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 derepressed 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 phosphate. 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.

Phospholipase B from the plasma membrane of Saccharomyces cerevisiae. Separation of two forms with different carbohydrate content.

Two forms of phospholipase B could be solubilized from the plasma membrane of Saccharomyces cerevisiae, separated by gel filtration with Sephacryl S-300 and identified by SDS-polyacrylamide gel electrophoresis as glycoproteins of the apparent molecular weights of about 220 000 (phospholipase B1) and 145 000 (phospholipase B2). The enzymes are very similar in respect to their catalytic properties. Both forms converted lysophosphatidylcholine to diacylphosphatidylcholine and unesterified fatty acids. The carbohydrate content of the glycoproteins could be reduced by treatment with endoglycosidase H and HF. By incubation of phospholipase B1 and phospholipase B2 with endoglycosidase H from Streptomyces griseus, one main protein with an apparent Mr of 67 000 and the same residual carbohydrate content was obtained. Treatment with HF reduced phospholipase B1 and phospholipase B2 to proteins with an apparent Mr of 52 000 and 67 000, respectively. These results could indicate that the two forms are similar in respect to their protein moieties. An antiserum raised in mice against phospholipase B2 showed no crossreactivity with phospholipase B1 as detected by immunoblot analysis. The reactivity of phospholipase B2 was diminished or abolished by progressive removal of carbohydrate. These results were taken as indications for differences in the carbohydrate component of the two enzyme forms.

Isolation and characterization of regulatory mutants from Schizosaccharomyces pombe involved in thiamine-regulated gene expression.

Mutants from Schizosaccharomyces pombe deficient in the regulation of thiamine-repressible acid phosphatase have been isolated. Mutants expressing derepressed levels of the enzyme in the presence and absence of thiamine map in three genes, tnr1, tnr2 and tnr3. mRNA levels of the pho4 gene (coding for thiamine repressible acid phosphatase) and another thiamine-regulatable gene, thi3 (coding for a thiamine biosynthetic enzyme and corresponding to nmt1) are constitutively synthesized in the mutants. The mutants also exhibit constitutive thiamine transport which is thiamine repressible in wild type. The tnr3 mutants reveal a 10-20-fold higher intracellular thiamine level than tnr1 and tnr2 mutants and wild type. Mutants expressing repressed levels of thiamine-repressible acid phosphatase map in gene thi1. No or little amounts of pho4- and nmt1-specific mRNA can be detected. These mutants are impaired in thiamine uptake and are thiamine auxotrophic due to the inability to synthesize the thiazole moiety of the thiamine molecule. All tested tnr and thi1 alleles are recessive, and thi1 mutations are epistatic over tnr mutations. We assume that the thi1 and tnr genes are involved in thiamine-mediated transcription control.

Thiamine-repressible genes in Schizosaccharomyces pombe are regulated by a Cys6 zinc-finger motif-containing protein.

Our previous genetic data indicate that the product of the Schizosaccharomyces pombe thi1 gene acts as an activator of several thiamine-repressible genes which are involved in the control of thiamine metabolism [Schweingruber et al., Genetics 130 (1992) 445-449; Zurlinden and Schweingruber, Gene 117 (1992) 141-143]. In this communication, we report the cloning and sequencing of thi1 and show that it carries an open reading frame which translates into a 775-amino-acid protein with the characteristics of a Cys6 zinc-finger-motif-containing transcription factor, as typified by Saccharomyces cerevisae GAL4. We, therefore, suggest that the thi1-encoded protein binds to upstream activator sequences of thiamine-repressible genes.

Cloning and regulation of Schizosaccharomyces pombe thi2, a gene involved in thiamine biosynthesis.

Biosyntheses of the pyrimidine and thiazole moieties of the thiamine molecule occur by separate pathways. In Schizosaccharomyces pombe, a gene, thi2, is responsible for thiazole synthesis [Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We have cloned a 3.1-kb genomic S. pombe fragment which can functionally complement a thi2 mutant. The fragment maps genetically at the thi2 site, indicating that it carries thi2. As shown by Northern hybridization analysis, the appearance of thi2 mRNA levels is repressed when cells are grown in the presence of thiamine and 5-(2-hydroxyethyl)-4-methylthiazole. The thi3 gene involved in the biosynthesis of the pyrimidine moiety, is also regulated by thiamine [Maundrell, J. Biol. Chem. 265 (1990) 10857-10864; Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We previously identified and analyzed four regulatory genes (tnr1, tnr2, tnr3, and thi1) that are responsible for the regulation of thi3 [Schweingruber et al., Genetics (1992) in press]. Mutants defective in these regulatory genes affect expression of thi2 in a similar way to thi3. This indicates that biosynthesis of the pyrimidine and thiazole moieties are under common genetic control in S. pombe.

