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

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 in Arabidopsis thaliana, 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. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild-type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2-hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre-reduced roGFP2 in the reverse reaction.

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 (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 induced by dithiothreitol was delayed in ero1 ero2. The characteristic signature of EGSH 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.

Visualizing Orientation and Topology of ER Membrane Proteins In Planta.

The orientation of membrane proteins within the lipid bilayer is key to understanding their molecular function. Similarly, the proper topology of multispanning membrane proteins is crucial for their function. Although bioinformatics tools can predict these parameters assessing the presence of hydrophobic protein domains sufficiently long to span the membrane and other structural features, the predictions from different algorithms are often inconsistent. Therefore, experimental analysis becomes mandatory. Redox-based topology analysis exploits the steep gradient in the glutathione redox potential (EGSH) across the ER membrane of about 80 mV to visualize the orientation of ER membrane proteins by fusing the EGSH biosensor roGFP2 to either the N- or the C-termini of the investigated protein sequence. Transient expression of these fusion proteins in tobacco leaves allows direct visualization of orientation and topology of ER membrane proteins in planta. The protocol outlined here is based on either a simple merge of the two excitation channels of roGFP2 or a colocalization analysis of the two channels and thus avoids ratiometric analysis of roGFP2 fluorescence.