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.     

Insertion of (dA-dT)n sequences into the regulatory region of the pho5 gene inhibits its expression

We have studied the effect of some regular sequences namely (dA-dT)n, (dA)n and (dG-dC)n on the Saccharomyces cerevisiae PHO5 gene expression. These sequences were inserted into the ClaI site, located between two phosphate-responsible upstream activating sequences (UAS's). A modified PHO5 gene cloned in vector plasmids was used to transform the pho3, pho5 yeast strain and the level of acid phosphatase expression in various conditions was measured. We show that the insertion of (dA-dT)n blocks, but not (dA)n or (dG-dC)n, leads to the dramatic decrease of PHO5 gene expression in a derepressed state. (dA-dT)n inserts also inhibit the expression of PHO5 gene which was put under the control of heterologous UASgal.     

Development of high-cell-density fermentation for heterologous interleukin 1\ production in Kluyveromyces lactis controlled by the PH05 promoter

The use of a phosphate organic form taken up by Kluyveromyces lactis removes repression of the PHO5 promoter and releases heterologous interleukin 1\ synthesis while providing sufficient phosphate for growth. The oxidative metabolism of high-cell-density fed-batch and chemostat cultures was thus maintained under derepressed protein synthesis conditions. Interleukin 1\ production was then growth-associated, an unusual mode of protein synthesis regulation under the control of the PHO5 promoter.     

Identification of a high-affinity phosphate transporter gene in a prasinophyte alga,Tetraselmis chui,and its expression under nutrient limitation

A high-affinity phosphate transporter gene, TcPHO, was isolated from a growth-dependent subtracted cDNA library of the marine unicellular alga Tetraselmis chui. The full-length cDNA of TcPHO obtained by 5' and 3' rapid amplification of cDNA ends was 1,993 bp long and encoded an open reading frame consisting of 610 amino acids. The deduced amino acid sequence of TcPHO exhibited 51.6 and 49.8% similarity to the amino acid sequences of PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa, respectively. In addition, hydrophobicity and secondary structure analyses revealed 12 conserved transmembrane domains that were the same as those found in PHO89 and PHO4. The expression of TcPHO mRNA was dependent on phosphate availability. With a low-phosphate treatment, the TcPHO mRNA concentration increased sharply to 2.72 fmol micro g of total RNA(-1) from day 1 to day 2 and remained at this high level from days 2 to 4. Furthermore, rescue treatment with either phosphate or p-nitrophenyl phosphate effectively inhibited TcPHO mRNA expression. In contrast, TcPHO mRNA expression stayed at a low level (range, 0.25 to 0.28 fmol micro g of total RNA(-1)) under low-nitrate conditions. The expression pattern suggests that TcPHO can be used as a molecular probe for monitoring phosphorus stress in T. chui.     

酵母PHO81蛋白的结构预测和功能分析

以Ｃｈｏｕ－Ｆａｓｍａｎ和ＧＯＲ方法预测酵母ＰＨＯ８１蛋白的二级结构。用Ｋｙｔｅ－Ｄｏｏｌｉｔｔｌｅ方法分析它的疏水性。分析了ＰＨＯ８１蛋白与其他蛋白的同源性,发现它包含５个ＳＷ１６／ＡＮＫ重复单位,这些重复单位可能是ＰＨＯ８１蛋白与负调控因子ＰＨＯ８０相互作用的位点。     

Functional analysis of the signal-sequence processing site of yeast acid phosphatase

A systematic study of the signal peptidase cleavage site of the main cell-wall-repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild-type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e. alanine, glycine, cysteine, serine or threonine. Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core-glycosylated protein accumulates in the cell. Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence. The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (-7 to -4 relative to the processing site) thus preserving the important -3 to -1 region.     

A deletion that includes the segment coding for the signal peptidase cleavage site delays release of Saccharomyces cerevisiae acid phosphatase from the endoplasmic reticulum.

We studied ultrastructural localization of acid phosphatase in derepressed Saccharomyces cerevisiae cells transformed with a multicopy plasmid carrying either the wild-type PHO5 gene or a PHO5 gene deleted in the region overlapping the signal peptidase cleavage site. Wild-type enzyme was located in the cell wall, as was 50% of the modified protein, which carried high-mannose-sugar chains. The remaining 50% of the protein was active and core glycosylated, and it accumulated in the endoplasmic reticulum cisternae. The signal peptide remained uncleaved in both forms. Cells expressing the modified protein exhibited an exaggerated endoplasmic reticulum with dilated lumen.     

Interrelationship between metabolic and genetic regulation of alkaline and acid phosphatases in E. coli cells]

The effect of exogenous orthophosphate and mutations in regulatory genes of alkaline phosphatase on the level of nonspecific acid phosphatase was studied. The level of this enzyme as well as the level of alkaline phosphatase were shown to be regulated by exogenous orthophosphate being derepressed under phosphate starvation. The derepression of acid phosphatase is accompanied by more rapid secretion of enzyme from membranes to soluble fraction. Mutations in all the four regulatory genes decrease the level of enzyme in cells. Genes phoR and phoS, participating in regulation of alkaline phosphatase, are required for the derepression of acid phosphatase under the conditions of phosphate starvation.