Identification and characterization of a mitochondrial thioredoxin system in plants

Abstract

Plants possess two well described thioredoxin systems: a cytoplasmic system including several thioredoxins and an NADPH-dependent thioredoxin reductase and a specific chloroplastic system characterized by a ferredoxin-dependent thioredoxin reductase. On the basis of biochemical activities, plants also are supposed to have a mitochondrial thioredoxin system as described in yeast and mammals, but no gene encoding plant mitochondrial thioredoxin or thioredoxin reductase has been identified yet. We report the characterization of a plant thioredoxin system located in mitochondria. Arabidopsis thaliana genome sequencing has revealed numerous thioredoxin genes among which we have identified AtTRX-o1, a gene encoding a thioredoxin with a potential mitochondrial transit peptide. AtTRX-o1 and a second gene, AtTRX-o2, define, on the basis of the sequence and intron positions, a new thioredoxin type up to now specific to plants. We also have characterized AtNTRA, a gene encoding a protein highly similar to the previously described cytosolic NADPH-dependent thioredoxin reductase AtNTRB but with a putative presequence for import into mitochondria. Western blot analysis of A. thaliana subcellular and submitochondrial fractions and in vitro import experiments show that AtTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their cleavable N-terminal signal. The two proteins truncated to the estimated mature forms were produced in Escherichia coli; AtTRX-o1 efficiently reduces insulin in the presence of DTT and is reduced efficiently by AtNTRA and NADPH. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.

Figure 1

Multiple sequence alignment of A. thaliana AtTRX-o1 (AAC12840) and AtTRX-o2 (AF396650) with different types of A. thaliana thioredoxins. The CLUSTALW software (npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html) was used to align AtTRX-h1 (P29448), AtTRX-f1 (Q9XFH8), AtTRX-m1 (O48737), and AtTRX-x (AAF15952). The arrows delimit protein regions encoded by different exons.

Figure 2

Phylogenetic tree of A. thaliana thioredoxins and plant thioredoxin o homologues. The DARWIN software was use to generate the phylogenetic tree of the following thioredoxins. A. thaliana thioredoxins h: AtTRX-h1 (P29448), AtTRX-h2 (S58123), AtTRX-h3 (S58118), AtTRX-h4 (S58119), AtTRX-h5 (S58120), AtTRX-h7 (AAD39316), AtTRX-h8 (AAG52561), and AtTRX-h9 (AAG51342). A. thaliana thioredoxins f: AtTRX-f1 (Q9XFH8) and AtTRX-f2 (Q9XFH9). A. thaliana thioredoxins m: AtTRX-m1 (O48737), AtTRX-m2 (AAF15949), AtTRX-m3 (AAF15950), and AtTRX-m4 (Q9SEU6). A. thaliana thioredoxin x: AtTRX-x (AAF15952). Plant thioredoxins o are translations of ESTs contigs: G. hirsutum GhTRX-o1 (GenBank ESTs AI725806 and AI729485); G. max GmTRX-o1 (AW423678, AW234806, BE210498, and AI941321); L. esculentum LeTRX-o1 (BE462179, AW037483, AW037392, and BE433326); Z. mays ZmTRX-o1 (BE345397, AI668273, BE519105, AI737410, and BE050128); O. sativa OsTRX-o1 (C27892 and AU100897).

Figure 3

Multiple sequence alignment of A. thaliana AtNTRA (AAB86519) and AtNTRB (CAB54874). Deduced amino acid sequences were aligned with the CLUSTALW program. Identical residues are indicated by black boxes, and similar residues are indicated by gray boxes.

Figure 4

Subcellular localization of AtTRX-o1 and AtNTRA in A. thaliana protoplasts. Western blot analyses of A. thaliana total protoplast (P), cytosol (Cy), mitochondrial (M), and chloroplast (C) fractions probed with antibodies directed against AtTRX-o1, AtNTRB, AtTRX-h3, NAD9, and the large subunit of the ribulose 1,5-bisphosphate carboxylase (RUBISCO-LSU) are shown.

Figure 5

In vitro protein import into isolated mitochondria. AtTRX-o1, AtTRX-o2 and AtNTRA were translated in vitro (lane 1) and incubated for 30 min at 25°C with potato mitochondria (lanes 2–5). Mitochondria were treated with valinomycin before import (lane 4) or with Triton X-100 (lane 5) and/or submitted to proteinase K digestion after import (lanes 3 and 5). Migration of the precursor (p) and mature (m) polypeptides is indicated.

Figure 6

Submitochondrial localization of AtTRX-o1 and AtNTRA. Western blot analysis of A. thaliana total mitochondria (M) and mitoplast (Mtp) fractions probed with anti-AtTRX-o1 and anti-AtNTRB sera is shown. Broken mitoplasts were centrifuged at 100,000 × g, giving rise to pellet (P) and supernatant (S) fractions. Western blots of the same protein fractions probed with antibodies against wheat NAD9 and tobacco Mn-SOD are shown on the bottom.

Figure 7

Activity of different A. thaliana thioredoxin systems determined by the insulin-disulfide reduction assay. (A) AtNTRA and (B) AtNTRB: ■, AtTRX-o1; ▴, AtTRX-o2; X, AtTRX-h3. (C) K_m values of AtNTRA and AtNTRB for the different recombinant thioredoxins at pH 7.0 and 20°C.