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- bis phosphatase, 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.

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B₁₂-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-α and 60-β, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.

Mitochondrial alterations are detected in most neurodegenerative disorders and may contribute to the dysfunction and demise of neuronal cells. Because glycogen synthase kinase-3 (GSK-3) is considered to be a critical factor in regulating neuronal cell survival and death, we studied the effects of modulating GSK-3 activity in cultured neurons treated with the mitochondrial inhibitor, rotenone. Interestingly, chronic inhibition of GSK-3 protects against rotenone-induced apoptosis in cultured neuronal cells. In an attempt to elucidate the molecular mechanisms underlying this neuroprotection, we demonstrated that chronic inhibition of GSK-3 reprograms the metabolism of neuronal cells, leading to an enhancement of glycolysis. This effect was accompanied by the induction and accumulation of hexokinase II (HKII) in the mitochondria. Interfering with either the mitochondrial binding of HKII or HKII expression significantly diminished the neuroprotection evoked by GSK-3 inhibition, and importantly, HKII overexpression is sufficient to protect against rotenone-induced cell death. Thus, mitochondrial HKII is a promoter of neuronal survival under the regulation of GSK-3. Furthermore, the neuroprotective effect of HKII may be relevant to neurodegenerative diseases in which glucose hypometabolism and mitochondrial dysfunction are prominent features.

Significance: A fundamental challenge for the plant life cycle is how to manage fluctuating light conditions. The thiol-based redox regulation in chloroplasts is a significant system for controlling chloroplast functions in response to light signals. Although chloroplasts likely possess a complex redox network supported by multiple redox-mediator proteins and target enzymes, the whole organization and biological significance have not been fully resolved. We performed biochemical and reverse-genetic studies and concluded that (i) two major redox systems via the ferredoxin-thioredoxin reductase/thioredoxin and NADPH-thioredoxin reductase C pathways differentially but cooperatively drive chloroplast redox regulation and (ii) their concerted action is critical for plant autotrophic growth. The regulatory circuits presented in this work contribute to the understanding of how plants survive in continuously changing environments.

Insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway, which takes place in plant chloroplasts. ATP hydrolysis by the CHLI subunit of magnesium chelatase is an essential component of this reaction, and the activity of this enzyme is a primary determinant of the rate of magnesium insertion into the chlorophyll molecule (tetrapyrrole ring). Higher plant CHLI contains highly conserved cysteine residues and was recently identified as a candidate protein in a proteomic screen of thioredoxin target proteins (Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., and Buchanan, B. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 370–375). To study the thioredoxin-dependent regulation of magnesium chelatase, we first investigated the effect of thioredoxin on the ATPase activity of CHLI1, a major isoform of CHLI in Arabidopsis thaliana. The ATPase activity of recombinant CHLI1 was found to be fully inactivated by oxidation and easily recovered by thioredoxin-assisted reduction, suggesting that CHLI1 is a target protein of thioredoxin. Moreover, we identified one crucial disulfide bond located in the C-terminal helical domain of CHLI1 protein, which may regulate the binding of the nucleotide to the N-terminal catalytic domain. The redox state of CHLI was also found to alter in a light-dependent manner in vivo. Moreover, we successfully observed stimulation of the magnesium chelatase activity in isolated chloroplasts by reduction. Our findings strongly suggest that chlorophyll biosynthesis is subject to chloroplast biogenesis regulation networks to coordinate them with the photosynthetic pathways in chloroplasts.