The Quaternary Structure of NADPH Thioredoxin Reductase C Is Redox-Sensitive

Abstract

NADPH thioredoxin reductase C (NTRC) is a chloroplast enzyme able to conjugate NADPH thioredoxin reductase (NTR) and thioredoxin (TRX) activities for the efficient reduction of 2-Cys peroxiredoxin (2-Cys PRX). Because NADPH can be produced in chloroplasts during darkness, NTRC plays a key role for plant peroxide detoxification during the night. Here, it is shown that the quaternary structure of NTRC is highly dependent on its redox status. In vitro, most of the enzyme adopted an oligomeric state that disaggregated in dimers upon addition of NADPH, NADH, or DTT. Gel filtration and Western blot analysis of protein extracts from Arabidopsis chloroplast stroma showed that native NTRC forms aggregates, which are sensitive to NADPH and DTT, suggesting that the aggregation state might be a significant aspect of NTRC activity in vivo. Moreover, the enzyme is localized in clusters in Arabidopsis chloroplasts. NTRC triple and double mutants, A164G-V182E-R183F and A164G-R183F, replacing key residues of NADPH binding site, showed reduced activity but were still able to dimerize though with an increase in intermediary forms. Based on these results, we propose that the catalytically active form of NTRC is the dimer, which formation is induced by NADPH.

Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in vivo function of reductase and thioredoxin domains

“…We report here that the full-length NTRC polypeptides extracted from Arabidopsis leaves in the presence or absence of DTT ( Figure 9 ) exist as homodimers that are resistant to detergent treatment without heating, suggesting that the dimeric structure of chloroplast NTRC is stable without any disulphide bridges between the monomers ( Figure 8B ). This conclusion is further confirmed by the observation that the mutation of both Cys residues either in the NTRd or in the TRXd active site does not abolish the formation of NTRC homodimers ( Figure 9 ; Pérez-Ruiz et al, 2009). The high conservation rate of the aromatic amino acids and those involved in salt bridges in the inter-monomeric interface of the NTRC domains ( Figure 8B ), suggests that these residues are important for forming stable homodimers.…”

“…These studies indicate that NTRC has a strong tendency to form oligomeric aggregates but it is still technically difficult to prove, whether oligomeric NTRC is the dominant form of the enzyme in vivo . Pérez-Ruiz et al (2009) demonstrated that stromal fraction of chloroplasts contained oligomeric NTRC aggregates with different masses that mostly disaggregated into dimers and monomers in the presence of DTT. We also detected slight smearing of NTRC polypeptides in the SDS-gel, if leaf proteins were extracted in DTT-free buffers ( Figure 9 ).…”

“…Pérez-Ruiz et al (2009) demonstrated that rice recombinant NTRC protein or NTRC extracted from Arabidopsis chloroplasts form oligomeric aggregates that dissociate into the dimeric form in the presence of NADPH, whereas NADPH did not influence the dissociation of barley NTRC aggregates in vitro (Wulff et al, 2011). Chae et al (2013) reported a heat-shock induced aggregation of NTRC and that the oligomeric NTRC acts as a chaperone preventing stress-induced aggregation of chloroplast proteins.…”

“…In NTRC, a TRX module is fused to the C-terminus of a reductase domain. The protein contains two redox-active 2-Cys motifs, CAIC in its NTR domain (NTRd) and CGPC in the TRX domain (TRXd; Serrato et al, 2004), and it is suggested to function as a dimer (Pérez-Ruiz and Cejudo, 2009; Pérez-Ruiz et al, 2009; Lee et al, 2012). Characterization of knockout lines of NTRC ( ntrc ) has indicated that NTRC is a crucial redox-regulator of a number of plastidial processes, including biogenesis of chloroplasts, biosynthetic pathways and ROS metabolism in chloroplasts (Pérez-Ruiz et al, 2006; Stenbaek et al, 2008; Michalska et al, 2009; Lepistö et al, 2009, 2012; Pulido et al, 2010; Kirchsteiger et al, 2012; Chae et al, 2013; Richter et al, 2013).…”

Functional analysis of the pathways for 2-Cys peroxiredoxin reduction in Arabidopsis thaliana chloroplasts

“…In addition, transcript and metabolite profiling revealed altered contents of amino acids and auxin in the ntrc mutant, a phenotype which was more severe under short-day conditions (Lepistö et al , 2009). It is therefore likely that the activity of NTRC as a protein disulphide reductase (Pérez-Ruiz and Cejudo, 2009; Pérez-Ruiz et al , 2009) adjusts the redox status of several target proteins involved in different metabolic and/or signalling pathways, thus explaining the pleiotropic phenotype of the NTRC knock-out plant.…”