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.

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.

A deletion map of cyc1 mutants and its correspondence to mutationally altered iso-1-cytochromes c of yeast.

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.

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.

Isolation and characterization of acid phosphatase mutants in Schizosaccharomycespombe.

72 mutants defective in the activity of the cell surface glycoprotein acid phosphatase were isolated and characterized. The mutants map in one genetic locus, phol. Many of them exhibit altered cell morphology. This characteristic cosegregates with acid phosphatase deficiency, implying that phol controls the activity of acid phosphatase and concomitantly cell morphology. Phol probably also influences growth rate and agglutination behaviour. By purifying acid phosphatase, two structurally related forms can be detected. One is inactive (form I) and one is the active acid phosphatase (form II). Mutant phol-270 and phol-277 lack the inactive form I. Mutant phol -38 exhibits mainly form I, form II being present only in minor amounts. Two other mutants examined differ only slightly from wildtype in their pattern of active and inactive forms. Tryptic peptide maps of the inactive and active acid phosphatase of the wildtype and the corresponding proteins of mutant phol-304 reveal similar structural alterations for the two mutant proteins. The results show that phol controls the expression of the active and inactive acid phosphatase. We conclude that phol represents the structural gene of the two forms of acid phosphatase.

Mutants of yeast initiating translation of iso-1-cytochrome c within a region spanning 37 nucleotides.

We used a specially constructed strain, cyc1-345, of the yeast Saccharomyces cerevisiae to isolate revertants that initiated translation of iso-1-cytochrome c at various sites along an extended region of the mRNA. Normal amounts of iso-1-cytochrome c occurred when translation initiated at the abnormal sites corresponding to amino acid positions -3, -2, 3 and 5, as well as the normal position -1; 20% of the normal amounts occurred when translation initiated at the abnormal position 9. These results with cyc1-345 revertants indicate that translation of iso-1-cytochrome c can initiate with the normal efficiency at any site within the region spanning 25 nucleotides. Furthermore, because the lower amount of the short iso-1-cytochrome c in the mutant initiating at position 9 may not necessarily reflect an inefficiency of translation, we believe that translation can initiate with normal or near-normal efficiencies at any site within a 37 nucleotide region, and presumably at any site preceding and following that of the normal initiation codon. These results establish that there is no absolute requirement for a particular sequence 5' to the initiation codon, and are consistent with our previous suggestion that translation starts at the AUG codon closest to the 5' end of the mRNA.

Thiamine in Schizosaccharomyces pombe: dephosphorylation, intracellular pool, biosynthesis and transport.

We have investigated the thiamine metabolism in Schizosaccharomyces pombe and shown that: (1) Thiamine-repressible acid phosphate, coded for by the gene pho4, dephosphorylates thiamine phosphates indicating that the enzyme acts as a thiamine phosphate phosphatase. (2) In vivo synthesized thiamine is present intracellularly mainly as thiamine diphosphate. Starving cells for glucose decreases the intracellular thiamine pool. (3) The genes thi2, thi3 and thi4 control thiamine biosynthesis and probably code for thiamine biosynthetic enzymes. Thi3, which is involved in the synthesis of the pyrimidine moiety of the thiamine molecule, is allelic to the thiamine repressible gene nmt1. (4) Thiamine uptake is a thiamine regulated process, probably occurs by active transport and is controlled by the gene ptr1.

Glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe.

We have purified secreted acid phosphatase of Schizosaccharomyces pombe. The enzyme is N-glycosylated, the associated carbohydrate accounts for 90% of the total molecular mass and the protein moiety has a molecular mass of 54 kDa. The deglycosylated enzyme still exhibits enzymatic activity. Using antibodies recognizing the protein moiety of the enzyme we have identified two intracellular precursors of acid phosphatase: an unglycosylated membrane-bound 54-kDa form that accumulates in the presence of tunicamycin and a partially glycosylated 72-kDa form that accumulates mostly in membranes of cells grown in rich medium. We further showed that the conversion of the 54-kDa and 72-kDa forms to partially glycosylated and fully glycosylated acid phosphatase is a regulated process. Growth conditions determine how much of translated 54-kDa acid phosphatase is glycosylated to the 72-kDa form and how much remains unglycosylated in membranes. When cells are grown in a rich medium, 5% of the total acid phosphatase protein remains as unglycosylated enzyme and 8% as partially glycosylated 72-kDa form. In cells grown in the minimal medium, however, all of the 54-kDa and 72-kDa forms of acid phosphatase are rapidly processed to fully glycosylated enzyme. The 72-kDa form and the unglycosylated form of acid phosphatase are not secreted or transported to the plasma membrane.

