The Aspergillus nidulans metE gene is regulated by a second system independent from sulphur metabolite repression.

Mutations in the Aspergillus nidulans metE gene lead to requirement for O-acetylhomoserine. The gene was cloned by complementation of the metE31 mutation. The coding sequence was found to be interrupted by two introns of 66 and 50 bp, respectively. metE codes for a peptide of 489 amino acids which belongs to the family of homoserine O-acetyltransferases and a well-defined superfamily of alpha/beta hydrolases. Transcription of the metE gene is strongly up-regulated by a severe limitation of methionine, but not of cysteine. This gene is the first sulphur metabolism gene described in A. nidulans which is not regulated by the sulphur metabolite repression system in which cysteine acts as the low-molecular-weight effector.

sconC, a gene involved in the regulation of sulphur metabolism in Aspergillus nidulans, belongs to the SKP1 gene family.

sconC, which encodes a negative regulator of sulphur metabolism in Aspergillus nidulans was cloned, sequenced, and found to belong to the highly conserved family of SKP1 genes essential for many cell functions, including cell cycle regulation. The ORF of 722 bp, encoding a protein of 161 amino acids, is interrupted by four introns. There is a fifth intron (135 bp long) in the upstream untranslated sequence. Two point mutations in conserved regions were identified in the mutant alleles sconC3 and sconC1, which result in relief of sulphur metabolite repression. The SCONC protein contains the PEST sequence common for proteins that are subject to rapid turnover. Transformation of the sconC3 mutant with sconB+ restores the wild-type phenotype. The sconB gene encodes a protein containing the F-box, a domain known to interact with Skp1 proteins. By analogy with other systems, it seems likely that the SCONC protein interacts with SCONB. sconC mRNA is present in the sconC3 and sconB2 mutants and the level of the sconC transcript seems not to be significantly regulated by supplementation of the medium with sulphur.

Cysteine biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation.

Using a Saccharomyces cerevisiae strain having the activities of serine O-acetyl-transferase (SATase), O-acetylserine/O-acetylhomoserine sulphydrylase (OAS/OAH SHLase), cystathionine beta-synthase (beta-CTSase) and cystathionine gamma-lyase (gamma-CTLase), we individually disrupted CYS3(coding for gamma-CTLase) and CYS4 (coding for beta-CTSase). The obtained gene disruptants were cysteine-dependent and incorporated the radioactivity of (35)S-sulphate into homocysteine but not into cysteine or glutathione. We concluded, therefore, that SATase and OAS/OAH SHLase do not constitute a cysteine biosynthetic pathway and that cysteine is synthesized exclusively through the pathway constituted with beta-CTSase and gamma-CTLase; note that OAS/OAH SHLase supplies homocysteine to this pathway by acting as OAH SHLase. From further investigation upon the cys3-disruptant, we obtained results consistent with our earlier suggestion that cysteine and OAS play central roles in the regulation of sulphate assimilation. In addition, we found that sulphate transport activity was not induced at all in the cys4-disruptant, suggesting that CYS4 plays a role in the regulation of sulphate assimilation.

Cloning and characterization of the Kluyveromyces lactis homocysteine synthase gene.

The Kluyveromyces lactis homocysteine synthase gene was cloned by complementation of the Saccharomyces cerevisiae met25 mutation. The coding sequence of the K. lactis gene shows a high similarity to the S. cerevisiae gene. Very little similarity is found in the 5' and 3' untranslated regions. However, one finds short DNA stretches in the promoter of the K. lactis gene which are identical to the nucleotide sequences implicated in the regulation of the S. cerevisiae homologue. This could explain strong transcriptional inhibition of the K. lactis gene by exogenous methionine in the S. cerevisiae host, and indicates a substantial conservation of the sulphur regulatory system between both yeast species.

The metG gene of Aspergillus nidulans encoding cystathionine beta-lyase: cloning and analysis.

The metG gene of Aspergillus nidulans encoding cystathionine beta-lyase, an enzyme of the main pathway of methionine synthesis, was cloned by complementation of a metG mutation. A comparison of metG genomic DNA and a cDNA copy derived from different A. nidulans strains revealed a marked DNA sequence polymorphism manifested mostly by silent point mutations. cDNA of the A. nidulans metG gene complemented the Escherichia coli metC69 mutation impairing cystathionine beta-lyase. This gene contains two introns and codes for a protein of 439 amino acids. The protein shows homology with bacterial, yeast and plant cystathionine beta-lyases, as well as with other enzymes belonging to a large family of pyridoxal 5'-phosphate binding proteins. Transcription of the metG gene is not appreciably regulated by the concentration of sulphur source in the growth medium.

