Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins.
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.
Stewart
E J
EJ
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Aslund
F
F
Beckwith
J
J
D000469:Alkaline Phosphatase / Q000378:metabolism*; D002451:Cell Compartmentation; D003593:Cytoplasm / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D013880:Thioredoxin-Disulfide Reductase / Q000172:deficiency / Q000235:genetics; D013879:Thioredoxins / Q000378:metabolism*
D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C491202:trxC protein, E coli; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D000469:Alkaline Phosphatase