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

Abstract

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. While many key players involved in oxidative protein folding are known, our understanding of how redox homeostasis in the ER is maintained and how EROs, the Cys residues of nascent proteins, and the luminal glutathione redox buffer interact is limited. Here, we isolated viable ero1 ero2 double mutants largely deficient in ERO activity, which rendered the mutants highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER lumen in vivo, we expressed the glutathione redox potential (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress, as induced by acute exposure to dithiothreitol, was delayed in ero1 ero2 mutants. The characteristic signature of EGSH dynamics in the ER lumen triggered by hypoxia was affected in the ero1 ero2 mutant 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.One sentence summaryDynamic monitoring the ER luminal glutathione redox potential highlights the role of EROs in defining redox conditions and the interplay between different redox inputs during hypoxia and reductive stress.

Reductive stress triggers ANAC017-mediated retrograde signaling to safeguard the endoplasmic reticulum by boosting mitochondrial respiratory capacity

“…They were strongly induced and their induction was UPR ER -specific since it was impaired in the UPR ER signaling backgrounds inositol requiring enzyme 1a and b ( ire1a ire1b ) and basic leucine zipper 28 and 60 ( bzip28 bzip60 ; Chen and Brandizzi, 2012 ; Deng et al, 2013 ). (3) Since impairment of root growth is a hallmark of ER stress in plants ( Deng et al, 2013 ; Lai et al, 2018 ), we tested the effect of lowering the endogenous cellular oxidation capacity in the ER using a line compromised in both ERO isoforms ( ero1 ero2 ; Ugalde et al, 2021a) . The ero1 ero2 seedlings showed arrested root growth in the presence of DTT, while that of WT seedlings decreased only moderately (−31%).…”

“…a reductive shift of key redox systems such as NADH/NAD + . While mETC is particularly affected, also oxidative protein folding in the ER relies on oxygen, as mediated by ERO ( Meyer et al, 2019 ; Ugalde et al, 2021a ). Hence hypoxia has been associated with reductive ER stress, similarly to the impact of DTT that was used as a model here.…”

“…The Arabidopsis thaliana T-DNA insertion lines aox1a-1 (SALK_084897; Giraud et al, 2008) , aox1a-2 (SAIL_030_D08; Col-0 background; Giraud et al, 2008) , ucp1-1 (SAIL_536_G01 and the corresponding complemented lines, cUCP1#9 and cUCP#14 , Sweetlove et al, 2006 ), anac017-1 (SALK_022174; Ng et al, 2013a , 2013b ), the EMS mutant rao2.1 ( Ng et al, 2013a , 2013b ), ero1-3 (SALK_096805; Fan et al, 2019 ; Ugalde et al, 2021a ) and the UPR ER double mutants ire1a ire1b (SALK_132285, SALK_050203; Chen and Brandizzi, 2012 ) and bzip28 bzip60 (WiscDsLox420D09, SAIL_238_F07; Deng et al, 2013 ) were described previously and validated by genotyping and sequencing before usage. As part of the molecular validation of the ucp1-1 background we mapped the T-DNA insertion site more upstream than previously reported ( Sweetlove et al, 2006 ), that is, in the promoter region of the UCP1 locus ( Supplemental Figure S13A ).…”

“…Hence, ucp1-1 represents a knockdown, rather than a knockout line, which is consistent with the immunoblotting analysis for UCP1 protein abundance reported previously ( Sweetlove et al, 2006 ) as well as a recent report ( Arcuri et al, 2021 ). For ero1 ero2 , the T-DNA insertion line ero1-3 was transformed to express an amiRNA targeted against ERO2 (amiRNA hairpin; Ugalde et al, 2021a) . A homozygous ero1 aox1a-1 line was generated by crossing the corresponding homozygous single mutants followed by selection of homozygous individuals in the F2 generation by genotyping.…”