn²⁺ even from normal medium when overexpressing PHO84, a phenotype which is rather restricted to metal-hyperaccumulating plants.     

Expression,glycosylation and secretion of yeast acid phosphatase in hamster BHK cells

The gene PHO5 coding for one of the repressible acid phosphatases of the yeastSaccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human -actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular massM _r=62000, indicating substitution of the polypeptide moiety by 2–3 asparagine-linked glycans. Analysis by sequential lectin affinity chromatography of glycopeptides obtained from r-APase with Pronase showed that the glycans are predominantly of the 2.2.4 triantennary and tetraantennary complex-type. These data suggest that the extensive glycosylation of yeast APase, which contains eight polymannose substituents, is not essential for secretion and expression of enzymatic activity of the transfected gene product.Abbreviations APase acid phosphatase - PBS phosphate buffered saline - TBS Tris buffered saline - con A concanavalin A - TCA Tetracarpidium conophorum agglutinin     

Genetic Analysis of Spontaneous Suppressors of the pho85 Mutation in Yeast Saccharomyces cerevisiae

Chaperones are known to play an important role in complexation of cyclin-dependent kinases with cyclins. In yeast cells growing in the presence of phosphate, cyclin-dependent kinase Pho85p and cyclin Pho80p form a complex and phosphorylate activator Pho4p. As a result, Pho4p is exported from the nucleus, and the PHO5 gene is not transcribed. The mutations suppressing thepho85 mutation were analyzed in order to identify genes which code for chaperones involved in the formation of the Pho80p–Pho85p complex in the presence of environmental phosphate. Dominant mutations DSP1, DSP2, and DSP4–6 were found. It is shown that the DSP1gene is 2.1 cM away from thePHO85 gene on chromosome XVI and probably coincides with the EGD1 gene coding for a chaperone.     

Two Alkaline Phosphatases from a Butirosin A Producer Bacillus vitellinus

In low-phosphate medium, a butirosin A producer B. vitellinus produced two alkaline phosphatases. These enzymes were fractionated by DEAE-cellulose column chromatography. One phosphatase (Pho I) was eluted with the lower concentration of NaCl compared with the other phosphatase (Pho II). In the wild type strain, Pho I was completely repressed in the high-phosphate medium, but 30% of the fully-derepressed level of Pho II was still produced.

The phosphatase-negative mutant, P-15, that was shown to accumulate butirosin A-6′-N-diphosphate in our previous study, produced only one phosphatase (Pho I) under the low-phosphate condition. Therefore, P-15 was characteristic of the deficiency in Pho II synthesis.

The partially purified preparations of Pho I and II were characterized. Although both enzymes had a similar molecular weight, they could be differentiated in control of synthesis, heat stability, substrate specificity and other properties. Kinetic properties showed that Pho-II was more specific than Pho I to aminoglycoside-phosphates; butirosin A-3′-phosphate, butirosin A-6′-N-diphosphate and 6′-deamino-6′-hydroxybutirosin A-6′-O-diphosphate. The roles of the two phosphatases in butirosin A biosynthesis were discussed.