Investigation of the relationship between oxidative stress and glucose signaling in Schizosaccharomyces pombe.

The invertase mutant defective in the glucose signaling pathway of Schizosaccharomyces pombe (ird11) is resistant to glucose repression. This mutant is able to consume sucrose alongside glucose and grows in glucose-containing media with a generation time close to that of the wild type. Intracellular oxidation, protein carbonyl, and reduced glutathione levels and catalase, superoxide dismutase, and glutathione peroxidase activity were investigated in ird11, to determine the relationship between oxidative stress response and glucose signaling. The expression profiles of some genes involved in regulation of glucose repression (fbp1, fructose-1,6-bis-phosphatase; hxk2, hexokinase) and stress response (atf1 and pap1 transcription factors; ctt1, catalase; sod1, Cu,Zn superoxide dismutase) were analyzed using the quantitative real-time PCR technique. Oxidative stress response in ird11 seems to be affected by glucose signaling in a manner different from that caused by glucose deprivation.

Intracellular scavenging activity of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) in the fission yeast, Schizosaccharomyces pombe.

The ability of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), a water-soluble vitamin E analogue, to prevent oxidative damages is well characterized, but the mechanisms underlying it remain unclear. The protective effect of Trolox pre-treatment on H(2)O(2)-induced toxicity might be attributed to the decreased cellular permeability to H(2)O(2) or in vitro scavenging activity of Trolox, induction of antioxidant enzymes or the direct scavenging activity of Trolox. The results obtained rule out the first and second possibilities and intracellular scavenging activity was found to be the mechanism whereby Trolox confers protection. This was confirmed by measuring protein oxidation (levels), and the observed decrease in proteasomal activity indicated that the decrease in protein carbonyls was due to Trolox scavenging activity rather than proteasome activation. In conclusion, the intracellular scavenging activity of Trolox is a key protective mechanism against H(2)O(2). These findings obtained in Schizosaccharomyces pombe, a good model organism for eukaryotic cells, can be used as standard protocols for investigating the antioxidant activity of pure or complex potential antioxidants.

Nitric oxide as a signaling molecule in the fission yeast Schizosaccharomyces pombe.

Nitric oxide synthases (NOS) catalyze the synthesis of ubiquitous signaling molecule nitric oxide (NO) which controls numerous biological processes. Using a spectrofluorometric NOS assay, we have measured the rate of total NO production in the crude cell extracts of Schizosaccharomyces pombe. NO production was reduced in the absence of NOS cofactors calmodulin and tetrahydrobiopterin, and a competitive NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was able to cause a statistically significant inhibition on the rate of total NO production. These results, for the first time, provide evidence that an enzyme with a NOS-like activity may be present in the fission yeast. In order to assess the possible regulatory roles of NO as a signaling molecule in this yeast, using the differential display technique, we screened for NO-responsive genes whose expression decreased upon exposure to L-NAME and increased in response to an NO donor, sodium nitroprusside treatment. Differential expression patterns of byr1, pek1, sid1, and wis1 genes were confirmed by quantitative real-time PCR. The physiological experiments performed based on the functions and molecular interactions of these genes have pointed to the possibility that NO production might be required for sporulation in S. pombe. Taken together, these findings suggest that NO may function as a signaling molecule which can induce both transcriptional and physiological changes in the fission yeast. Hence, these data also imply that S. pombe can be used as a model system for investigating the mechanisms underlying NO-related complex signaling pathways.

Nickel resistance in fission yeast associated with the magnesium transport system.

We isolated and characterized a nickel (Ni2+)-resistant mutant (GA1) of Schizosaccharomyces pombe. This mutant strain displayed resistance to both Ni2+ and Zn2+, but not to Cd2+, Co2+, and Cu2+. The growth rate of GA1 increased proportionally with increasing Mg2+ concentrations until 50 mM Mg2+. The GA1 mutation phenotype suggests a defect in Mg2+ uptake. Sequence analysis of the GA1 open reading frame (ORF) O13779, which is homologous to the prokaryotic and eukaryotic CorA Mg2+ transport systems, revealed a point mutation at codon 153 (ccc to acc) resulting in a Pro153Thr substitution in the N-terminus of the CorA domain. Our results provide novel genetic information about Ni2+ resistance in fission yeast. Specifically, that reducing Mg2+ influx through the CorA Mg2+ transport membrane protein confers Ni2+ resistance in S. pombe. We also report that Ni2+ ion detoxification of the fission yeast is related to histidine metabolism and pH.

The 3' terminal sequence of the inosine monophosphate dehydrogenase gene encodes an active domain in the yeast Schizosaccharomyces pombe

The gua1 gene encoding inosine monophosphate dehydrogenase (IMPDH), which catalyses the first step in de novo biosynthesis of guanosine monophosphate (GMP), was cloned in the yeast Schizosaccharomyces pombe by functional complementation of a gua1ura4-D18 mutant strain from a S. pombe DNA genomic library. Complementation analysis revealed a 1.2 kb fragment which segregation analysis confirmed did not code for a suppressor gene. Only 446 nucleotides of the gua1 gene encoding the IMPDH C-terminal residues were found within this 1.2 kb sequence (GenBank, AJ293460). The comparison of this wild-type fragment with the same fragment from the gua1ura4-D18 mutant revealed that there was a point mutation at position 1261 (guanine -> adenine) from the 5' end, corresponding to the amino acid residue 421 (glycine -> serine) of the enzyme. Dot and Northern analyses showed that the gua1 gene was expressed in transformants as well as in the wild-type and the gua1ura4-D18 mutant, but enzyme activity was only detected in wild-type and transformant cells. It seems likely that a 446 bp fragment from the 3' end of the gua1 gene abolished the point mutation in the mutant strain, suggesting that this fragment participates in the sequences encoding the active domain of IMPDH in S. pombe.

Isolation and characterization of glucose derepressed invertase mutants from Schizosaccharomyces pombe.

We have isolated 14 different Schizosaccharomyces pombe mutants that synthesize invertase enzyme constitutively. Analyses of invertase activities revealed that the degrees of resistance to glucose repression were not similar among different complementation groups. One of the complementation groups appeared to be associated with functional and/or regulatory defects in hexose transport. Another complementation group appeared to be specific for the regulation of the inv1 gene alone, implying that these mutations might be associated with different genes acting on the glucose sensing and signaling pathway. In addition, we found that the wild-type level glucose uptake is essential for the full-level repression of inv1 expression.