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

Abstract

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. 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. Malate dehydrogenase (MDH) 3The abbreviations used are: MDH, malate dehydrogenase; cytMDH, cytosolic MDH; cytMDHred, the reduced form of cytMDH; cytMDHox, the oxidized form of cytMDH; Trx, thioredoxin; DTT, dithiothreitol; HPLC, high pressure liquid chromatography. catalyzes the reversible reaction of oxaloacetate to malate utilizing the NAD+/NADH or NADP+/NADPH cofactor system and is known to occur ubiquitously in mammals, plants, and most prokaryotes. NAD-dependent MDHs (EC 1.1.1.37) generally exist as a homodimer with a subunit of 32–37 kDa (1Walk R.A. Hock B. Biochem. Biophys. Res. Commun. 1978; 81: 636-643Crossref PubMed Scopus (43) Google Scholar, 2Hock B. Gietl C. Ann. N. Y. Acad. Sci. 1982; 386: 350-376Crossref PubMed Scopus (17) Google Scholar), whereas NADP-dependent MDH (EC 1.1.1.82) possesses a larger subunit of ∼42 kDa (3Cretin C. Luchetta P. Joly C. Decottignies P. Lepiniec L. Gadal P. Sallantin M. Huet J.C. Pernollet J.C. Eur. J. Biochem. 1990; 192: 299-303Crossref PubMed Scopus (46) Google Scholar, 4Scheibe R. Kampfenkel K. Wessels R. Tripier D. Biochim. Biophys. Acta. 1991; 1076: 1-8Crossref PubMed Scopus (19) Google Scholar). In plants, MDH isoforms have been reported in the cytosol and in organelles and have been found to carry out important roles in a number of metabolic pathways (5Gietl C. Biochim. Biophys. Acta. 1992; 1100: 217-234Crossref PubMed Scopus (162) Google Scholar). In general, MDH proteins are grouped into five classes according to their location within the cell; (i) cytosolic NAD-dependent MDH (cytMDH); (ii) mitochondrial NAD-dependent MDH, which is part of the tricarboxylic acid cycle; (iii) glyoxisomal and peroxisomal NAD-dependent MDHs, which are involved in photorespiration; (iv) chloroplast NADP-dependent MDH, which is required for the transfer of reducing equivalents from chloroplast stroma to cytosol; and (v) chloroplast NAD-dependent MDH (6Scheibe R. Physiol. Plant. 2004; 120: 21-26Crossref PubMed Scopus (361) Google Scholar). Among the classes listed, chloroplast NADP-dependent MDH is a thiol enzyme that has been subject of intense study (4Scheibe R. Kampfenkel K. Wessels R. Tripier D. Biochim. Biophys. Acta. 1991; 1076: 1-8Crossref PubMed Scopus (19) Google Scholar, 7Scheibe R. Anderson L.E. Biochim. Biophys. Acta. 1981; 636: 58-64Crossref PubMed Scopus (109) Google Scholar, 8Crawford N.A. Droux M. Kosower N.S. Buchanan B.B. Arch. 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Trxs are an important class of regulatory proteins that modulate the activity of a wide variety of thiol-containing chloroplast enzymes by the reduction of specific disulfide bonds (15Buchanan B.B. Arch. Biochem. Biophys. 1991; 288: 1-9Crossref PubMed Scopus (335) Google Scholar). In the light, excess NADPH (produced by photosynthesis but surplus to CO2 fixation requirements) induces the conversion of oxaloacetate to malate, resulting in the regeneration of the electron acceptor NADP+. In chloroplasts, the activated form of MDH is responsible for this reaction. The resulting malate is translocated to the cytosol via the dicarboxylate transporter (16Taniguchi M. Taniguchi Y. Kawasaki M. Takeda S. Kato T. Sato S. Tabata S. Miyake H. Sugiyama T. Plant Cell Physiol. 2002; 43: 706-717Crossref PubMed Scopus (81) Google Scholar), where cytosolic MDH (cytMDH) converts malate back to oxaloacetate, with NAD+ acting as an oxidant to form NADH. This mechanism allows chloroplast reducing equivalents to be transferred to the cytosol, and the transport system described above is known as the “malate valve” (6Scheibe R. Physiol. Plant. 2004; 120: 21-26Crossref PubMed Scopus (361) Google Scholar). NADH accumulated in the cytosol is then used in the mitochondria for respiratory ATP generation or may alternatively be used for cytosolic nitrate reduction. Hence cytMDH functions as a key player in the transfer of reducing equivalents from the chloroplast to other destinations in plant cells. In the cytosol, a number of Trx-h isoforms have been reported, although the function of each of these Trx-h isoforms has not been determined very well (17Rivera-Madrid R. Mestres D. Marinho P. Jacquot J.P. Decottignies P. Miginiac-Maslow M. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5620-5624Crossref PubMed Scopus (113) Google Scholar, 18Brehelin C. Laloi C. Setterdahl A.T. Knaff D.B. Meyer Y. Photosynth. 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Therefore, determination of the target proteins of Trx is of significant importance in our understanding of the physiological roles of Trx-h as transducer of reducing equivalents. Recent enrichment of genome data bases has allowed the screening and molecular characterization of possible target proteins of Trx using a proteomic approach (22Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 23Yano H. Wong J.H. Lee Y.M. Cho M.J. Buchanan B.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4794-4799Crossref PubMed Scopus (205) Google Scholar, 24Balmer Y. Koller A. del Val G. Manieri W. Schurmann P. Buchanan B.B. