Stewart E J, Aslund F, Beckwith J
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
EMBO J. 1998 Oct 1;17(19):5543-50. doi: 10.1093/emboj/17.19.5543.
Cytoplasmic proteins do not generally contain structural disulfide bonds, although certain cytoplasmic enzymes form such bonds as part of their catalytic cycles. The disulfide bonds in these latter enzymes are reduced in Escherichia coli by two systems; the thioredoxin pathway and the glutathione/glutaredoxin pathway. However, structural disulfide bonds can form in proteins in the cytoplasm when the gene (trxB) for the enzyme thioredoxin reductase is inactivated by mutation. This disulfide bond formation can be detected by assessing the state of the normally periplasmic enzyme alkaline phosphatase (AP) when it is localized to the cytoplasm. Here we show that the formation of disulfide bonds in cytoplasmic AP in the trxB mutant is dependent on the presence of two thioredoxins in the cell, thioredoxins 1 and 2, the products of the genes trxA and trxC, respectively. Our evidence supports a model in which the oxidized forms of these thioredoxins directly catalyze disulfide bond formation in cytoplasmic AP, a reversal of their normal role. In addition, we show that the recently discovered thioredoxin 2 can perform many of the roles of thioredoxin 1 in vivo, and thus is able to reduce certain essential cytoplasmic enzymes. Our results suggest that the three most effective cytoplasmic disulfide-reducing proteins are thioredoxin 1, thioredoxin 2 and glutaredoxin 1; expression of any one of these is sufficient to support aerobic growth. Our results help to explain how the reducing environment in the cytoplasm is maintained so that disulfide bonds do not normally occur.
细胞质蛋白一般不含有结构性二硫键，不过某些细胞质酶在其催化循环过程中会形成此类键。在大肠杆菌中，后一类酶中的二硫键可通过两个系统还原：硫氧还蛋白途径和谷胱甘肽/谷氧还蛋白途径。然而，当硫氧还蛋白还原酶基因（trxB）因突变而失活时，细胞质中的蛋白质会形成结构性二硫键。当通常位于周质的碱性磷酸酶（AP）定位于细胞质时，可通过评估其状态来检测这种二硫键的形成。在此我们表明，trxB突变体细胞质AP中二硫键的形成取决于细胞中两种硫氧还蛋白的存在，即硫氧还蛋白1和硫氧还蛋白2，它们分别是trxA和trxC基因的产物。我们的证据支持一种模型，即这些硫氧还蛋白的氧化形式直接催化细胞质AP中的二硫键形成，这与其正常作用相反。此外，我们表明最近发现的硫氧还蛋白2在体内能够发挥硫氧还蛋白1的许多作用，因此能够还原某些必需的细胞质酶。我们的结果表明，三种最有效的细胞质二硫键还原蛋白是硫氧还蛋白1、硫氧还蛋白2和谷氧还蛋白1；其中任何一种的表达都足以支持有氧生长。我们的结果有助于解释细胞质中的还原环境是如何维持的，从而使二硫键通常不会形成。
