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Toru Hisabori

Publications and source records attributed to Toru Hisabori.

At least 19 recordsLinked to original sources
PubMed2006

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.

Arabidopsis↗
PubMed2006

The regulator of the F1 motor: inhibition of rotation of cyanobacterial F1-ATPase by the epsilon subunit.

The chloroplast-type F(1) ATPase is the key enzyme of energy conversion in chloroplasts, and is regulated by the endogenous inhibitor epsilon, tightly bound ADP, the membrane potential and the redox state of the gamma subunit. In order to understand the molecular mechanism of epsilon inhibition, we constructed an expression system for the alpha(3)beta(3)gamma subcomplex in thermophilic cyanobacteria allowing thorough investigation of epsilon inhibition. epsilon Inhibition was found to be ATP-independent, and different to that observed for bacterial F(1)-ATPase. The role of the additional region on the gamma subunit of chloroplast-type F(1)-ATPase in epsilon inhibition was also determined. By single molecule rotation analysis, we succeeded in assigning the pausing angular position of gamma in epsilon inhibition, which was found to be identical to that observed for ATP hydrolysis, product release and ADP inhibition, but distinctly different from the waiting position for ATP binding. These results suggest that the epsilon subunit of chloroplast-type ATP synthase plays an important regulator for the rotary motor enzyme, thus preventing wasteful ATP hydrolysis.

Adenosine Triphosphate↗
PubMed2006

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 Cys(330) 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.

Arabidopsis↗
PubMed2006

In vitro reconstitution of monogalactosyldiacylglycerol (MGDG) synthase regulation by thioredoxin.

Monogalactosyldiacylglycerol (MGDG), a major membrane lipid of chloroplasts, is synthesized by MGDG synthase (MGD) localized in chloroplast envelope membranes. We investigated whether MGD activity is regulated in a redox-dependent manner using recombinant cucumber MGD overexpressed in Escherichia coli. We found that MGD activity is reversibly regulated by reduction and oxidation in vitro and that an intramolecular disulfide bond(s) is involved in MGD activation. Because thioredoxin efficiently reduced disulfide bonds to enhance MGD activity in vitro, MGD is potentially an envelope-bound thioredoxin target protein in higher plants.

Chloroplasts↗
PubMed2005

Thioredoxin affinity chromatography: a useful method for further understanding the thioredoxin network.

Thioredoxin affinity chromatography can be used to recognize the target proteins of thioredoxin or thioredoxin-related proteins in whole cells or certain cellular compartments. In the last couple of years, many potential target proteins have been identified from various organelles and organisms by this method. Based on the information on the target proteins provided by these studies, the complete thioredoxin-related redox networks can now be efficiently described.

Chloroplasts↗
PubMed2004

Anti-oxidative stress system in cyanobacteria. Significance of type II peroxiredoxin and the role of 1-Cys peroxiredoxin in Synechocystis sp. strain PCC 6803.

Two antioxidant proteins, SLL1621 and SLR1198, were captured in the cyanobacteria Synechocystis sp. PCC 6803 using thioredoxin affinity chromatography, which was first applied to the survey of thioredoxin target proteins in chloroplasts (Motohashi, K., Kondoh, A., Stumpp, M. T., and Hisabori, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11224-11229). They are annotated as AhpC/TSA family protein (SLL1621) and antioxidant protein (SLR1198) in CyanoBase (Nakamura, Y., Kaneko, T., Hirosawa, M., Miyajima, N., and Tabata, S. (1998) Nucleic Acids Res. 26, 63-67). Based on sequence homology analysis SLL1621 and SLR1198 are categorized into type II peroxiredoxin and 1-Cys type peroxiredoxin, respectively. In vitro interaction between SLL1621 and thioredoxin was confirmed using the recombinant proteins expressed in Escherichia coli. Furthermore, we found that SLL1621 shows remarkable glutathione-dependent peroxidase activity. Disruption of the sll1621 gene had a dramatic effect on the viability of the cyanobacterial cells even under weak light conditions (50 micromol.m(-2).s(-1)), suggesting this peroxiredoxin is essential for this cyanobacterium. In contrast, although the peroxidase activity of SLR1198 was scarcely detected, disruption of the gene, slr1198, certainly affected the growth rate of the cells. The results indicate the physiological significance of two different peroxiredoxins as an anti-oxidative stress system in cyanobacteria.

Bacterial Proteins↗
PubMed2004

Two cAMP receptor proteins with different biochemical properties in the filamentous cyanobacterium Anabaena sp. PCC 7120.