Structure and regulation of cysD, the homocysteine synthase gene of Aspergillus nidulans.

The A. nidulans cysD gene encoding homocysteine synthase (O-acetyl-L-homoserine sulphydrylase) has been isolated by functional complementation of a cysD11 mutation. The gene contains five short introns and codes for a protein of 437 amino acids. The protein shows homology with bacterial and yeast O-acetyl- and O-succinyl-homoserine sulphydrylases, particularly from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Kluyveromyces lactis. The cysD cDNA is able to complement a S. cerevisiae mutation impairing homocysteine synthase. Synthesis of the cysD mRNA is down-regulated by a high concentration of methionine in growth medium without sulphate and up-regulated under sulphur limitation. A comparison of cysD genomic and cDNA copies, derived from different A. nidulans strains, revealed a marked DNA-sequence polymorphism manifested mostly by silent point mutations. There was, however, much less polymorphism in the protein sequence.

The Aspergillus nidulans sulphur regulatory gene sconB encodes a protein with WD40 repeats and an F-box.

The Aspergillus nidulans gene sconB, one of the four identified genes controlling sulphur metabolite repression, was cloned and analysed. It encodes a polypeptide of 678 amino acids containing seven WD repeats characteristic of the large WD40 family of eukaryotic regulatory proteins. The SCONB protein has nuclear localisation signals and is very similar to the Neurospora crassa SCON2 and Saccharomyces cerevisiae Met30 proteins, both of which are involved in the regulation of sulphur metabolism. The N. crassa scon-2 gene complements the sconB2 mutation. All three proteins also contain a newly identified motif, the F-box, found in a number of eukaryotic regulatory proteins. This motif is responsible, at least in some cases, for ubiquitin-mediated proteolysis. The sconB transcript is derepressed under sulphur limitation conditions and partly repressed by high methionine.

Cloning and characterization of the Aspergillus nidulans cysB gene encoding cysteine synthase.

The cysB gene of A. nidulans was cloned by complementation of a cysB mutation. This is the first cloned eukaryotic genomic sequence coding for cysteine synthase. The gene contains one 71-bp intron and codes for a protein of 370 amino acids. Its N-terminal region has characteristic features of transit peptides, suggesting mitochondrial localisation of the enzyme. The protein shows homology with bacterial and plant cysteine synthases among which it occupies a remote phylogenetic position and apparently represents a distinct subfamily. Transcription of the cysB gene is not appreciably regulated by the concentration of methionine in the growth medium.

Regulation of sulphate assimilation in Saccharomyces cerevisiae.

We examined how the activity of O-acetylserine and O-acetylhomoserine sulphydrylase (OAS/OAH) SHLase of Saccharomyces cerevisiae is affected by sulphur source added to the growth medium and genetic background of the strain. In a wild-type strain, the activity was repressed if methionine, cysteine or glutathione was added to the growth medium. However, in a strain deficient of cystathionine gamma-lyase, cysteine and glutathione were repressive, but methionine was not. In strains deficient of serine O-acetyltransferase (SATase), OAS/OAH SHLase activity was low regardless of sulphur source and was further lowered by cysteine and glutathione, but not by methionine. From these observations, we concluded that S-adenosylmethionine should be excluded from being the effector for regulation of OAS/OAH SHLase. Instead, we suspected that S. cerevisiae would have the same regulatory system as Escherichia coli for sulphate assimilation; i.e. cysteine inhibits SATase to lower the cellular concentration of OAS which is required for induction of the sulphate assimilation enzymes including OAS/OAH SHLase. Subsequently, we obtained data supporting this speculation.

Regulation of folate-dependent enzyme levels in Aspergillus nidulans: studies with regulatory mutants.

The synthesis of folate-dependent enzymes in Aspergillus nidulans appears to be regulated by intracellular pools of homocysteine and methionine. The results are consistent with the view that homocysteine acts as an inducer and methionine as a corepressor, but the molecular mechanism of the regulation is still unknown. Methionine-requiring mutants, metH2 and metD10, apparently allelic, show deregulation of folate-dependent enzymes. Most characteristic of the mutants is a repressed level of folylpolyglutamate synthetase. New mutations suppressing the metH2 lesion which render folate enzymes insensitive to methionine-mediated repression have been isolated. These mutations are likely to identify new regulatory genes in folate metabolism.