Amiloride toxicity in the fission yeast Schizosaccharomyces pombe is released by thiamine and mutations in the thiamine-repressible gene car1.

Amiloride (Am) inhibits growth in the fission yeast Schizosaccharomyces pombe. We show that the toxic effect of this drug is relieved by low concentrations of thiamine (Th) and that the pyrimidine moiety of the Th molecule is responsible for growth inhibition release. A putative membrane protein encoded by the car1 gene is the target for Am action. It is responsible for Am sensitivity and is involved in the utilization of Th and its biosynthetic precursor, 4-amino-5-hydroxymethyl-2-methylpyrimidine. Its expression is repressed by Th and is under the genetic control of the genes, thi1, tnr1, tnr2 and tnr3, which have previously been shown to be responsible for the transcriptional control of genes involved in the biosynthesis and dephosphorylation of Th.

Cloning and characterization of two genes restoring acid phosphatase activity in pho1- mutants of Schizosaccharomyces pombe.

Schizosaccharomyces pombe acid phosphatase (APh) is a secreted cell surface glycoprotein which is deficient in pho1 mutants. By screening an S. pombe gene bank for sequences which can functionally rescue the pho1-44 mutation, we have isolated two genomic clones carried in plasmids pSp4B and pSp4C/2. These two sequences map of different genetic loci and show no cross hybridization by Southern blotting. pSp4C/2 was found to contain the PHO1 gene, and cells transformed with this plasmid produce a protein which cross-reacts with antibodies raised against the protein moiety of APh. Data from Northern blotting experiments show that pSp4C/2 encodes a 1.6-kb transcript, and that mRNA levels are increased when cells are grown in low concentrations of inorganic phosphate. The results indicate that pSp4C/2 contains the structural gene for APh, PHO1, whereas pSp4B appears to carry a gene coding for a minor species of APh, PHO4 which is not regulated by extracellular phosphate.

The tRNA N2,N2-dimethylguanosine-26 methyltransferase encoded by gene trm1 increases efficiency of suppression of an ochre codon in Schizosaccharomyces pombe.

In the majority of eukaryotic tRNAs, the guanosine at position 26 is modified by a dimethyl group, but so far a function of this modification has not been detected. We isolated the Schizosaccharomyces pombe gene, trm1, encoding the tRNA N2, N2-dimethylguanosine-26 methyltransferase. Strains having the gene deleted completely lack N2,N2-dimethylguanosine. In strains carrying the weak ochre tRNA suppressor sup3-i, deletion of trm1 abolishes suppression indicating that the trm1 deletion acts as an antisuppressor mutation. The result suggests that in vivo N2, N2-dimethylguanosine-26 increases the capacity of the sup3-i serine tRNA to translate the UAA (ochre) codon.

The antitrypanosomal drug melarsoprol competitively inhibits thiamin uptake in mouse neuroblastoma cells.

Melarsoprol is the main drug used for the treatment of late-stage sleeping sickness, although it causes severe side-effects such as encephalopathy and polyneuropathy leading to death in some patients. Recent data suggest that melarsoprol and its active metabolite melarsenoxide interfere with thiamin transport and metabolism in E. coli and yeast, but there are no data concerning their possible effects on thiamin metabolism in mammalian cells. We tested both drugs on thiamin transport in cultured mouse neuroblastoma cells using (14)C-labeled thiamin. Melarsoprol, competitively inhibits high-affinity thiamin transport in mouse neuroblastoma cells with a K(i) of 44 micromol/L. However, the active compound melarsenoxide has no inhibitory effect. This suggests that the side effects of melarsoprol treatment are unlikely to be due to inhibition of thiamin transport by melarsenoxide, its main metabolite in the brain.

Identification of a DNA element in the fission yeast Schizosaccharomyces pombe nmt1 (thi3) promoter involved in thiamine-regulated gene expression.

To define DNA elements involved in thiamine-regulated transcription of the Schizosaccharomyces pombe gene nmt1 (thi3), we analyzed several nmt1 promoter constructs. We detected a DNA element which is required for promoter activation in the absence of thiamine. It is located 54 to 62 bp upstream of the TATA box and matches the consensus sequence of the binding site for the mammalian transcription factor C/EBP (CAAT/enhancer binding protein). We show that the element specifically binds proteins.