Two open reading frames (ORFs), alr0295 and alr2325, are found to encode putative cAMP receptor proteins (CRPs) in the genome of the filamentous cyanobacterium Anabaena sp. PCC 7120. These ORFs were named cAMP receptor protein-like gene A in Anabaena sp. PCC 7120 (ancrpA) and cAMP receptor protein-like gene B in Anabaena sp. PCC 7120 (ancrpB), respectively, and those translated products were investigated. The equilibrium dialysis measurements revealed that AnCrpA bound with cAMP specifically, while AnCrpB bound with both cAMP and cGMP, though the affinity for cGMP was weak. The binding affinity for cAMP of AnCrpA showed the lowest dissociation constant, approximately 0.8 microM, among bacterial CRPs. A gel mobility shift assay elucidated that AnCrpA and AnCrpB formed a complex with the consensus DNA sequence in the presence of cAMP, although AnCrpB did not have ordinary DNA-binding motifs.

Amino Acid Sequence↗
PubMed2004

Significance of the epsilon subunit in the thiol modulation of chloroplast ATP synthase.

To understand the regulatory function of the gamma and epsilon subunits of chloroplast ATP synthase in the membrane integrated complex, we constructed a chimeric FoF1 complex of thermophilic bacteria. When a part of the chloroplast F1 gamma subunit was introduced into the bacterial FoF1 complex, the inverted membrane vesicles with this chimeric FoF1 did not exhibit the redox sensitive ATP hydrolysis activity, which is a common property of the chloroplast ATP synthase. However, when the whole part or the C-terminal alpha-helices region of the epsilon subunit was substituted with the corresponding region from CF1-epsilon together with the mutation of gamma, the redox regulation property emerged. In contrast, ATP synthesis activity did not become redox sensitive even if both the regulatory region of CF1-gamma and the entire epsilon subunit from CF1 were introduced. These results provide important features for the regulation of FoF1 by these subunits: (1) the interaction between gamma and epsilon is important for the redox regulation of FoF1 complex by the gamma subunit, and (2) a certain structural matching between these regulatory subunits and the catalytic core of the enzyme must be required to confer the complete redox regulation mechanism to the bacterial FoF1. In addition, a structural requirement for the redox regulation of ATP hydrolysis activity might be different from that for the ATP synthesis activity.

Adenosine Triphosphate↗
PubMed2004

Inverse regulation of rotation of F1-ATPase by the mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase.

In F1-ATPase, the rotation of the central axis subunit gamma relative to the surrounding alpha3beta3 subunits is coupled to ATP hydrolysis. We previously reported that the introduced regulatory region of the gamma subunit of chloroplast F1-ATPase can modulate rotation of the gamma subunit of the thermophilic bacterial F1-ATPase (Bald, D., Noji, H., Yoshida, M., Hirono-Hara, Y., and Hisabori, T. (2001) J. Biol. Chem. 276, 39505-39507). The attenuated enzyme activity of this chimeric enzyme under oxidizing conditions was characterized by frequent and long pauses of rotation of gamma. In this study, we report an inverse regulation of the gamma subunit rotation in the newly engineered F1-chimeric complex whose three negatively charged residues Glu210-Asp211-Glu212 adjacent to two cysteine residues of the regulatory region derived from chloroplast F1-ATPase gamma were deleted. ATP hydrolysis activity of the mutant complex was stimulated up to 2-fold by the formation of the disulfide bond at the regulatory region by oxidation. We successfully observed inverse redox switching of rotation of gamma using this mutant complex. The complex exhibited long and frequent pauses in its gamma rotation when reduced, but the rotation rates between pauses remained unaltered. Hence, the suppression or activation of the redox-sensitive F1-ATPase can be explained in terms of the change in the rotation behavior at a single molecule level. These results obtained by the single molecule analysis of the redox regulation provide further insights into the regulation mechanism of the rotary enzyme.

Amino Acid Sequence↗
PubMed2004

Complete inhibition and partial Re-activation of single F1-ATPase molecules by tentoxin: new properties of the re-activated enzyme.

During hydrolysis of ATP, the gamma subunit of the rotary motor protein F(1)-ATPase rotates within a ring of alpha(3)beta(3) subunits. Tentoxin is a phyto-pathogenic cyclic tetrapeptide, which influences F(1)-ATPase activity of sensitive species. At low concentrations, tentoxin inhibits ATP hydrolysis of ensembles of F(1) molecules in solution. At higher concentrations, however, ATP hydrolysis recovers. Here we have examined how tentoxin acts on individual molecules of engineered F(1)-ATPase from the thermophilic Bacillus PS3 (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119). We found that inhibition by tentoxin caused a virtually complete stop of rotation, which was partially relieved at higher tentoxin concentrations. Re-activation, however, was not simply a reversal of inhibition; while the torque appears unaffected as compared with the situation without tentoxin, F(1) under re-activating conditions was less susceptible to inhibitory ADP binding but displayed a large number of short pauses, indicating infringed energy conversion.

