Journal of Microbiology

Research Support, Non-U.S. Gov't

The Role and Regulation of Trx1, a Cytosolic Thioredoxin in Schizosaccharomyces pombe: Ji-Yoon Song , Jung-Hye Roe; J. Microbiol. 2008;46(4):408-414. Published online August 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0076-4

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The genome of fission yeast Schizosaccharomyces pombe harbors two genes for thioredoxins, trx1+ and trx2+, which encode cytosolic and mitochondrial thioredoxins, respectively. The Δtrx1 mutant was found sensitive to diverse external stressors such as various oxidants, heat, and salt, whereas Δtrx2 mutant was not sensitive except to paraquat, a superoxide generator. Both Δtrx1 and Δtrx2 mutants were more resistant to diamide, a thiol-specific oxidant, than the wild type. The trx1+ gene expression was induced by H2O2 and menadione, being mediated through a stress-responsive transcription factor Pap1. In Δtrx1 cells, the basal expression of Pap1-regulated genes were elevated, suggesting a role for Trx1 as a reducer for oxidized (activated) Pap1. The Δtrx1 mutant exhibited cysteine auxotrophy, which can be overcome by adding sulfite. This suggests that Trx1 serves as a primary electron donor for 3’-phosphoadenosine-5’-phosphosulfate (PAPS) reductase and thus is an essential protein for sulfur assimilation in S. pombe. These results suggest that, in contrast to Trx2 whose role is more confined to mitochondrial functions, Trx1 plays a major role in protecting S. pombe against various stressful conditions and enables proper sulfur metabolism.

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Journal Article

Carbon Source-Dependent Regulation of the Schizosaccharomyces pombe pbh1 Gene: Su-Jung Kim , Nam-Chul Cho , In Wang Ryu , Kyunghoon Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2006;44(6):689-693.; DOI: https://doi.org/2454 [pii]

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Abstract: Pbh1, from the fission yeast Schizosaccharomyces pombe, is a baculoviral inhibitor of apoptosis (IAP) repeat (BIR) domain-containing protein. Its unique encoding gene was previously found to be regulated by nitric oxide and nitrogen starvation. In the current work, the Pbh1-lacZ fusion gene was used to elucidate the transcriptional regulation of the pbh1 gene under various carbon sources. When fermentable carbon sources, such as glucose (at a low concentration of 0.2%), sucrose (2.0%) and lactose (2.0%), were the sole carbon source, the synthesis of β-galactosidase from the Pbh1-lacZ fusion gene was reasonably enhanced. However, the induction by these fermentable carbon sources was abolished in the Pap1-negative S. pombe cells, implying that this type of induction of the pbh1 gene is mediated by Pap1. Ethanol (2.0%), a nonfermentable carbon source, was also able to enhance the synthesis of β-galactosidase from the fusion gene in wild-type cells but not in Pap1-negative cells. The results indicate that the S. pombe pbh1 gene is up-regulated under metabolic oxidative stress in a Pap1-dependent manner.

Research Support, Non-U.S. Gov'ts

Transcriptional Analysis and Pap1-Dependence of the Unique Gene Encoding Thioredoxin Reductase from the Fission Yeast: Hyun-Jung Kang , Sung-Min Hong , Byung-Chul Kim , Kyunghoon Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2006;44(1):35-41.; DOI: https://doi.org/2339 [pii]

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Abstract: The unique gene encoding thioredoxin reductase (TrxR) was previously cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its expression was induced by oxidative stress. To elucidate the regulatory mechanism of the S. pombe TrxR gene, three fusion plasmids were generated using polymerase chain reaction: pYUTR20, pYUTR30, and pYUTR40. Plasmid pYUTR20 has an upstream region of 891 base pairs, pYUTR30 has 499 in this region, and pYUTR40 has an 186 bp upstream region. Negatively acting sequence is located between ‒1,526 ~ ‒891 bp upstream of the gene. The upstream sequence, responsible for the induction of TrxR by menadione (MD), is situated on the ‒499 ~ ‒186 bp region, which is also required for TrxR induction by mercuric chloride. The same region also appeared to be required for Pap1-mediated transcriptional regulation of the TrxR gene, which contains the two plausible Pap1 binding sites, TTACGAAT and TTACGCGA. Consistently, basal and inducible expression of the TrxR gene was markedly lower in the Pap1-negative TP108-3C cells than in wild-type yeast cells. In summary, up-regulation of the S. pombe TrxR gene is mediated by Pap1 via the transcriptional motif( s) located on the ‒499 ~ ‒186 bp region.

