Dataset Information

The Mammalian Cytosolic Thioredoxin Reductase Pathway Acts via a Membrane Protein to Reduce ER-localised Proteins

ABSTRACT: Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfide bonds. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is critical 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 lumen. These results provide compelling evidence for the critical role of the cytosol in regulating ER redox homeostasis to ensure correct protein folding and to facilitate the degradation of misfolded ER proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Sergio Lilla

LAB HEAD: Sara Rossana Zanivan

PROVIDER: PXD017923 | Pride | 2020-04-09

REPOSITORIES: Pride

ACCESS DATA

Publications

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

Cao Xiaofei X Lilla Sergio S Cao Zhenbo Z Pringle Marie Anne MA Oka Ojore B V OBV Robinson Philip J PJ Szmaja Tomasz T van Lith Marcel M Zanivan Sara S Bulleid Neil J NJ

Journal of cell science 20200430 8

Folding 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 prot ...[more]

PMID: 32184267

Similar Datasets

Project description:In Arabidopsis thaliana the evolutionary conserved N-terminal acetyltransferase (Nat) complexes NatA and NatB co-translationally acetylate 60% of the proteome. Both have recently been implicated in the regulation of plant stress responses. While NatA mediates drought tolerance, NatB is required for pathogen resistance and the adaptation to high salinity and high osmolarity. Salt and osmotic stress impair protein folding and result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER-membrane resident E3 ubiquitin ligase DOA10 targets misfolded proteins for degradation during ER stress and is conserved among eukaryotes. In yeast, DOA10 recognizes conditional degradation signals (Ac/N-degrons) created by NatA and NatB. Assuming that this mechanism is preserved in plants, the lack of Ac/N-degrons required for efficient removal of misfolded proteins might explain the sensitivity of NatB mutants to protein harming conditions. In this study, we investigate the response of NatB mutants to dithiothreitol (DTT) and tunicamycin (TM) induced ER stress. We report that NatB mutants are hypersensitive to DTT but not TM, suggesting that the DTT hypersensitivity is caused by an over-reduction of the cytosol rather than an accumulation of unfolded proteins in the ER. In line with this hypothesis, the cytosol of NatB depleted plants is constitutively over-reduced and a global transcriptome analysis reveals that their reductive stress response is permanently activated. Moreover, we demonstrate that doa10 mutants are susceptible to neither DTT nor TM, ruling out a substantial role of DOA10 in ER-associated protein degradation (ERAD) in plants. Contrary to previous findings in yeast, our data indicates that N-terminal acetylation (NTA) does not inhibit ER targeting of a substantial amount of proteins in plants. In summary, we provide further evidence that NatB-mediated imprinting of the proteome is vital for the response to protein-harming stress and rule out DOA10 as the sole recognin for substrates in the plant ERAD pathway.

Dataset Information

The Mammalian Cytosolic Thioredoxin Reductase Pathway Acts via a Membrane Protein to Reduce ER-localised Proteins

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure