Constitutive Acid Phosphatase Research Articles

Surfaceome of pathogenic yeasts, Candida parapsilosis and Candida tropicalis, revealed with the use of cell surface shaving method and shotgun proteomic approach.

In the course of infections caused by pathogenic yeasts from the genus Candida, the fungal cell surface is the first line of contact with the human host. As the surface-exposed proteins are the key players in these interactions, their identification can significantly contribute to discovering the mechanisms of pathogenesis of two emerging pathogens from this genus, C. parapsilosis and C. tropicalis. Therefore, the aim of the present study was to identify the cell wall-attached proteins of these two species with the use of cell surface shaving and a shotgun proteomic approach. Different morphological forms of C. parapsilosis and C. tropicalis cells obtained after growth under various conditions were subjected to this treatment. This allowed to indicate the most abundant cell surface proteins on the basis of the normalized spectral abundance factors. In case of yeast-like forms these were, among others, proteins similar to a chitinase, glyceraldehyde-3-phosphate dehydrogenase and an inducible acid phosphatase for C. parapsilosis, and a constitutive acid phosphatase, pyruvate decarboxylase and glyceraldehyde-3-phosphate dehydrogenase for C. tropicalis. In case of pseudohyphal forms, proteins similar to a cell surface mannoprotein Mp65, chitinase and glycosylphosphatidylinositol-anchored transglycosylase Crh11 were identified at the cell surface of C. parapsilosis. The Rbt1 cell wall protein, a hyphally regulated cell wall protein and proteins from agglutinin-like sequence protein family were found as the most abundant on C. tropicalis pseudohyphae. Apart from the abovementioned proteins, several additional covalently bound and atypical cell wall proteins were also identified. These results extend the current knowledge regarding the molecular basis of virulence of these two non-albicans Candida species.

Genetic instability of constitutive acid phosphatase in shochu and sake yeast

Genetic instability of constitutive acid phosphatase (cAPase) activity was observed in a shochu brewer's yeast strain (Ko), which consistently produced 0.3-1% progeny without cAPase when it had been subcultured for a long period of time in barley shochu mash or in conventional complete medium. Genetic analysis showed that the cAPase-negative phenotype was associated with a single mutation in the PHO3 gene and that the Ko strain had heteroallelic PHO3/pho3 genes, while the PHO3⁻ mutants had the homoallelic pho3/pho3 defect. Some sake yeast strains that are cAPase negative, such as K6, K7 and K9, also had the same homoallelic defect, whereas another sake yeast strain K3, with heteroallelic PHO3/pho3 genes, displayed similar genetic instability of cAPase activity. In all cases, the pho3-defective genes were generated by deletion of an approximately 1.9 kb region between the PHO5-PHO3 tandem genes on chromosome II, resulting in chimeric PHO5/3 fusion genes with different fusion points. By integrating a lys2 marker, which is linked with the pho3 allele on the arm of chromosome II in the Ko strain, we demonstrated that the pho3/pho3 defect originated either from a loss of heterozygosity at the heteroallelic PHO3/pho3 locus or from a looping out of the PHO3 region. Although fermentation experiments have not yet indicated any correlation between cAPase activity and alcohol production, the PHO3⁻ mutation itself could prove to be a useful selective marker for yeast strains carrying a number of advantageous mutations for fermentation and which display phenotypic diversity and stability.

Researching the mechanisms of PHO3 gene regulation depending on the nitrogen source in medium in yeast Saccharomyces cerevisiae

Delicate regulation of gene expression is performed through competition of transcriptional factors for promoters that provides punctual cell response to environmental changes. Gene PHO3 of yeast S. cerevisiae encodes the constitutive acid phosphatase. In this work we have researched the genetic control of PHO3 gene expression depending on the source of nitrogen in medium. The PHO3 expression level was proven to decrease with yeast using a poor nitrogen source like urea. Possible regulatory mechanisms for gene PHO3 were revealed involving a repressor of nitrogen regulated genes Gzf3p and ubiquitin ligase Rsp5p.

Researching the mechanisms of gene РНО3 regulation de pending on nitrogen source in medium in yeast Sacharomyces cere visiae

Delicate regulation of gene expression is performed through transcriptional factors competition for promoters that provides punctual cell response to environmental changes. Gene PHO3 of yeast S. cerevisiae encodes the constitutive acid phosphatase. In this work we researched genetic control of the gene PHO3 expression in response to nitrogen source in medium. PHO3 expression level was proved to decrease while yeast using poor nitrogen source like urea. Possible regulatory mechanisms for gene PHO3 were revealed involving repressor of nitrogen regulated genes Gzf3p and ubiquitin ligase Rsp5p

Vesicular transport of extracellular acid phosphatases in yeast Saccharomyces cerevisiae.

A method for isolation of secretory vesicles from the yeast Saccharomyces cerevisiae based on the disintegration of protoplasts by osmotic shock followed by separation of the vesicles by centrifugation in a density gradient of Urografin was developed in this study. Two populations of the secretory vesicles that differ in density and shape were separated. Acid phosphatases (EC 3.1.3.2) were used as markers of the secretory vesicles. It was shown that the constitutive acid phosphatase (PHO3 gene product) is mainly transported to the cell surface by a lower density population of vesicles, while the repressible acid phosphatase (a heteromer encoded by PHO5, PHO10, and PHO11 genes) by a vesicle population of higher density. These data provide evidence that at least two pathways of transport of yeast secretory proteins from the place of their synthesis and maturation to the cell surface may exist. To reveal the probable reasons for transport of Pho3p and Pho5p/Pho10p/Pho11p enzymes by two different kinds of vesicles, we isolated vesicles from strains that synthesize the homomeric forms of the repressible acid phosphatase. It was demonstrated that glycoproteins encoded by the PHO10 and/or PHO11 genes could be responsible for the choice of one of the alternative transport pathways of the repressible acid phosphatase. A high correlation coefficient between bud formation and secretion of Pho5p phosphatase and the absence of correlation between bud formation and secretion of minor phosphatases Pho10p and Pho11p suggests different functional roles of the polypeptides that constitute the native repressible acid phosphatase.

The influence of microcystin-LR and hepatotoxic cyanobacterial extract on the water plant Spirodela oligorrhiza.

The eutrophication of the Sulejów Reservoir dam in Poland is related to toxicity from cyanobacterial blooms. The main species responsible for hepatotoxic bloom formation is Microcystis aeruginosa. The aim of this study was to evaluate the influence of toxic cyanobacterial extract on the growth and morphology of the water plant Spirodela oligorrhiza, compared with commercial-grade microcystin-LR (MC-LR). It was found that after 96 h of incubation the highest concentration of cyanobacterial extract, containing 0.344 mg MC-LR/L, reduced the number of fronds by about 50% in comparison with the control. The extract effected a reduction in the frond mass and a decrease in chlorophyll (a + b) concentration. A reduction in the number of fronds was also observed after the first 24 h of incubation in the presence of 0.2 and 0.1 microg/L of commercial-grade MC-LR. Changes in activity of constitutive acid phosphatase and RNase after 7 days of incubation with commercial-grade MC-LR were observed. The results confirm the toxicity of cyanobacterial hepatotoxins to Spirodela oligorrhiza, which can be used as a sensitive bioindicator.

Purification, characterization and exit routes of two acid phosphatases secreted by Botrytis cinerea

The two forms of acid phosphatase secreted by Botrytis cinerea were purified, characterized and found to possess fundamentally different properties. The phosphate-repressible acid phosphatase was a monomer of clearly defined molecular weight (55–56 kDa), with a polypeptide component of 43 kDa. The enzyme was secreted through the hyphal, apex and accumulated in the culture fluid. Its high affinity for a wide range of substrates rendered it suitable for a putative role as an extracellular phosphate-scavenging enzyme. Secretion of a constitutive acid phosphatase occurred at low levels in phosphate-enriched culture; at phosphate starvation, the activity of this enzyme was enhanced ten-fold in the culture fluid and two-fold in the cell wall. The constitutive enzyme had strikingly different properties from those of the repressible form. It consisted of a 93 kDa polypeptide which was heavily glycosylated such that the secreted protein displayed size heterogeneity of 140–200 kDa by SDS-polyacrylamide gel electrophoresis and 320–450 kDa by native Sephacryl S-300 chromatography. Irrespective of the phosphate status, most of the secreted constitutive enzyme activity was retained by the cell wall. These results were consistent with ultrastructural histochemical observations which suggested that this enzyme form was secreted by a novel pathway via the fusion of vacuoles with the plasma membrane in mature hyphal segments.

Acid phosphatase secretion by Botrytis cinerea

Botrytis cinerea secreted low levels of a constitutive acid phosphatase into liquid culture when grown in the condition of phosphate starvation or in the presence of high concentrations (4 MM) of organic phosphate esters or inorganic phosphate. During phosphate starvation the overall acid phosphatase activity secreted into the culture fluid was increased up to 80-fold due to the appearance of a second (phosphate-repressible) enzyme form. On native polyacrylamide electrophoresis gels, the constitutive enzyme displayed size heterogeneity, whereas the repressible enzyme was resolved as a discrete band. Both forms of the enzyme reacted with p -nitrophenylphosphate under such conditions, but only the constitutive form was found to hydrolyse β-glycerophosphate. Histochemical staining with these two substrates with the Gomori lead-capture method revealed significant staining of secretory vesicles in the hyphal tip region only in phosphate-starved mycelium with p -nitrophenylphosphate as the substrate. By contrast, vacuolar structures were stained with both substrates under conditions of phosphate starvation as well as abundance of inorganic phosphate. The implications of these observations for the likely exit route of the two acid phosphatase forms are discussed.

