Ken Motohashi

Comprehensive survey of proteins targeted by chloroplast thioredoxin

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 NH2-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.

HCF164 Receives Reducing Equivalents from Stromal Thioredoxin across the Thylakoid Membrane and Mediates Reduction of Target Proteins in the Thylakoid Lumen

HCF164 is a membrane-anchored thioredoxin-like protein known to be indispensable for assembly of cytochrome b6 f in the thylakoid membranes. In this study, we report the finding that chloroplast stroma m-type thioredoxin is the source of reducing equivalents for reduction of HCF164 in the thylakoid lumen, providing strong evidence that higher plant chloroplasts possess a trans-membrane reducing equivalent transfer system similar to that found in bacteria. To probe the function of HCF164 in the lumen, a screen to identify the reducing equivalent acceptor proteins of HCF164 was carried out by using a resin-immobilized HCF164 single cysteine mutant, leading to the isolation of putative target thylakoid proteins. Among the newly identified target proteins, the reduction of the PSI-N subunit of photosystem I by HCF164 was confirmed both in vitro and in isolated thylakoids. Two components of the cytochrome b6 f complex, the cytochrome f and Rieske FeS proteins, were also identified as novel potential target proteins. The data presented here suggest that HCF164 serves as an important transducer of reducing equivalents to proteins in the thylakoid lumen.

The CHLI1 Subunit of Arabidopsis thaliana Magnesium Chelatase Is a Target Protein of the Chloroplast Thioredoxin

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.

ε Subunit, an Endogenous Inhibitor of Bacterial F1-ATPase, Also Inhibits F0F1-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980)Biochemistry 19, 526–531), the ε subunit, an endogenous inhibitor of bacterial F1-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F0F1-ATPase. However, we report here that the ε subunit is exerting inhibition in F0F1-ATPase. We prepared a C-terminal half-truncated ε subunit (εΔC) of the thermophilicBacillus PS3 F0F1-ATPase and reconstituted F1- and F0F1-ATPase containing εΔC. Compared with F1- and F0F1-ATPase containing intact ε, those containing εΔC showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F0F1-ATPase containing εΔC was capable of ATP-driven H+ pumping. The time-course of pumping at low ATP concentration was faster than that by the F0F1-ATPase containing intact ε. Thus, the comparison with noninhibitory εΔC mutant shed light on the inhibitory role of the intact ε subunit in F0F1-ATPase.

A novel factor required for the assembly of the DnaK and DnaJ chaperones of Thermus thermophilus.

We previously reported the isolation of T.DnaK·DnaJ chaperone complex from Thermus thermophilus. Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex. A dnaK gene cluster of T. thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB. Interestingly, T.DnaJ lacks the whole “cysteine-rich region” that has been postulated to be necessary to bind unfolded proteins. The orf4 gene encodes a novel 78-amino acid protein. Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex. Careful reexamination of the T.DnaK·DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4. As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK·DnaJ complex in E. coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein. The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK·DnaJ assembly factor A). The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA. Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.

Identification of lysophospholipid receptors in human platelets: the relation of two agonists, lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1‐phosphate (Sph‐1‐P) are known as structurally related bio‐active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph‐1‐P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph‐1‐P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph‐1‐P, and high concentrations of Sph‐1‐P specifically desensitized LPA‐induced intracellular Ca2+ mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph‐1‐P receptors which might be expressed in human platelets, by RT‐PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets.

Systematic Exploration of Thioredoxin Target Proteins in Plant Mitochondria

The thioredoxin (Trx) system is known to play a pivotal role in cellular redox regulation, but its target proteins in plant mitochondria remain largely uncharacterized. In this study, we systemically screened Trx target candidates in plant mitochondria. Mitochondrial protein extracts were prepared from Arabidopsis shoots, spinach leaves and potato tubers, and then subfractionated into soluble matrix and insoluble membrane fractions. Protein extracts were loaded onto an affinity column immobilizing Arabidopsis mitochondria-localized o-type Trx mutant protein, in which one of two internal cysteines at the active site was substituted by serine. Proteins forming mixed-disulfide intermediates with the mutated Trx were identified by proteomic approaches. This procedure allowed the determination of 101 Trx target candidate proteins involved in a broad spectrum of mitochondrial processes. Furthermore, biochemical assay revealed that one of the potential Trx target proteins, alternative oxidase, is actually redox regulated by Trx. This study provides insights into the regulatory mechanism of diverse functions in mitochondrial biology that are mediated through the Trx system.

Towards a Functional Dissection of Thioredoxin Networks in Plant Cells

Thioredoxins are a ubiquitous family of redox equivalent mediators, long considered to possess a limited number of target enzymes. Recent progress in proteomic research has allowed the identification of a wide variety of candidate proteins with which this small protein may interact in vivo. Moreover, the activity of thioredoxin itself has been recently found to be subject to regulation by posttranslational modifications, adding an additional level of complexity to the function of this intriguing enzyme family. The current review charts the technical progress made in the continuing discovery of the numerous and diverse roles played by these proteins in the regulation of redox networks in plant cells.

Thioredoxin-h1 Reduces and Reactivates the Oxidized Cytosolic Malate Dehydrogenase Dimer in Higher Plants

Cytosolic malate dehydrogenase (cytMDH) was captured by thioredoxin affinity chromatography as a possible target protein of cytosolic thioredoxin (Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., and Hisabori, T. (2004) Plant Cell Physiol. 45, 18–27). To further dissect this interaction, we aimed to determine whether cytMDH can interact with the cytosolic thioredoxin and whether its activity is redox-regulated. We obtained the active recombinant cytMDH that could be oxidized and rendered inactive. Inactivation was reversed by incubation with low concentrations of dithiothreitol in the presence of recombinant Arabidopsis thaliana thioredoxin-h1. Inactivation of cytMDH was found to result from formation of a homodimer. By cysteine mutant analysis and peptide mapping analysis, we were able to determine that the cytMDH homodimer occurs by formation of a disulfide bond via the Cys330 residue. Moreover, we found this bond to be efficiently reduced by the reduced form of thioredoxin-h1. These results demonstrate that the oxidized form cytMDH dimer is a preferable target protein of the reduced form thioredoxin-h1 as suggested by thioredoxin affinity chromatography.

Regulation of Translation by the Redox State of Elongation Factor G in the Cyanobacterium Synechocystis sp. PCC 6803

Elongation factor G (EF-G), a key protein in translational elongation, was identified as a primary target of inactivation by reactive oxygen species within the translational machinery of the cyanobacterium Synechocystis sp. PCC 6803 (Kojima, K., Oshita, M., Nanjo, Y., Kasai, K., Tozawa, Y., Hayashi, H., and Nishiyama, Y. (2007) Mol. Microbiol. 65, 936–947). In the present study, we found that inactivation of EF-G (Slr1463) by H2O2 was attributable to the oxidation of two specific cysteine residues and formation of a disulfide bond. Substitution of these cysteine residues by serine residues protected EF-G from inactivation by H2O2 and allowed the EF-G to mediate translation in a translation system in vitro that had been prepared from Synechocystis. The disulfide bond in oxidized EF-G was reduced by thioredoxin, and the resultant reduced form of EF-G regained the activity to mediate translation in vitro. Western blotting analysis showed that levels of the oxidized form of EF-G increased under strong light in a mutant that lacked NADPH-thioredoxin reductase, indicating that EF-G is reduced by thioredoxin in vivo. These observations suggest that the translational machinery is regulated by the redox state of EF-G, which is oxidized by reactive oxygen species and reduced by thioredoxin, a transmitter of reducing signals generated by the photosynthetic transport of electrons.