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 370-375Crossref PubMed Scopus (354) Google Scholar, 25Lindahl M. Florencio F.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 16107-16112Crossref PubMed Scopus (140) Google Scholar, 26Marchand C. Le Marechal P. Meyer Y. Miginiac-Maslow M. Issakidis-Bourguet E. Decottignies P. Proteomics. 2004; 4: 2696-2706Crossref PubMed Scopus (169) Google Scholar, 27Lemaire S.D. Guillon B. Le Marechal P. Keryer E. Miginiac-Maslow M. Decottignies P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7475-7480Crossref PubMed Scopus (206) Google Scholar, 28Balmer Y. Vensel W.H. Tanaka C.K. Hurkman W.J. Gelhaye E. Rouhier N. Jacquot J.P. Manieri W. Schurmann P. Droux M. Buchanan B.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2642-2647Crossref PubMed Scopus (272) Google Scholar). We recently used Trx affinity chromatography (29Hisabori T. Hara S. Fujii T. Yamazaki D. Hosoya-Matsuda N. Motohashi K. J. Exp. Bot. 2005; 56: 1463-1468Crossref PubMed Scopus (50) Google Scholar) in an attempt to capture the potential cytosolic target proteins of Arabidopsis thaliana Trx-h and identified a number of proteins including anti-oxidative stress-related proteins, proteins involved in biosynthesis and degradation of proteins, and several metabolic enzymes (30Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (174) Google Scholar). Within these captured enzymes, we sought to verify and characterize Trx-dependent regulation of cytMDH in this study. Materials—The Bradford protein assay system was from BioRad. DTT was from Sigma. All of the other chemicals were of the highest grade commercially available. Preparation of Recombinant Trx-h1—Recombinant Trx-h1 was expressed in Escherichia coli using an expression plasmid and the Trx-h1 gene sequence from A. thaliana (30Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (174) Google Scholar), purified as described K. F. Y. H. Hisabori T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and at The Trx was determined by the assay Preparation of the for was from A. thaliana as described in M.T. Motohashi K. Hisabori T. Biochem. J. PubMed Scopus Google Scholar) and used as a for The for cytMDH was by using with the and The for the enzyme in are The was into the and of the expression and the were by and of resulting expression was into E. coli and were at was by the of 1 by further at for of the recombinant cytMDH, of the were out at The were in by and at for at The was then to a that been with and the proteins were with a of in The cytMDH were and was to a of This protein was to a and with The obtained the protein was and to using molecular The protein was determined by Bradford assay using as and of the Recombinant the recombinant cytMDH was obtained in the reduced form the protein was oxidized by incubation with for 1 at cytMDH was to to the in was for 1 at with DTT in the presence or of the of cytMDH was by using Preparation of the of of cytMDH were by using the G. 1990; Google Scholar) using and the and of the mutant proteins were as mutant was of the mutant the was found to be MDH activity was at in a reaction 1 and NADH. The activity was as a in at of NADH for NADH of was used for the of the of the NADH. In this MDH activity is as the of of determine the disulfide was with grade for at in with a ratio of The obtained were by or reduced reduction of the obtained from cytMDHox, the of was with DTT for 1 at The were then by at a of with and and using the at at at and at The peptide were at acid of the were by peptide and their molecular were determined by using or oxidized cytMDH was a that been with and were out with an using a at The were out in and and by the at The specific was the acid using the which was by J. The results were by using the P. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar), and the function of was of the of Recombinant cytMDH by of the activity of purified recombinant cytMDH in the presence of NADH and 1 acid of to cytosolic MDHs from other and organelles Plant J. PubMed Scopus Google Scholar, F. A. A. M. A. 2001; PubMed Scopus (11) Google Scholar). Therefore, of in cytMDH activity were out as described above in of the We whether the enzyme activity of cytMDH is by conditions. recombinant cytMDH expressed in E. coli was purified as a reduced the protein could be oxidized by incubation with low concentrations of resulting in cytMDH reduction of oxidized cytMDH by incubation with 1 DTT, enzyme activity was that cytMDH could be activated and by a change in cytMDH an reported of MDH proteins of that this protein a homodimer G. 1989; PubMed Scopus Google Scholar, J. Biol. 1992; PubMed Scopus Google Scholar, J. L. PubMed Scopus Google Scholar, K. S. M. Lemaire-Chamley M. Issakidis-Bourguet E. Miginiac-Maslow M. H. PubMed Scopus Google Scholar, B. M. P. K. A. S. A. B. K. R. H. J. Biol. 2002; PubMed Scopus Google Scholar, A. D. G. A. G. R. T. J. Biol. 2004; PubMed Scopus Google Scholar). To the formation of the disulfide bond within the dimer we used The reduced form of cytMDH to the whereas oxidized cytMDH as a that cytMDH was able to form an disulfide conditions. cytMDH by between oxidized cytMDH and the reduced