13] Thiamin transporters in yeast

This chapter examines the thiamin transporters in yeast. The initial rate of [ 14 C]thiamin uptake by Saccharomyces cerevisiae ( S. cerevisiae )grown at exponential phase in thiamin-free Wickerham's synthetic medium is approximately 10-fold higher than that by commercially available bakers' yeast. The uptake by the former cells is linear for about 2 min, whereas that by the latter cells is for about 20 min. Thiamin accumulation is mediated by a single active transport system operating with a pH optimum of 4.5–5.0 and an apparent K m value for thiamin of 0.18 μ M. Two thiamin-binding proteins have been found in S. cerevisiae —one is a soluble thiamin-binding protein (sTBP) in the periplasmic space, and the other is bound to plasma membrane (mTBP). The thiamin-binding activities of these proteins can be assessed by a standard equilibrium dialysis method. The so-called constitutive acid phosphatase localized in the yeast periplasmic space is repressed by exogenous thiamin. Because both thiamin transport and the activity of sTBP are repressed by pregrowth of yeast cells in thiamin-containing medium, the activity of the thiamin-repressible acid phosphatase is also measured throughout the purification of sTBP.

Molecular analysis of the PHO81 gene ofSaccharomyces 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.

Reciprocal regulation of the tandemly duplicated PHO5/PHO3 gene cluster within the acid phosphatase multigene family of Saccharomyces cerevisiae.

We characterized the organization and expression of PHO5 and PHO3, the tightly linked repressible and constitutive acid phosphatase genes of Saccharomyces cerevisiae. The "constitutive" gene, PHO3, is expressed only when PHO5 is not. Altering PHO5 expression, either through promoter deletions or through mutations in trans-acting regulatory genes, showed that PHO5 expression is sufficient to block transcription of PHO3. An active genomic copy of PHO5 was able to block expression of PHO3 from a high-copy-number plasmid, showing that some trans-acting product of PHO5 is involved. This is probably a translation product, since the presence of a nontranslatable PHO5 RNA did not inhibit transcription of PHO3.

Structural analysis of the two tandemly repeated acid phosphatase genes in yeast.

We have sequenced the genetically linked genes for repressible (PHO5) and and constitutive (PHO3) acid phosphatase from S. cerevisiae. Both genes are located on a 3.91 Kb BamHI and HpaI fragment, in the order (5') PHO5, PHO3 (3'). The mRNA transcripts have been analysed by S1-nuclease mapping. They show heterogenous initiation sites. Each of the PHO5 and PHO3 genes codes for 467 amino acids as deduced from the DNA sequence. The coding regions of the two genes show homology both at the nucleotide (82%) and the amino acid (87%) level. In the coding sequences, long stretches of homologous regions are flanked by small non-homologous regions. The nucleotide homology (65%) extends to some length into the 5' and 3' non-coding flanking sequences. Further upstream sequences are unrelated. The comparison of the NH2-terminal amino acid sequence deduced from the nucleotide sequence, with that of purified repressible acid phosphatase revealed the presence of a putative signal peptide.

RNA and homology mapping of two DNA fragments with repressible acid phosphatase genes from Saccharomyces cerevisiae.

Two EcoRI restriction fragments carrying Saccharomyces cerevisiae repressible acid phosphatase genes were analyzed. Transcripts were mapped by restriction endonuclease cleavage of glyoxal-stabilized R-loops and by gel blot hybridizations to cDNA. Homology between the two fragments was examined by gel blots and heteroduplex analysis. Each fragment carried a region of about 1.5 kilobases that coded for a repressible acid phosphatase, and these regions showed homology to one another. In addition, one fragment carried a second region of somewhat lower homology that probably codes for the so-called constitutive acid phosphatase.

RNA and homology mapping of two DNA fragments with repressible acid phosphatase genes from Saccharomyces cerevisiae.

Two EcoRI restriction fragments carrying Saccharomyces cerevisiae repressible acid phosphatase genes were analyzed. Transcripts were mapped by restriction endonuclease cleavage of glyoxal-stabilized R-loops and by gel blot hybridizations to cDNA. Homology between the two fragments was examined by gel blots and heteroduplex analysis. Each fragment carried a region of about 1.5 kilobases that coded for a repressible acid phosphatase, and these regions showed homology to one another. In addition, one fragment carried a second region of somewhat lower homology that probably codes for the so-called constitutive acid phosphatase.

