Endoplasmic reticulum oxidoreductin provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. Here, we isolated viable Arabidopsis thaliana ero1 ero2 double mutants that are highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER in vivo, we expressed the glutathione redox potential (E_GSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of −240 mV in the ER of Arabidopsis plants. We found E_GSH values of −241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal E_GSH was reduced further to −253 mV. Recovery to reductive ER stress induced by dithiothreitol was delayed in ero1 ero2. The characteristic signature of E_GSH dynamics in the ER lumen triggered by hypoxia was affected in ero1 ero2 reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.

Dynamic monitoring of the ER luminal glutathione redox potential highlights the role of ER oxidoreductins in defining redox conditions and the interplay between different redox inputs during hypoxia and reductive stress.

IN A NUTSHELL Background: Most secreted proteins contain disulfide bridges that are essential for their structure and function. Those disulfides are introduced into the nascent polypeptide through the oxidation of cysteines in the endoplasmic reticulum (ER) lumen. Oxidative protein folding requires molecular oxygen (O₂) as ultimate electron acceptor. The final electron transfer is catalyzed by thiol oxidases called ER oxidoreductins (EROs). Question: What is the role of EROs in maintaining ER redox homeostasis at steady state and when oxygen supply is limiting? Finding: Arabidopsis thaliana contains two ERO genes. An ero1 ero2 double mutant generated by combining a null allele for ERO1 with a knockdown allele for ERO2 showed enhanced sensitivity towards thiol-based reductive challenge and hypoxia. By monitoring the glutathione redox potential E_GSH in the ER lumen using the redox biosensor variant roGFP2iL we measured −241 mV in the wild-type, which is a less oxidizing value than previously thought. A good match between the midpoint potential of the biosensor variant and the physiological E_GSH in the ER lumen enabled dynamic measurements indicating ERO activity in vivo. Diminished ERO activity in ero1 ero2 caused a reductive shift to −253 mV and delayed recovery after reductive challenge. The dynamics of luminal E_GSH under hypoxia in ero1 ero2 differed from the response obtained in wild-type plants, indicating that ERO activity plays a key role in luminal redox homeostasis. Next steps: Monitoring luminal E_GSH represents a platform for evaluating ER redox dynamics and allows assessing other candidates for their potential contribution to oxidative protein folding and maintaining luminal redox homeostasis. Future research may focus on the integration of ER redox homeostasis and phytohormone signaling especially under stress situations or during developmental phases associated with hypoxic conditions.