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A second protein disulfide isomerase plays a protective role against nitrosative and nutritional stresses in Schizosaccharomyces pombe

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Abstract

In the present work, a second gene encoding protein disulfide isomerase (PDI2) was cloned and characterized from Schizosaccharomyces pombe, and its regulation was studied. The structural gene encoding PDI2 was amplified from the genomic DNA using PCR, and ligated into the E. coli-yeast shuttle vector pRS316 to generate the recombinant plasmid pYPDI2. The determined DNA sequence carries 2,578 bp and is able to encode a protein of 726 amino acid sequence with CGAC at the putative active site. The fission yeast cells harboring pYPDI2 contained 1.62- and 2.73-fold higher PDI activity than the control yeast cells in exponential and stationary phases, respectively, indicating that the cloned gene is in vivo functioning. The PDI2 mRNA levels in both vector control and pYPDI2-containing yeast cells were found to be significantly higher in the stationary phase than in the exponential phase, suggesting that expression of the PDI2 gene is under stationary control. The yeast cells harboring pYPDI2 showed enhanced survival on minimal media plates containing nitric oxide (NO)-generating sodium nitroprusside (SNP) and no nitrogen. The synthesis of β-galactosidase from the PDI2-lacZ fusion gene was markedly enhanced in the Pap1-positive KP1 cells by SNP and nitrogen starvation. However, the enhancement in the synthesis of β-galactosidase from the PDI2-lacZ fusion gene by SNP and nitrogen starvation appeared to be relatively reduced in the Pap1-negative TP108-3C cells than in the Pap1-positive KP1 cells. The PDI2 mRNA level was elevated by SNP and nitrogen starvation in the Pap1-positive cells but not in the Pap1-negative cells. In brief, the S. pombe PDI2 plays a protective role against nitrosative and nutritional stresses, and is positively regulated by NO and nitrogen starvation in a Pap1-dependent manner.

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Abbreviations

NO:

Nitric oxide

ONPG:

ο-Nitrophenyl-β-d-galactopyranoside

PCR:

Polymerase chain reaction

PDI:

Protein disulfide isomerase

RT–PCR:

Reverse transcriptase-polymerase chain reaction

SNP:

Sodium nitroprusside

S. pombe :

Schizosaccharomyces pombe

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Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0072536). We wish to acknowledge Ms Hee Jin for her technical assistance.

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Authors and Affiliations

  1. Division of Life Sciences and Research Institute of Life Sciences, College of Natural Sciences, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon, 200-701, Korea

    Eun-Hye Lee & Chang-Jin Lim

  2. Department of Life Science, Ewha Womans University, Seoul, 120-750, Korea

    Dong-Hoon Hyun

  3. College of Pharmacy, Sookmyung Women’s University, 52 Hyochangwon-gil, Seoul, 140-742, Korea

    Eun-Hee Park

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  1. Eun-Hye Lee
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  2. Dong-Hoon Hyun
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  4. Chang-Jin Lim
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Corresponding author

Correspondence to Chang-Jin Lim.

Additional information

The sequence reported in this paper has been deposited in the GenBank database with the accession number GQ507432.

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Lee, EH., Hyun, DH., Park, EH. et al. A second protein disulfide isomerase plays a protective role against nitrosative and nutritional stresses in Schizosaccharomyces pombe . Mol Biol Rep 37, 3663–3671 (2010). https://doi.org/10.1007/s11033-010-0018-1

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