Sulfur amino acid metabolism in Schizosaccharomyces pombe: occurrence of two O-acetylhomoserine sulfhydrylases and the lack of the reverse transsulfuration pathway.

The fission yeast Schizosaccharomyces pombe has a unique organization of sulfur amino acid metabolism: it has two distinct O-acetylhomoserine sulfhydrylases (homocysteine synthases). Similar to Enterobacteriaceae, S. pombe lacks cystathionine beta-synthase and cystathionine gamma-lyase-the enzymes of the reverse transsulfuration pathway, by which methionine is readily metabolized to cysteine-a likely effector in the sulfur metabolite repression system. Consequently no repression of sulfate assimilation is observed when methionine is added to the growth medium.

Role of O-acetylhomoserine sulfhydrylase in sulfur amino acid synthesis in various yeasts.

Mutants defective in O-acetylhomoserine sulfhydrylase (OAH-SHLase) were obtained in five yeast strains representative of different yeast genera: Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe and Trichosporon cutaneum. In vitro, in all five strains, the enzyme also had O-acetylserine (OAS) sulfhydrylase activity so it is a 'bifunctional' OAH/OAS-SHLase (Yamagata, 1989). The enzyme was only found to be essential in S. cerevisiae (OAH SHLase-negative mutants are auxotrophs). Its impairment in K. lactis caused a slower growth rate and a decrease of the sulfur amino acid pool. In T. cutaneum only the pool was affected whereas in Y. lipolytica and S. pombe the lesion caused no change in the growth rate nor in the pool. In all strains where OAH SHLase-negative mutants were prototrophs, a monofunctional OAS sulhydrylase was detected. The results indicate that OAH SHLase may play different physiological roles in various yeasts.

At least four regulatory genes control sulphur metabolite repression in Aspergillus nidulans.

Mutations in four genes: sconA (formerly suA25meth, mapA25), sconB (formerly mapB1), sconC and sconD, the last two identified in this work, relieve a group of sulphur amino acid biosynthetic enzymes from methionine-mediated sulphur metabolite repression. Exogenous methionine has no effect on sulphate assimilation in the mutant strains, whereas in the wild type it causes almost complete elimination of sulphate incorporation. In both mutant and wild-type strains methionine is efficiently taken up and metabolized to S-adenosylmethionine, homocysteine and other compounds, scon mutants also show elevated levels of folate-metabolizing enzymes which results from the large pool of homocysteine found in these strains. The folate enzymes appear to be inducible by homocysteine and repressible by methionine (or S-adenosylmethionine).

Comparative studies on O-acetylhomoserine sulfhydrylase: physiological role and characterization of the Aspergillus nidulans enzyme.

O-acetylhomoserine sulfhydrylase (OAH SHLase) from Aspergillus nidulans is an oligomeric protein with a broad substrate specificity with regard to sulfhydryl compounds. As its Saccharomyces cerevisiae counterpart the enzyme also reacts with O-acetylserine and is inhibited by carbonyl reagents but not by antiserum raised against the yeast enzyme. In contrast to Saccharomyces cerevisiae the enzyme is not essential for Aspergillus nidulans as indicated by the completely prototrophic phenotype of OAH SHLase-negative mutants. Its major physiological role in Aspergillus nidulans seems to be recycling of the thiomethyl group of methylthio-adenosine but it is also a constituent of the alternative pathway of cysteine synthesis.

Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae: regulatory roles of methionine and S-adenosylmethionine reassessed.

cys4-1, a mutation in the reverse trans-sulphuration pathway, relieves the sulphate assimilation pathway and homocysteine synthase from methionine-mediated repression. Since the mutation blocks the synthesis of cysteine from methionine downstream from homocysteine, this indicates that neither methionine nor S-adenosylmethionine serve as low-molecular-mass effectors in this regulatory system, contradicting earlier hypotheses.

Generation of genetic recombinants in Trichosporon cutaneum by spontaneous segregation of protoplast fusants.

Auxotrophic mutants were isolated in two strains of Trichosporon cutaneum. Complementing auxotrophs were hybridized by protoplast fusion. Some of the fusants were apparently transient diploids and segregated to give recombinant marker combinations.