The Schizosaccharomyces pombe Gene Encoding [gamma]-Glutamyl Transpeptidase I Is Regulated by Non-fermentable Carbon Sources and Nitrogen Starvation: Hong-Gyum Kim , Hey-Jung Park , Hyun-Jung Kang , Hye-Won Lim , Kyunghoon Kim , Eun-Hee Park , Kisup Ahn , Chang-Jin Lim; J. Microbiol. 2005;43(1):44-48.; DOI: https://doi.org/2139 [pii]

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Abstract: In our previous study, the first structural gene (GGTI) encoding g-glutamyl transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of [beta]-galactosidase from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.

Transcriptional Regulation of the Schizosaccharomyces pombe Gene Encoding Glutathione S-Transferase I by a Transcription Factor Pap1: Hong-Gyum Kim , Byung-Chul Kim , Kyunghoon Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2004;42(4):353-356.; DOI: https://doi.org/2099 [pii]

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Abstract: In a previous study, a gst gene was isolated from the fission yeast Schizosaccharomyces pombe. This gene was dubbed gst I, and was characterized using the gstI-lacZ fusion plasmid pYSH2000. In this work, four additional fusion plasmids, pYSHSD1, pYSHSD2, pYSHSD3 and pYSHSD4, were constructed, in order to carry (respectively) 770, 551, 358 and 151 bp upstream regions from the translational initiation point. The sequence responsible for induction by aluminum, mercury and hydrogen peroxide was located in the range between -1,088 and -770 bp upstream of the S. pombe gst I gene. The same region was identified to contain the nucleotide sequence responsible for regulation by Pap1, and has one putative Pap1 binding site, TTACGTAT, located in the range between -954 ~ -947 bp upstream of the gst I gene. Negatively acting sequences are located between -1,088 and -151 bp. These findings imply that the Pap1 protein is involved in basal and inducible transcription of the gst I gene in the fission yeast S. pombe.

Transcriptional Regulation of the Gene Encoding g-Glutamylcysteine Synthetase from the Fission Yeast Schizosaccharomyces pombe: Su-Jung Kim , Hong-Gyum Kim , Byung-Chul Kim , Kyunghoon Kim , Eun-Hee Park , Chang-Jin Lim; J. Microbiol. 2004;42(3):233-238.; DOI: https://doi.org/2083 [pii]

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Abstract: Transcriptional regulation of the Schizosaccharomyces pombe [gamma]-glutamylcysteine synthetase (GCS) gene was examined using the two GCS-lacZ fusion plasmids pUGCS101 and pUGCS102, which harbor 607 bp and 447 bp upstream regions, respectively. The negatively-acting sequence was located in the -607 ~ -447 bp upstream region of the GCS gene. The upstream sequence responsible for induction by menadione (MD) and L-buthionine-(S, R)-sulfoximine (BSO) resides in the -607 ~ -447 bp region, whereas the sequence which codes for nitric oxide induction is located within the -447 bp region, measured from the translational initiation point. Carbon source-dependent regulation of the GCS gene appeared to be dependent on the nucleotide sequence within -447 bp region. The transcription factor Pap1 is involved in the induction of the GCS gene by MD and BSO, but not by nitric oxide. Induction of the GCS gene occurring due to low glucose concentration does not depend on the presence of Pap1. These data imply that induction by MD and BSO may be mediated by the Pap1 binding site, probably located in the -607 ~ -447 region, and also that the nitric oxide-mediated regulation of the S. pombe GCS gene may share a similar mechanism with its carbon-dependent induction.

Nitrogen Depletion Causes Up-Regulation of Glutathione Content and γ-Glutamyltranspeptidase in Schizosaccharomyces pombe: Seung-Hyun Song , Chang-Jin Lim; J. Microbiol. 2008;46(1):70-74.; DOI: https://doi.org/10.1007/s12275-007-0244-y

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Abstract: This work aims to elucidate the relationship between nitrogen depletion and Glutathione (GSH) level in Schizosaccharomyces pombe. The total GSH level was much higher in the Pap1-positive KP1 cells than in the Pap1-negative TP108-3C cells, suggesting that synthesis of GSH is dependent on Pap1. When the Pap1-positive KP1 cells were transferred to the nitrogen-depleted medium, total GSH level significantly increased up to 6 h and then slightly declined after 9 h. Elevation of the total GSH level was observed to be much less with the Pap1-negative cells. However, glucose deprivation was not able to enhance the GSH level in the KP1 cells. Activity of γ-glutamyltranspeptidase (γ-GT), an enzyme in the first step of GSH catabolism, also increased during nitrogen depletion. The total GSH level was more significantly enhanced in the KP1 cells overexpressing γ-GT2 than γ-GT1 during nitrogen starvation. Reactive oxygen species (ROS) levels were not changed during nitrogen starvation in both Pap1-positive and Pap1-negative cells. Collectively, nitrogen depletion causes up-regulation of GSH synthesis and γ-GT in a Pap1-dependent manner.

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