The mammalian cytosolic thioredoxin reductase pathway acts via a membrane protein to reduce ER-localised proteins.

Xiaofei Cao,Philip J Robinson,Tomasz Szmaja,Sergio Lilla,Ojore B V Oka,Sara Zanivan,Neil J Bulleid,Zhenbo Cao,Marie Anne Pringle,Marcel van Lith

doi:10.1242/jcs.241976

Abstract

ABSTRACTFolding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is crucial for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the crucial role of the cytosol in regulating ER redox homeostasis, ensuring correct protein folding and facilitating the degradation of misfolded ER proteins.

Highlights

Proteins entering the secretory pathway undergo several modifications that are unique to the ER including glycosylation and disulfide formation (Braakman & Bulleid, 2011)
We recently showed that the regeneration of NADPH within the cytosol is required to ensure correct disulfide formation in the ER lumen (Poet, Oka et al, 2017) raising the possibility that the cytosolic reductive pathways are responsible for ensuring correct disulfide formation in the ER lumen
Our previous work provided the first indication that a cytosolic reductive pathway was required to ensure correct disulfide formation within the ER (Poet et al, 2017)

Summary

Introduction

Proteins entering the secretory pathway undergo several modifications that are unique to the ER including glycosylation and disulfide formation (Braakman & Bulleid, 2011). The consequence of these modifications is often increased stability of the protein fold in preparation for secretion into the extracellular milieu. PDI exchanges its disulfide with substrate proteins during and following their translocation into the ER lumen (Chen, Helenius et al, 1995) During this process, disulfides may form that are not present in the final native structure (Jansens, van Duijn et al, 2002). Disulfides may form that are not present in the final native structure (Jansens, van Duijn et al, 2002) Such non-native disulfides need to be removed to allow correct folding or to facilitate protein degradation (Ushioda, Hoseki et al, 2008). In addition to the reduction of structural disulfides, there is a requirement for a reductive pathway to recycle enzymes such as methionine sulfoxide reductase (Cao, Mitchell et al, 2018), vitamin K epoxide reductase (Rishavy, Usubalieva et al, 2011), peroxiredoxin IV (Tavender, Springate et al, 2010) and formyl glycine generating enzyme (Dierks, Dickmanns et al, 2005) that contain active site thiols which become oxidised during catalysis

Methods

Results

Conclusion