Cloning, nucleotide sequence, and regulation of Schizosaccharomyces pombe thi4, a thiamine biosynthetic gene.

thi4 mutants of Schizosaccharomyces pombe exhibit defective thiamine biosynthesis, and thi4 mutations define a gene which is believed to be involved in the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine or 5-(2-hydroxyethyl)-4-methylthiazole and/or in the coupling of the two phosphorylated precursors to thiamine monophosphate (A. M. Schweingruber, J. Dlugonski, E. Edenharter, and M. E. Schweingruber, Curr. Genet. 19:249-254, 1991). The thi4 gene was cloned by functional complementation of a thi4 mutant and physically mapped on the left arm of chromosome I close to the genetic marker gln1. The thi4-carrying DNA fragment shows an open reading frame encoding a protein of 518 amino acids and a calculated molecular mass of 55.6 kDa. The appearance of thi4 mRNA is strongly repressed by thiamine and to a lesser extent by 5-(2-hydroxyethyl)-4-methylthiazole. thi4 mRNA production is under the control of the thi1 gene-encoded transcription factor and of the negative regulators encoded by genes tnr1, tnr2, and tnr3. thi4 is expressed and regulated in manners similar to those of other S. pombe genes involved in thiamine metabolism, including thi2, thi3, and pho4.

Posttranslational regulation of repressible acid phosphatase in yeast.

On the basis of genetic data it has been suggested that repressible acid phosphatase of Saccharomyces cerevisiae is regulated by a control circuit involving operator-repressor mechanisms (Toh-e et al., 1978). We measured no significant difference in the amount of translatable mRNA of repressed and derepressed cells in the reticulocyte in vitro translation system. We find a 25 fold difference in specific enzyme activity in repressed versus derepressed cells whereas the amount of 35S-methionine labelled enzyme protein as measured by antibody precipitation varies only 2-3 fold. This argues for posttranslational regulation of preexisting inactive acid phosphatase. Minor regulatory effects at the transcriptional or translational level cannot be excluded.

Differential regulation of the active and inactive forms of Saccharomyces cerevisiae acid phosphatase.

Acid phosphatase in S. cerevisiae exists as an enzymatically active, cell wall associated form and as an enzymatically inactive, probably membrane-bound form (Schweingruber and Schweingruber, in press). Orthophosphate dependent and independent regulation determines the level of acid phosphatase activity. To deduce the regulation mechanisms we purified and quantified active and inactive acid phosphatase from cells grown under different physiological conditions and displaying variable levels of enzyme activity. Orthophosphate dependent regulation does not include significant changes in the amount of total (active and inactive) acid phosphatase protein synthesized. Under the experimental conditions chosen increased activity is achieved by preferential synthesis of the active form and by increasing the specific activity of the active enzyme. Orthophosphate independent regulation seems to occur by similar mechanisms.

Modulation of a cell surface glycoprotein in yeast: acid phosphatase.

Upon inorganic phosphate starvation the cell wall glycoprotein acid phosphatase of yeast Saccharomyces cerevisiae is derepressed. Purified acid phosphatase isolated from early log phase cells differs in reactivity and stability from acid phosphatase from late log phase cells indicating that the two enzymes are structurally different. This demonstrates that the yeast cell has not only the capacity to regulate the amount of acid phosphatase but also the ability to vary (modulate) the structure of the secreted enzyme. Modulation of acid phosphatase may be a mechanism which is involved in morphogenetic and behavioral differentiation of the yeast cell.

A Schizosaccharomyces pombe gene, ksg1, that shows structural homology to the human phosphoinositide-dependent protein kinase PDK1, is essential for growth, mating and sporulation.

Fission yeast (Schizosaccharomyces pombe) requires inositol for growth, mating and sporulation. To define putative genes that are involved in the processing and transduction of the inositol signal, mutants that are temperature sensitive for growth and sporulation were selected on a medium containing non-limiting amounts of inositol. Two such mutants (ksg1-208 and ksg1-358) were analyzed, which are impaired in mating and sporulation at 30 degrees C and undergo growth arrest in the G2 phase of the cell cycle at 35 degrees C. The ksg1 gene was isolated by functional complementation. It maps on the left arm of chromosome II and encodes a putative 592-amino acid protein which exhibits good structural homology to a human 3-phosphoinositide-dependent protein kinase (PDK1) and its rat and Drosophila homologues. The two mutants have the same substitution at amino acid position 159: a glycine residue is replaced by glutamic acid. Deletion of the gene is lethal for haploid cells. We propose that ksg1 is involved in one or several phosphoinositide signalling processes that are responsible for control of the life cycle.

The structural gene coding for thiamin-repressible acid phosphatase in Schizosaccharomyces pombe.

The pho4 gene of the fission yeast Schizosaccharomyces pombe is regulated by thiamin. The nucleotide sequence of this gene is given here and it is shown that it matches the amino acid sequence of thiamin-repressible acid phosphatase, corroborating genetic evidence that pho4 represents the structural gene of this enzyme. The gene codes for a protein of 463 amino acids in length and shows regions of strong similarity with the phosphate-repressible acid phosphatase of Schizosaccharomyces pombe. The enzyme has a cleavable signal sequence 18 amino acids long and carries nine potential N-glycosylation sites.