Comprehensive survey of proteins targeted by chloroplast thioredoxin

Abstract

Possible target proteins of chloroplast thioredoxin (Trx) have been investigated in the stroma lysate of spinach chloroplasts. For that purpose, we immobilized a mutant of m-type Trx in which an internal cysteine at the active site was substituted with serine, on cyanogen bromide-activated resin. By using this resin, the target proteins in chloroplast were efficiently acquired when they formed the mixed-disulfide intermediates with the immobilized Trxs. We could acquire Rubisco activase (45 kDa) and 2-Cys-type peroxiredoxin (Prx), which were recently identified as targets of chloroplast Trxs. Glyceraldehyde-3-phosphate dehydrogenase and sedoheputulose 1,7-bisphosphatase, well-known thiol enzymes in the Calvin cycle, also were recognized among the collected proteins, suggesting the method is applicable for our purpose. Furthermore, four proteins were identified from a homology search of the NH(2)-terminal sequence of the acquired proteins: glutamine synthetase, a protein homologous to chloroplast cyclophilin, a homolog of Prx-Q, and the Rubisco small subunit. The Trx susceptibilities of the recombinant cyclophilin and Prx-Q of Arabidopsis thaliana were then examined. The method developed in the present study is thus applicable to investigate the various redox networks via Trxs and the related enzymes in the cell.

Figure 1

Model of the process to capture the target proteins by Trx mutant. The internal cystein at the active site of Trx was substituted with serine and immobilized on the resin. The mixed-disulfide intermediate should be broken by the addition of DTT, thus eluting the target proteins.

Figure 2

Elution of proteins from the Trx-immobilized resin with DTT. The target proteins in the chloroplast stroma lysate were initially retained on the resin and subsequently eluted by the addition of DTT. Each of the protein bands was assigned by NH₂-terminal sequencing. White triangles show the proteins for which an NH₂-terminal sequence was determined and assigned in the database. Black triangles show the mutant Trx-m that was released from resin. The bars on the right side of the gel indicate the positions of the molecular markers. The protein bands shown with gray triangles remained unassigned based on their NH₂-terminal sequences. SBPase, sedoheptulose 1,7-bis phosphatase; PPIase, peptide-propyl cis-trans isomerase.

Figure 3

(A–C) Reduction levels of Prx-Q_At under the redox conditions were determined by AMS labeling. The purified Prx-Q_At was in the oxidized form. A total of 3 μM of Prx-Q_At was incubated with 50 μM CuCl₂ or the indicated concentration of DTT in the absence (A) or presence of 4 μM (B) or 0.04 μM (C) Trx-m. Then the reduction level of Prx-Q_At was determined by the incorporation of AMS and the change of the mobility during SDS/PAGE. (D) Trx-m dependent peroxidase activity of Prx-Q_At was measured with H₂O₂ as a substrate and NADPH as a reductant. The decrease in the absorbance at 340 nm was monitored (trace e). In traces a–c, Trx-m, Trx reductase, or Prx-Q_At was omitted from the reaction mixture, respectively. In trace d, Trx-m_C41S was used instead of Trx-m.

Figure 4

Reduction levels of ChCyP_At under the redox conditions were determined by AMS labeling. The oxidized form ChCyP_At (3 μM) was incubated with 50 μM CuCl₂ or the indicated concentration of DTT in the absence (A) or presence of 3 μM (B) Trx-m. Then the reduction level of ChCyP_At was determined by the incorporation of AMS and the change of the mobility in SDS/PAGE.