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.

Schizosaccharomyces pombe thiamine pyrophosphokinase is encoded by gene tnr3 and is a regulator of thiamine metabolism, phosphate metabolism, mating, and growth.

The Schizosaccharomyces pombe gene tnr3 has been genetically defined as a negative regulator of genes involved in thiamine metabolism (Schweingruber, A. M., Frankhauser, H., Dlugonski, J., Steinmann-Loss, C., and Schweingruber, M. E. (1992) Genetics 130, 445-449). We have isolated and sequenced the gene and show that it codes for a putative protein of 569 amino acids which exhibits, in its carboxyl-terminal half, good homology to Saccharomyces cerevisiae thiamine pyrophosphokinase (TPK). tnr3 mutants have reduced levels of intracellular thiamine diphosphate, show impaired TPK activity, which is enhanced by introducing the tnr3 wild type gene on a plasmid, and can be complemented by the S. cerevisiae TPK-encoding gene TH180. These data strongly suggest that tnr3 encodes S. pombe TPK. We present evidence that TPK also acts as a negative regulator for gene pho1, which is derepressed when cells are starved for phosphate and show that in contrast to wild type cells, tnr3 mutants mate constitutively in response to thiamine, indicating that TPK is also involved in regulation of mating. Disruption of the tnr3 gene is lethal, and a tnr3 mutant expressing only residual TPK activity grows slowly and shows aberrant morphology.

Growth of a mutant defective in a putative phosphoinositide-specific phospholipase C of Schizosaccharomyces pombe is restored by low concentrations of phosphate and inositol.

A mutant (plc1-1) of Schizosaccharomyces pombe unable to grow on a minimal medium containing high amounts of phosphate was selected. On yeast-extract agar its growth is temperature sensitive. Tests in liquid synthetic medium show that growth of the mutant is partially restored by lowering the phosphate and inositol concentrations in the growth medium. The growth defect is fully suppressed by a plasmid encoding a putative protein having the structural features of phosphoinositide-specific phospholipases C (PI-PLC). This protein, of 899 amino-acids, contains the characteristic X and Y domains found in all PI-PLCs of higher and lower eucaryotes and reveals, in addition, an EF-hand motif (putative Ca(2+)-binding site). Like the corresponding enzyme from Saccharomyces cerevisiae, the S. pombe PI-PLC is most similar to the delta form of PI-PLC isoenzymes. The cloned gene integrates at the plc1 site indicating that plc1 codes for a putative PI-PLC. Plc1 physically maps on the left arm of chromosome II between rad11 and mei3.

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.

Gene sam1 encoding adenosylmethionine synthetase: effects of its expression in the fission yeast Schizosaccharomyces pombe.

By screening gene libraries of Schizosaccharomyces pombe with a DNA fragment encoding part of the Saccharomyces cerevisiae S-adenosylmethionine synthetase (SAMS), we isolated the fission yeast sam1 gene. Its sequence exhibits good homology to SAMSs of other organisms and reveals the motifs characteristic for SAMSs. SAMS activity and sam1 mRNA levels decrease when cells enter stationary phase. In haploid strains, gene sam1 is essential for growth; if weakly expressed, cells mate and sporulate at a reduced rate. Strains overexpressing sam1 exhibit methionine-sensitive growth. This methionine-induced growth inhibition is partially relieved by adenine. We assume that methionine reduces the level of one or several adenine nucleotides by a SAMS-mediated mechanism. Intracellular SAM levels increase drastically by exogenously added methionine. This increase predicts that mutants exhibiting methionine revertible phenotypes can be indicative for mutations in proteins exhibiting SAM-dependent functions. In agreement with this prediction, we show that mutant pmt2-5 has this phenotype and that gene pmt2 encodes a potential SAM-dependent enzyme.

