Biochemical insight into redox regulation of plastidial 3-phosphoglycerate dehydrogenase from Arabidopsis thaliana

Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redox-regulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography-based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins from Arabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under non-reducing conditions, which lowers PGDH enzyme activity. Mass spectrometry and site-directed mutagenesis analyses allowed us to identify the redox-active Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of and expand our understanding of the Trx-mediated redox-regulatory network in plants.


Introduction
To tune cellular physiology, a number of proteins in the cell undergo several post-translational modifications. Thiol-based redox regulation is one of such mechanisms; it controls enzyme activity by switching the oxidation/reduction states of Cys residues (e.g., formation/cleavage of disulfide bonds). A small ubiquitous protein thioredoxin (Trx) is largely responsible for the redox regulation. Trx contains the highly conserved amino acid sequence WCGPC at the active site. By using two Cys residues in this motif, Trx catalyzes a dithiol-disulfide exchange reaction with its target proteins, allowing modulation of their enzyme activities. Trx is thus critical for transmitting reducing power to redoxregulated proteins and adjusting cellular functions in response to changes in local redox environments (1,2).
Trx-mediated redox-regulatory system is ubiquitously found in all kingdoms of life.
Among them, the system in plant chloroplasts has attracted much attention due to its unique mode of action related to photosynthesis. Upon illumination, photochemical reactions are triggered in the thylakoid membrane, generating the reducing power. Trx receives a part of reducing power from ferredoxin (Fd) via Fd-Trx reductase (FTR), and in turn transfers it to several redox-regulated proteins. This redox cascade ensures light-responsive coordination of chloroplast functions, which has long been recognized as the molecular basis of the redoxregulatory system in chloroplasts (3,4).
Another characteristic of the chloroplast system is the emergence of multiple Trx subtypes, 3 categorized into f-, m-, x-, y-, and z-types (5,6).
They have different midpoint redox potentials and protein surface charges, conferring functional diversity to each of the Trx subtypes (e.g., distinct target selectivity) (7)(8)(9)(10)(11). In addition, other proteins serving as the mediator of reducing

PGDH physically interacts with Trx
We first investigated whether PGDH has a crosstalk with redox-regulatory factors, such as Trx. For this purpose, we applied the affinity chromatography-based screening method (25).
One of Arabidopsis plastidial Trx isoforms, Trx-f1, or Arabidopsis NTRC, each of which was prepared in the form of monocysteinic variant (13), was used as bait in this experiment.
Chloroplast soluble proteins extracted from spinach leaves were loaded onto a Trx-f1-or NTRC-immobilized affinity chromatography column. The proteins associated with Trx-f1 or NTRC via the mixed-disulfide bond were eluted by a reducing agent dithiothreitol (DTT). As shown in Fig. 2A (for Trx-f1) and B (for NTRC), different SDS-PAGE profiles of DTT-eluted proteins were evident between assays using Trx-f1 and NTRC, owing to their distinct target selectivity (13). Immunoblotting analyses indicated that PGDH was bound to Trx-f1, but not to NTRC (Fig. 2C, D). After eluted by DTT, the molecular weight of PGDH seems to be slightly varied (Fig. 2C) These results give an implication that all of PGDH isoforms in Arabidopsis are redoxregulated as the target of Trx. However, we cannot conclude the possibility for PGDH redox regulation at this stage, because it is known that affinity chromatography-based method often traps pseudo-target proteins of Trx during the incubation process (43). To verify this issue, it is necessary to clarify (i) Trx-dependent reduction, (ii) redox-dependent change in the activity, and (iii) Cys residues responsible for the redox regulation. Following studies were designed to address these points.
We prepared PGDH1-3 from Arabidopsis as the purified recombinant proteins (Fig. S1). We examined whether the redox state of PGDH is 5 variable or not (Fig. 3D). Redox shift assays using a thiol-modifying reagent indicated that PGDH1 mainly existed as two different redox states under control (DTT-free) conditions. PGDH1 was converted to a fully reduced state in the presence of DTT. It was thus suggested that PGDH1 can form at least one disulfide bond in the molecule. By contrast, the redox states of PGDH2 and PGDH3 were unaltered by DTT. We then examined the effects of DTT on PGDH enzyme activity (Fig. 3E). PGDH1 was largely activated by DTT, while PGDH2 and PGDH3 were not. Taken together, these results suggest that PGDH1 is a redox-sensitive protein whose activity is enhanced upon reduction, whereas PGDH2 and PGDH3 are not. To directly evaluate this expectation, we performed peptide mapping analysis based on the mass spectrometry. PGDH1 protein was in-gel digested using trypsin, and mass spectra of the resulting peptides were compared between control and DTT treatments. As expected, the overall mass spectra were apparently similar between the control and DTT-treated samples ( Fig. S4), but a few differences were observed. Alignment of PGDH from several photosynthetic 6 organisms shows that this Cys pair is largely conserved in land plants, but not in algae or cyanobacteria (Fig. 4A, Fig. S2).

Some Trx subtypes in plastids support PGDH1 reductive activation
We finally studied the involvement of Trx and NTRC in PGDH1 redox regulation ( Fig. 6A-D).

PGDH redox shift assay
For the reducing reaction, PGDH was reacted at             Statistical analyses were performed using the Tukey-Kramer multiple comparison test, but significant difference was not observed (denoted as same letters, P > 0.05).   Figure 3. Redox sensitivity of Arabidopsis PGDH. (A-C) Affinity chromatography-based test for binding of PGDH1 (A), PGDH2 (B), or PGDH3 (C) to Arabidopsis Trx-f1. Proteins extracted from each PGDHtransformed E. coli cells were loaded as input. Protein elution profiles were analyzed by SDS-PAGE, followed by silver staining. PGDH was detected by immunoblotting analysis using a PGDH antibody. (D) Redox shift assay of PGDH using the thiol-modifying reagent. Each PGDH (1.2 μM) was incubated with or without 10 mM DTT for 15 min. PGDH was then labeled with the maleimide-PEG 11 -biotin and loaded on non-reducing SDS-PAGE. Ox, oxidized form; Red, reduced form. (E) Enzyme activity measurement of PGDH. Each PGDH (1.2 μM) was incubated with or without 1 mM DTT for 15 min. PGDH activity was then monitored. Data are shown as the mean ± SD (n = 4). Statistical analyses were performed using the Student t-test. N.S., not significant.    Figure 5. Identification of redox-active Cys residues in Arabidopsis PGDH1. (A, B) Peptide mapping analysis based on mass spectrometry. Before the Cys alkylation and following in-gel digestion with trypsin, the protein sample was incubated in the absence (control) or presence of 10 mM DTT. Overall mass spectra are shown in Fig. S4. (C) Redox shift assay of PGDH1 wild type (WT) and Cys-substituted mutants (C102S and C86S/C102S) using the thiol-modifying reagent. Each PGDH1 was incubated with or without 10 mM DTT for 15 min. PGDH was then labeled with the methyl-PEG 24 -maleimide and loaded on non-reducing SDS-PAGE.