Abstract
Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.
Highlights
The formation of disulfides within the mammalian endoplasmic reticulum (ER) is a highly orchestrated process that occurs during the folding and assembly of proteins entering the secretory pathway (Braakman & Bulleid, 2011)
This result was unexpected as protein disulfides do not normally form within the cytosol of mammalian cells and indicated that the reducing capacity of the reticulocyte cytosol extract had become compromised during its preparation
The results from this work demonstrate that the cytosolic reductive pathway, cytosolic TrxR1, is required for the reduction in non-native disulfides within proteins entering the ER
Summary
The formation of disulfides within the mammalian endoplasmic reticulum (ER) is a highly orchestrated process that occurs during the folding and assembly of proteins entering the secretory pathway (Braakman & Bulleid, 2011). The introduction of disulfides is catalyzed by a family of disulfide exchange proteins typified by protein disulfide isomerase (PDI; Ellgaard & Ruddock, 2005). The disulfide within their active sites is exchanged with free thiols within a folding polypeptide chain as it enters the lumen of the ER. The disulfide formed within the folding polypeptide may be the same as that found in the native state, but can form between two thiols not linked in the final structure (Jansens et al, 2002). In parallel to an oxidative pathway, a reductive pathway must exist in the ER to ensure correct protein folding and allow dislocation of proteins for degradation
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