Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification

Abstract

Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC1 and GRXC2 being better reductants than GRXC3 and GRXC4 with bis(2-hydroxyethyl) disulfide and oxidized roGFP2 as substrates. Vice versa, GRXC3 and GRXC4 are better oxidants of reduced roGFP2 in the reverse reaction. Analysis of electrostatic surface potentials mirrors the phylogenetic classification of class I GRXs but cannot fully account for the observed kinetic differences in their interaction with roGPF2. Despite localization of two class I GRXs each in the cytosol and the endomembrane system, the respective double null mutants are viable without obvious phenotypes.

Diverse INOSITOL PHOSPHORYLCERAMIDE SYNTHASE mutant alleles of Physcomitrium patens offer new insight into complex sphingolipid metabolism

“…roGFP2 can be used as a topology sensor due to the strong glutathione redox difference between the cytosol (Fig. 9e) and endomembrane lumen (Brach et al, 2009;Schl€ oßer et al, 2024) (Fig. 9f).…”

Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge

“…In addition, we found a trend for increased total GSH content, which would according to the Nernst equation suggest more negative E GSH , although HPLC measurements do not allow for subcellular resolution. A shift of roGFP2 thiol/disulfide redox state to more oxidized values usually reveals an oxidative shift in E GSH , as roGFP2 specificity for the glutathione/GRX system was tested in vitro and in vivo [ 29 , 41 , 65 , 75 ]. Particularly, inefficient reduction of GSSG caused by absence of GR leads to an increase of OxD roGFP2 in the same subcellular compartment [ 39 , 40 , 45 ].…”

Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge

“…In addition, we found a trend for increased total GSH content, which would according to the Nernst equation suggest more negative E GSH , although HPLC measurements do not allow for subcellular resolution. A shift of roGFP2 thiol/disulfide redox state to more oxidized values usually reveals an oxidative shift in E GSH , as roGFP2 specificity for the glutathione/GRX system was tested in vitro and in vivo (Meyer et al, 2007; Begas et al, 2017; Trnka et al, 2020; Schlößer et al, 2023). Thus, inefficient reduction of GSSG caused by absence of glutathione reductase (GR) leads to an increase of OxD roGFP2 in the same subcellular compartment (Marty et al, 2009; Marty et al, 2019; Müller-Schüssele et al, 2020).…”

“…Partial redundancy between the GSH/GRX and TRX redox systems was also demonstrated in plants, regarding the cytosol and mitochondrial matrix (Marty et al, 2009), but not for the plastid stroma (Marty et al, 2019). Regarding mutants lacking all class I GRX of one compartment, double null mutants of A. thaliana lacking cytosolic GRXC1 and GRXC2 were originally described to be lethal (Riondet et al, 2012) but have recently been reported to be viable and with a WT-like phenotype (Schlößer et al, 2023). In the secretory pathway, which lacks a GR and thus possesses a less reducing E GSH , double mutants of GRXC3/GRXC4 grow normally (Schlößer et al, 2023) but showed decreased hypocotyl length compared to WT when grown in elevated temperature (Dard et al, 2022).…”

“…Regarding mutants lacking all class I GRX of one compartment, double null mutants of A. thaliana lacking cytosolic GRXC1 and GRXC2 were originally described to be lethal (Riondet et al, 2012) but have recently been reported to be viable and with a WT-like phenotype (Schlößer et al, 2023). In the secretory pathway, which lacks a GR and thus possesses a less reducing E GSH , double mutants of GRXC3/GRXC4 grow normally (Schlößer et al, 2023) but showed decreased hypocotyl length compared to WT when grown in elevated temperature (Dard et al, 2022). Regarding plastid-targeted class I GRX, no null mutants were described yet.…”