ATP Synthetase Complexes↗
PubMed2004

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana.

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 B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, 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.

Antioxidants↗
PubMed2004

VOZ; isolation and characterization of novel vascular plant transcription factors with a one-zinc finger from Arabidopsis thaliana.

A 38-bp pollen-specific cis-acting region of the AVP1 gene is involved in the expression of the Arabidopsis thaliana V-PPase during pollen development. Here, we report the isolation and structural characterization of AtVOZ1 and AtVOZ2, novel transcription factors that bind to the 38-bp cis-acting region of A. thaliana V-PPase gene, AVP1. AtVOZ1 and AtVOZ2 show 53% amino acid sequence similarity. Homologs of AtVOZ1 and AtVOZ2 are found in various vascular plants as well as a moss, Physcomitrella patens. Promoter-beta-glucuronidase reporter analysis shows that AtVOZ1 is specifically expressed in the phloem tissue and AtVOZ2 is strongly expressed in the root. In vivo transient effector-reporter analysis in A. thaliana suspension-cultured cells demonstrates that AtVOZ1 and AtVOZ2 function as transcriptional activators in the Arabidopsis cell. Two conserved regions termed Domain-A and Domain-B were identified from an alignment of AtVOZ proteins and their homologs of O. sativa and P. patens. AtVOZ2 binds as a dimer to the specific palindromic sequence, GCGTNx7ACGC, with Domain-B, which is comprised of a functional novel zinc coordinating motif and a conserved basic region. Domain-B is shown to function as both the DNA-binding and the dimerization domains of AtVOZ2. From highly the conservative nature among all identified VOZ proteins, we conclude that Domain-B is responsible for the DNA binding and dimerization of all VOZ-family proteins and designate it as the VOZ-domain.

Arabidopsis↗
PubMed2003

Molecular evolution of the modulator of chloroplast ATP synthase: origin of the conformational change dependent regulation.

Chloroplast ATP synthase synthesizes ATP by utilizing a proton gradient as an energy supply, which is generated by photosynthetic electron transport. The activity of the chloroplast ATP synthase is regulated in several specific ways to avoid futile hydrolysis of ATP under various physiological conditions. Several regulatory signals such as Delta mu H(+), tight binding of ADP and its release, thiol modulation, and inhibition by the intrinsic inhibitory subunit epsilon are sensed by this complex. In this review, we describe the function of two regulatory subunits, gamma and epsilon, of ATP synthase based on their possible conformational changes and discuss the evolutionary origin of these regulation systems.

Chloroplast Proton-Translocating ATPases↗
PubMed2003

Chloroplast cyclophilin is a target protein of thioredoxin. Thiol modulation of the peptidyl-prolyl cis-trans isomerase activity.

Chloroplast cyclophilin has been identified as a potential candidate of enzymes in chloroplasts that are regulated by thioredoxin (Motohashi, K., Kondoh, A., Stumpp, M. T., and Hisabori, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 11224-11229). In the present study we found that the peptidyl-prolyl cis-trans isomerase activity of cyclophilin is fully inactivated in the oxidized form. Reduction of cyclophilin by thioredoxin-m recovered the isomerase activity. Two crucial disulfide bonds were determined by disulfide-linked peptide mapping. The relevance of these cysteines for isomerase activity was confirmed by the mutagenesis studies. Because four cysteine residues in Arabidopsis thaliana cyclophilin were conserved in the isoforms from several organisms, it appears that this redox regulation must be one of the common regulation systems of cyclophilin.

Chloroplast Thioredoxins↗
PubMed2003

Conformational change of the chloroplast ATP synthase on the enzyme activation process detected by the trypsin sensitivity of the gamma subunit.

Delta mu H(+) is known to stimulate the enzyme activity of chloroplast ATP synthase in addition to its important role as energy supply for ATP synthesis. In the present study, we focused on the relationship between the proton translocation via the membrane sector of ATP synthase, F(o), and the conformational change of the central stalk subunit gamma. The conformational change of CF(1) mainly at the gamma subunit was induced by the proton flow via F(o) in the absence of substrates. The effects of inhibitors on CF(o) or CF(1) for this conformational change were also examined. The observed conformational change was partially suppressed by ADP binding. From these results, we propose the Delta mu H(+)-dependent conformational change of CF(1) on the enzyme activation process, which is affected by both ADP binding to the catalytic sites and proton flow via F(o) portion.

Adenosine Diphosphate↗