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.
Mol Biol Rep. 2010 Dec;37(8):3663-71. doi: 10.1007/s11033-010-0018-1. Epub 2010 Mar 4.
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.
在本工作中，我们从裂殖酵母中克隆并鉴定了第二个编码蛋白二硫键异构酶（PDI2）的基因，并研究了其调控机制。使用 PCR 从基因组 DNA 中扩增编码 PDI2 的结构基因，并将其连接到大肠杆菌-酵母穿梭载体 pRS316 中，生成重组质粒 pYPDI2。确定的 DNA 序列携带 2578bp，能够编码一个由 726 个氨基酸序列组成的蛋白质，在假定的活性位点处含有 CGAC。在指数期和静止期，携带 pYPDI2 的裂殖酵母细胞中的 PDI2 活性分别比对照酵母细胞高 1.62 倍和 2.73 倍，表明克隆基因在体内发挥作用。在载体对照和含有 pYPDI2 的酵母细胞中，PDI2mRNA 的水平在静止期显著高于指数期，表明 PDI2 基因的表达受静止期调控。在含有 SNP 和无氮的最小培养基平板上，携带 pYPDI2 的酵母细胞的存活率增加。在 Pap1 阳性的 KP1 细胞中，来自 PDI2-lacZ 融合基因的β-半乳糖苷酶的合成通过 SNP 和氮饥饿显著增强。然而，在 SNP 和氮饥饿下，来自 PDI2-lacZ 融合基因的β-半乳糖苷酶的合成在 Pap1 阴性的 TP108-3C 细胞中比在 Pap1 阳性的 KP1 细胞中相对降低。SNP 和氮饥饿导致 Pap1 阳性细胞中的 PDI2mRNA 水平升高，但在 Pap1 阴性细胞中则没有。总之，裂殖酵母 PDI2 对硝化和营养胁迫具有保护作用，并且以依赖于 Pap1 的方式被 NO 和氮饥饿正向调节。
