Here we show that the enzyme is required for proper folding of disulfide-containing proteins and has a direct effect on generating the oxidizing environment of the ER. We recently showed that the lone FMO in yeast (yFMO) can catalyze the O 2- and NADPH-dependent oxidation of a range of biological thiols, including GSH, cysteine, and cysteamine ( 12). It has also been suggested that flavin-containing monooxygenases (FMOs), known to oxidize certain thiols, may be involved in maintaining the cellular redox balance, perhaps through the disulfide exchange with cystamine ( 11). It has been suggested that there may be a preferential transport of GSSG from the cytoplasm to the ER, although the apparent K m for such transport, 9 mM, may be too high to be physiologically meaningful ( 2). Until now it has not been clear how the necessary oxidizing equivalents are generated or how the oxidizing environment of the ER is created and maintained. There are no data to suggest how ERO1 is itself oxidized or to suggest that it generates oxidizing potential.Ĭytoplasmic GSH is maintained in its reduced form through the action of the ubiquitous enzyme GSH reductase, which catalyzes the reaction: GSSG + NADPH + H + → 2 GSH + NADP +. ERO1 is thought to be part of the redox machinery of the ER and to help maintain its oxidizing potential. Deletion of the gene renders cells hypersensitive to exogenous DTT, and overexpression confers resistance to the reducing agent. It has recently been shown that the lumen of the ER contains a 65-kDa glycoprotein called ERO1, which is essential for oxidative folding of proteins with disulfide bonds ( 5, 10). For these purposes, the ER contains molecular chaperones ( 6, 7), peptidyl-proline isomerase ( 8), and protein disulfide isomerase ( 9). The ER is specialized for maturation of secretory and membrane proteins and is the site of disulfide-bond formation. It has also been shown that such mutants can properly fold, in the ER, disulfide-containing proteins, suggesting that although GSSG may be the principal form of oxidizing equivalents in the ER, it can be replaced by other chemicals ( 5). However, yeast mutants unable to synthesize GSH can grow if supplied with exogenous reducing agents such as GSH or DTT ( 4). This redox potential is similar to that shown in vitro to be optimal for refolding of proteins with disulfide bonds ( 3). The endoplasmic reticulum (ER) of eukaryotes is a more oxidizing environment, with a GSH/GSSG ratio of 1:1–3:1 and a total concentration likely to be ≈1 mM ( 2). The proper redox potential is buffered by glutathione (GSH) and glutathione disulfide (GSSG) at a ratio of about 100:1 and a total concentration ≈10 mM. The cytoplasm of eukaryotic cells is generally a reducing environment. Protein folding is essential for cell function and is a specialized and compartmentalized activity ( 1).
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