a b s t r a c tThe identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.
Using a Saccharomyces cerevisiae strain having the activities of serine O-acetyl-transferase (SATase), O-acetylserine/ O-acetylhomoserine sulphydrylase (OAS/OAH SHLase), cystathionine-synthase (-CTSase) and cystathionine-lyase (-CTLase), we individually disrupted CYS3 (coding for-CTLase) and CYS4 (coding for-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-CTSase and-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.
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.
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.
Schizosaccharomyces pombe, in contrast to Saccharomyces cerevisiae and Aspergillus nidulans, lacks cystathionine beta-synthase and cystathionine gamma-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.
Sulfate uptake, the first step of sulfate assimilation in all organisms, is a highly endoergic, ATP requiring process. It is under tight control at the transcriptional level and is additionally modulated by posttranslational modifications, which are not yet fully characterized. Sulfate anion is taken up into the cell by specific transporters, named sulfate permeases, located in the cell membrane. Bacterial sulfate permeases differ significantly from the eukaryotic transporters in their evolutionary origins, structure and subunit composition. This review focuses on the diversity and regulation of sulfate permeases in various groups of organisms.
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