Marzena Sieńko

The Aspergillus nidulans metR gene encodes a bZIP protein which activates transcription of sulphur metabolism genes

The identification, isolation and characterization of a new Aspergillus nidulans positive‐acting gene metR, which encodes a transcriptional activator of sulphur metabolism, is reported. metR mutants are tight auxotrophs requiring methionine or homocysteine for growth. Mutations in the metR gene are epistatic to mutations in the negative‐acting sulphur regulatory scon genes. The metR coding sequence is interrupted by a single intron of 492 bp which is unusually long for fungi. Aspergillus nidulans METR is a member of bZIP family of DNA‐binding proteins. The bZIP domains of METR and the Neurospora crassa CYS3 transcriptional activator of sulphur genes are highly similar. Although Neurospora cys‐3 gene does not substitute for the metR function, a chimeric metR gene with a cys‐3 bZIP domain is able to transform the ΔmetR mutant to methionine prototrophy. This indicates that METR recognizes the same regulatory sequence as CYS3. The metR gene is not essential, as deletion mutants are viable and have similar phenotype as point mutants. In contrast to the Neurospora cys‐3, transcription of the metR gene was found to be regulated neither by METR protein nor by sulphur source. Transcription of metR gene is derepressed in the sconB2 mutant. Transcription of genes encoding sulphate permease, homocysteine synthase, cysteine synthase, ATP‐sulphurylase, and sulphur controller –sconB is strongly regulated by the metR gene product and depends on the character of the metR mutation and sulphur supplementation.

Sulphur amino acid synthesis in Schizosaccharomyces pombe represents a specific variant of sulphur metabolism in fungi

Schizosaccharomyces pombe, in contrast to Saccharomyces cerevisiae and Aspergillus nidulans, lacks cystathionine β‐synthase and cystathionine γ‐lyase, two enzymes in the pathway from methionine to cysteine. As a consequence, methionine cannot serve as an efficient sulphur source for the fungus and does not bring about repression of sulphur assimilation, which is under control of the cysteine‐mediated sulphur metabolite repression system. This system operates at the transcriptional level, as was shown for the homocysteine synthase encoding gene. Our results corroborate the growing evidence that cysteine is the major low‐molecular‐weight effector in the regulation of sulphur metabolism in bacteria, fungi and plants. The Sz. pombe homocysteine synthase gene sequence was submitted to GenBank under Accession No. AF012876. Copyright

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

Abstract The metG gene of Aspergillus nidulans encoding cystathionine β-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 β-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 β-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

Abstract 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.

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

Abstract 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.

Aspergillus nidulans genes encoding reverse transsulfuration enzymes belong to homocysteine regulon

Homocysteine is an intermediate in methionine synthesis in Aspergillus nidulans, but it can also be converted to cysteine by the reverse transsulfuration pathway involving cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL). Because homocysteine is toxic to the cell at high concentrations, this pathway also functions as a means of removal of its excess. We found that the transcription of the mecA and mecB genes encoding CBS and CGL was upregulated by excess of homocysteine as well as by shortage of cysteine. Homocysteine induced transcription of both genes when added to the growth medium or overproduced in a regulatory mutant. The derepressing effect of cysteine shortage was observed in some mutants and in the wild-type strain during sulfur starvation. An increase in the level of mecA or mecB transcript roughly parallel with the elevation of the respective enzyme activity. On the basis of the mode of mecA and mecB regulation by homocysteine, these genes may be classified in a group of genes upregulated directly or indirectly by this amino acid. We call this group of genes the “homocysteine regulon”.

Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress.