Gene ste20 controls amiloride sensitivity and fertility in Schizosaccharomyces pombe.

It has been shown previously that amiloride, a widely used diuretic drug, inhibits growth in Schizosaccharomyces pombe. Here we show that the drug also alleviates repression by various nutrients of mating and sporulation in fission yeast. We selected spontaneous mutants that are amiloride-resistant and unable to mate and sporulate. One of them defines the gene ste20. This gene has been cloned and sequenced. It codes for a putative protein of 1309 amino acids. Its sequence does not provide any clues to its function. In contrast to the wild-type, mutants defective in this gene can grow in a medium containing 40 microm amiloride, do not arrest in G(1), and do not induce ste11 expression upon nitrogen starvation and thus are sterile. In addition the ste20 mutants are methylamine-sensitive, exhibit enhanced medium acidification and are defective in the utilization of gycerol as a carbon source.

Methionine induces sexual development in the fission yeast Schizosaccharomyces pombe via an ste11-dependent signalling pathway.

Methionine added to minimal medium overcomes the repressing effects of ammonium and cyclic AMP (cAMP) on sexual development and efficiently induces mating and sporulation in homothallic strains of Schizosaccharomyces pombe. In heterothallic strains it induces G1 arrest when cells enter stationary phase. We show that methionine reduces the intracellular cAMP pool and induces the expression of at least two cAMP-repressible genes, including fbp1 and ste11. The easiest interpretation of the results is that methionine induces sexual development via a cAMP-dependent ste11 signalling pathway.

Cordycepin in Schizosaccharomyces pombe: effects on the wild type and phenotypes of mutants resistant to the drug.

The adenosine analogue cordycepin (3'-deoxyadenosine) inhibits growth and causes aberrant cell morphology in the fission yeast, Schizosaccharomyces pombe. Exogenously added thiamine, the pyrimidine moiety of the thiamine molecule, and adenine alleviate its growth-disturbing effect. At concentrations that do not inhibit growth, the drug reduces mating and sporulation and causes a decrease in the mRNA level of gene ste11 and the ste11-dependent gene, mei2. The mating- and sporulation-inhibiting effect of cordycepin is overcome by adenine. A mutant disrupted for the ado1 gene encoding adenosine kinase exhibits a cordycepin-resistant and methionine-sensitive phenotype, excretes adenosine into the medium and mates and sporulates poorly in the presence of adenine. A S. pombe mutant containing a frameshift mutation at the beginning of the carboxy-terminal half of gene ufd1 (the Saccharomyces cerevisiae UFD1 homologue) is cordycepin-resistant and sterile. Strains disrupted for the ufd1 gene only form microcolonies.

Intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae.

To elucidate intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae we prepared a monoclonal antibody that recognizes specifically the protein moiety of this cell surface glycoprotein. With this antibody membranes and soluble fractions of wild-type cells, grown in low-phosphate medium in the presence and absence of tunicamycin, were examined by the immunoblot technique. Similarly, secretory mutants, blocked at distinct steps in the secretory pathway at the restrictive temperature as well as a strain harboring several copies of the structural gene PHO5 for repressible acid phosphatase, were analyzed. The data suggest the following sequence of events in acid phosphatase maturation and secretion: three unglycosylated precursors with molecular masses of 60 kDa, 58 kDa and 56 kDa are synthesized into membranes of the endoplasmic reticulum, where these are core glycosylated in a membrane-bound form. They appear on sodium dodecyl sulfate gels as bands with molecular masses of 76 kDa and 80 kDa. Owing to a rate-limiting maturation step, occurring after core glycosylation, they can accumulate in a membrane-bound form. At the Golgi apparatus outer carbohydrate chains are attached to the core and the enzyme appears in a soluble form, indicating a release of acid phosphatase from the membrane between the endoplasmic reticulum and the Golgi. Pulse-chase experiments suggest that the time for acid phosphatase synthesis and its transport to the Golgi is about 5 min.