ERO1-independent production of H(2)O(2) within the endoplasmic reticulum fuels Prdx4-mediated oxidative protein folding

The endoplasmic reticulum (ER)–localized peroxiredoxin 4 (PRDX4) supports disulfide bond formation in eukaryotic cells lacking endoplasmic reticulum oxidase 1 (ERO1). The source of peroxide that fuels PRDX4-mediated disulfide bond formation has remained a mystery, because ERO1 is believed to be a major producer of hydrogen peroxide (H(2)O(2)) in the ER lumen. We report on a simple kinetic technique to track H(2)O(2) equilibration between cellular compartments, suggesting that the ER is relatively isolated from cytosolic or mitochondrial H(2)O(2) pools. Furthermore, expression of an ER-adapted catalase to degrade lumenal H(2)O(2) attenuated PRDX4-mediated disulfide bond formation in cells lacking ERO1, whereas depletion of H(2)O(2) in the cytosol or mitochondria had no similar effect. ER catalase did not effect the slow residual disulfide bond formation in cells lacking both ERO1 and PRDX4. These observations point to exploitation of a hitherto unrecognized lumenal source of H(2)O(2) by PRDX4 and a parallel slow H(2)O(2)-independent pathway for disulfide formation.