Pedrajas José R, Porras Pablo, Martínez-Galisteo Emilia, Padilla C Alicia, Miranda-Vizuete Antonio, Bárcena J Antonio
Departamento de Biología Experimental, Facultad de Ciencias Experimentales, University of Jaén, 23071-Jaén, Spain.
Biochem J. 2002 Jun 15;364(Pt 3):617-23. doi: 10.1042/BJ20020570.
Glutaredoxin (Grx)2 from Saccharomyces cerevisiae is a member of the two-cysteine (dithiol) subfamily of Grxs involved in the defence against oxidative stress in yeast. Recombinant yeast Grx2p, expressed in Escherichia coli, behaves as a 'classical' Grx that efficiently catalyses the reduction of hydroxyethyl disulphide by GSH. Grx2p also catalyses the reduction of GSSG by dihydrolipoamide with even higher efficiency. Western blot analysis of S. cerevisiae crude extracts identifies two isoforms of Grx2p of 15.9 and 11.9 kDa respectively. The levels of these two isoforms reach a peak during the exponential phase of growth in normal yeast extract/peptone/dextrose ('YPD') medium, with the long form predominating over the short one. From immunochemical analysis of subcellular fractions, it is shown that both isoforms are present in mitochondria, but only the short one is detected in the cytosolic fraction. On the other hand, only the long form is prominent in microsomes. Mitochondrial isoforms should represent the processed and unprocessed products of an open reading frame (YDR513W), with a putative start codon 99 bp upstream of the GRX2 start codon described thus far. These results indicate that GRX2 contains two in-frame start codons, and that translation from the first AUG results in a product that is targeted to mitochondria. The cytosolic form would result either by initiation from the second AUG, or by differential processing of one single translation product.
酿酒酵母中的谷氧还蛋白（Grx）2是参与酵母抗氧化应激防御的双半胱氨酸（二硫醇）亚家族Grx的成员。在大肠杆菌中表达的重组酵母Grx2p表现为一种“经典”的Grx，能有效地催化谷胱甘肽（GSH）还原羟乙基二硫化物。Grx2p还能更高效地催化二氢硫辛酰胺还原氧化型谷胱甘肽（GSSG）。对酿酒酵母粗提物的蛋白质印迹分析鉴定出分别为15.9 kDa和11.9 kDa的两种Grx2p同工型。在正常酵母提取物/蛋白胨/葡萄糖（“YPD”）培养基中生长的指数期，这两种同工型的水平达到峰值，其中长型占主导地位。通过对亚细胞组分的免疫化学分析表明，两种同工型都存在于线粒体中，但只有短型在胞质组分中被检测到。另一方面，只有长型在微粒体中较为突出。线粒体同工型应该代表一个开放阅读框（YDR513W）的加工和未加工产物，其推定的起始密码子位于迄今描述的GRX2起始密码子上游99 bp处。这些结果表明GRX2包含两个框内起始密码子，并且从第一个AUG开始翻译会产生一种靶向线粒体的产物。胞质形式可能是由第二个AUG起始翻译产生，或者是由单一翻译产物的差异加工产生。