Purification and identification of inactive forms of repressible and constitutive acid phosphatase in yeast

Acid phosphatase (EC 3.1.3.2, orthophosphoric-monoester phosphohydrolase, (acid optimum)) from the budding yeast Saccharomyces cerevisiae was purified from repressed and depressed cells. Without Triton X-100 in the extraction buffer only the constitutive or repressible active enzyme eluted from a Sepharose CL-6B column, the last step of the purification procedure. When Triton X-100 was included in the extraction buffer, an additional protein peak eluted prior to the active acid phosphatase. The material from this new peak, a glycoprotein, had no acid phosphatase activity but cross-reacted with antibodies raised against repressible acid phosphatase. The tryptic fingerprints of the inactive proteins are very similar to the ones of the corresponding active enzymes. We conclude that this new glycoprotein represents an inactive form of repressible and constitutive acid phosphatase. The fact that inactive acid phosphatase can be recovered only in the presence of Triton X-100 indicates that it is membrane-bound.

Two yeast acid phosphatase structural genes are the result of a tandem duplication and show different degrees of homology in their promoter and coding sequences.

We have cloned the structural genes for a regulated ( PHO5 ) and a constitutive ( PHO3 ) acid phosphatase from yeast by transformation and complementation of a yeast pho3 , pho5 double mutant. Both genes are located on a 5.1-kb BamHI fragment. The cloned genes were identified on the basis of genetic evidence and by hybrid selection of mRNA coupled with in vitro translation and immunoprecipitation. Subcloning of partial Sau3A digests and functional in vivo analysis by transformation together with DNA sequence analysis showed that the two genes are oriented in the order (5') PHO5 , PHO3 (3'). While the nucleotide sequences of the two coding regions are quite similar, the putative promoter regions show a lower degree of sequence homology. Partly divergent promoter sequences may explain the different regulation of the two genes.

Acid phosphatase isoenzymes of Chlamydomonas reinhardii.

Acid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wild-type and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are related to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P2 and their total absence in mutant Pa. The pI values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either alpha-naphthyl phosphate or different acetate esters as substrates.

Mapping of a centromere-linked gene responsible for constitutive acid phosphatase synthesis in yeast.

From a chemostat in which the activity of acid phosphatase was limiting, a mutant with a constitutive acid phosphatase was selected. This gene (pho80) responsible for the production of a repressor was mapped 5.3 centimorgans apart from the centromere on the right arm of chromosome XV.

Mode of Action of p-Fluorophenylalanine in Aspergillus nidulans: Effect on the Synthesis and Activity of Phosphatase Isoenzymes

The polyacrylamide disc gel electrophoretic pattern of five isoenzymes of phosphomono-esterases (both alkaline and acid) of Aspergillus nidulans has been established. Bands I and V are derepressible acid phosphatases, band II is constitutive acid phosphatase, band III a derepressible alkaline phosphatase and band IV a constitutive alkaline phosphatase. Isoenzyme III can be completely inhibited by p-fluorophenylalanine (FPA). Experiments using FPA, cycloheximide and actinomycin D indicated that transcription for isoenzyme III (inhibited by actinomycin D) took place between 20 and 21 h incubation, and translation of the mRNA so formed occurred between 21 and 22 h. FPA and cycloheximide were both inhibitory at the time of translation. However, cycloheximide caused a gradual reduction in the quantity of all isoenzymes, whereas only isoenzyme III was affected by FPA. The different effect of FPA was confirmed by incorporation of radioactive FPA into isoenzyme III. FPA apparently did not inhibit synthesis of isoenzyme III, but inactivated the isoenzyme III formed in its presence. Constitutive isoenzymes synthesized in the presence of FPA showed normal activity. It is suggested that FPA may not necessarily affect the activity of all proteins synthesized in its presence but whenever it is incorporated into indispensable sites in proteins, it inactivates them. This may be the mechanism of growth inhibition by FPA in Aspergillus nidulans.

Some properties of phosphate-repressible and -constitutive acid phosphatases of baker's yeast.

Inorganic phosphate-repressible and -constitutive acid phosphatases of baker's yeast were examined to determine whether these two acid phosphatases are the same or different molecular species. In DEAE–cellulose chromatography, the repressible enzyme was elated by 50 mm malonate buffer (pH 5.5), whereas the constitutive one was eluted by a much higher buffer concentration. Both enzymes showed non-specific acid phosphatase activities but no phosphodiesterase activity. The repressible enzyme was more sensitive to heat treatment and to chemicals such as Zn2+, Hg2+, ICH2CONH2, PCMB, NaF, urea and Triton X−100 than the constitutive one. Optimum pH of the repressible enzyme was 4.3 whereas that of the constitutive one was 3.6, and Km of the repressible enzyme for p-nitrophenylphosphate was 0.74 × 10−3 m and that of the constitutive one was 1.1 × 10−3 m; both of them were competitively inhibited by inorganic phosphate. These results suggest that both enzymes represent different molecular species.