Human Nedd4 ubiquitin ligase, or its variants, inhibit yeast cell growth by disturbing the actin cytoskeleton organization and dynamics, and lead to an increase in levels of ubiquitinated proteins. In a screen for multicopy suppressors which rescue growth of yeast cells producing Nedd4 ligase with an inactive WW4 domain (Nedd4w4), we identified a fragment of ATG2 gene encoding part of the Atg2 core autophagy protein. Expression of the Atg2-C1 fragment (aa 1074-1447) improved growth, actin cytoskeleton organization, but did not significantly change the levels of ubiquitinated proteins in these cells. The GFP-Atg2-C1 protein in Nedd4w4-producing cells primarily localized to a single defined structure adjacent to the vacuole, surrounded by an actin filament ring, containing Hsp42 and Hsp104 chaperones. This localization was not affected in several atg deletion mutants, suggesting that it might be distinct from the phagophore assembly site (PAS). However, deletion of ATG18 encoding a phosphatidylinositol-3-phosphate (PI3P)-binding protein affected the morphology of the GFP-Atg2-C1 structure while deletion of ATG14 encoding a subunit of PI3 kinase suppressed toxicity of Nedd4w4 independently of GFP-Atg2-C1. Further analysis of the Atg2-C1 revealed that it contains an APT1 domain of previously uncharacterized function. Most importantly, we showed that this domain is able to bind phosphatidylinositol phosphates, especially PI3P, which is abundant in the PAS and endosomes. Together our results suggest that human Nedd4 ubiquitinates proteins in yeast and causes proteotoxic stress and, with some Atg proteins, leads to formation of a perivacuolar structure, which may be involved in sequestration, aggregation or degradation of proteins.

Regulatory mutations affecting sulfur metabolism induce environmental stress response in Aspergillus nidulans.

Mutations in the cysB, sconB and sconC genes affect sulfur metabolism in Aspergillus nidulans in different ways. The cysB mutation blocks synthesis of cysteine by the main pathway and leads to a shortage of this amino acid. The sconB and sconC mutations affect subunits of the SCF ubiquitin ligase complex, which inactivates the MetR transcription factor in the presence of an excess of cysteine. In effect, both cysB and scon mutations lead to permanent derepression of MetR-dependent genes. We compared transcriptomes of these three mutants with that of a wild type strain finding altered expression of a few hundred genes belonging to various functional categories. Besides those involved in sulfur metabolism, many up-regulated genes are related to stress responses including heat shock and osmotic stress. However, only the scon strains are more resistant to exogenous stress agents than the wild type strain while cysB is more sensitive. The two-component signal transduction system is a functional category, which is most enriched among genes up-regulated in the cysB, sconB and sconC mutants. A large group of up-regulated genes are involved in carbohydrate and energy metabolism, including genes coding for enzymes of trehalose and glycerol synthesis. The altered expression of these genes is accompanied by changes in sugar and polyol accumulation in conidia of the mutants. Genes encoding enzymes of the glyoxylate bypass and the GABA shunt are also up-regulated along with genes coding for enzymes of alcohol fermentation. Among the down-regulated genes the most numerous are those encoding membrane proteins and enzymes involved in secondary metabolism, including the penicillin biosynthesis cluster.

The budding yeast orthologue of Parkinson's disease-associated DJ-1 is a multi-stress response protein protecting cells against toxic glycolytic products

Saccharomyces cerevisiae Hsp31p is a DJ-1/ThiJ/PfpI family protein that was previously shown to be important for survival in the stationary phase of growth and under oxidative stress. Recently, it was identified as a chaperone or as glutathione-independent glyoxalase. To elucidate the role played by this protein in budding yeast cells, we investigated its involvement in the protection against diverse environmental stresses. Our study revealed that HSP31 gene expression is controlled by multiple transcription factors, including Yap1p, Cad1p, Msn2p, Msn4p, Haa1p and Hsf1p. These transcription factors mediate the HSP31 promoter responses to oxidative, osmotic and thermal stresses, to potentially toxic products of glycolysis, such as methylglyoxal and acetic acid, and to the diauxic shift. We also demonstrated that the absence of the HSP31 gene sensitizes cells to these stressors. Overproduction of Hsp31p and its homologue Hsp32p rescued the sensitivity of glo1Δ cells to methylglyoxal. Hsp31p also reversed the increased sensitivity of the ald6Δ strain to acetic acid. Since Hsp31p glyoxalase III coexists in S. cerevisiae cells with thousand-fold more potent glyoxalase I/II system, its biological purpose requires substantiation. We postulate that S. cerevisiae Hsp31p may have broader substrate specificity than previously proposed and is able to eliminate various toxic products of glycolysis. Alternatively, Hsp31p might be effective under high concentration of exogenous methylglyoxal present in some natural environmental niches populated by budding yeast, when glyoxalase I/II system capacity is saturated.