form of Trx-h1 was identified by Trx affinity chromatography (30Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (174) Google Scholar). To this interaction, oxidized cytMDH was with concentrations of DTT in the presence or the of cytMDH activity was found to be by concentrations of DTT most was by the of of the reduction of disulfide was also by Trx-h1 and The between cytMDH activity and its reduction level that cytMDH activity is by formation of an disulfide of the reduction by Trx-h1 was in the presence of Trx-h1 and DTT suggesting that the reduced form of Trx-h1 can efficiently reduce of the of cytMDH for thaliana cytMDH cysteine of which are in plant cytosolic MDH isoforms To determine the regulatory cysteine responsible for dimer we cytMDH and and their The was found to both the of MDH activity and disulfide bond formation These results that the oxidized cytMDH dimer is by disulfide formation between Cys330 the mapping analysis K. F. Y. H. Hisabori T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus (119) Google Scholar) was used to characterize both the and disulfide bonds in the protein conditions. To was by and the resulting peptide were by or incubation with DTT for reduction peptide were identified in the oxidized form and which were not in the reduced form In a peptide the peptide were reduced in These peptide were by and The was found to have a of and the sequence from the C-terminal of This to that of a dimer of the peptide which was by the disulfide was also in the peptide and peptide including and including this result the of a disulfide the that the and in the oxidized form that the suggested disulfide bond does not a regulatory in the cytMDH protein to was found in reduced peptide and was found to cysteine we have been to the of the peptide in the reduced form mapping analysis of using and the acid in the sequence of the The acid determined by was by of a was the peptide of the homodimer by disulfide The the acid in the sequence of the The acid determined by The was by The of a was the peptide The of the homodimer by disulfide in a of the the molecular of the reduced and the oxidized cytMDH were The oxidized form cytMDH molecular in the chromatography in the presence of whereas the for the was the as that for the reduced which was as a of were of cytMDH disulfide dimer and although the of the of the was found to be in the In the MDH activity of the was in these in in and in that both the reduced form dimer but the in the may be of a of dimer and The molecular of the reduced form cytMDH was and that for the of the oxidized form cytMDH was These were the for dimer and larger that for suggesting that the molecular of these cytMDH were very from of the proteins or that these cytMDH proteins have affinity to the To determine the molecular in the oxidized and the reduced we out The analysis of the of the oxidized form cytMDH molecular were and The analysis for the reduced form cytMDH with the of which is to the for the of the oxidized these the conversion of to the molecular of which is to the for dimer for the reduced and that of for the of the oxidized cytosolic Trx-h isoforms have been reported in the A. thaliana genome Y. J.P. F. Photosynth. Res. 2005; PubMed Scopus Google Scholar), at five of which have been found to be expressed (17Rivera-Madrid R. Mestres D. Marinho P. Jacquot J.P. Decottignies P. Miginiac-Maslow M. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5620-5624Crossref PubMed Scopus (113) Google the physiological of each Trx is to be Trx affinity chromatography was used in an attempt to the target proteins of the Trx-h to the of several potential metabolic enzyme (29Hisabori T. Hara S. Fujii T. Yamazaki D. Hosoya-Matsuda N. Motohashi K. J. Exp. Bot. 2005; 56: 1463-1468Crossref PubMed Scopus (50) Google Scholar, D. Motohashi K. Kasama T. Hara Y. Hisabori T. Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (174) Google Scholar). Among the captured proteins, cytMDH was found to be of that regulation of its has been the subject of intense study. Moreover, in to the cytosolic form was thought to be a the chloroplast MDH is by N- and C-terminal The although these N- and C-terminal extensions, cytMDH is also a redox-sensitive in 1 and we were able to that recombinant cytMDH an disulfide bond conditions. a target of the reduced form of this disulfide bond was found to be responsible for the regulation of the enzyme activity of recombinant The MDH activity in the cytosol obtained from the Arabidopsis was of and of this activity was incubation of the with not that (i) the activity is by of cytMDH and (ii) the specific activity of cytMDH in is equivalent to that of the recombinant cytMDH, the of cytMDH protein was to be of the cytosolic Trx-h is known to be a and expressed and several Trx-h have been (17Rivera-Madrid R. Mestres D. Marinho P. Jacquot J.P. Decottignies P. Miginiac-Maslow M. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5620-5624Crossref PubMed Scopus (113) Google Scholar, C. N. Y. G. Meyer Y. Proc. Natl. Acad. 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A. 2004; 101: PubMed Scopus Google are to the physiological of cytMDH and In to the characterization of cytMDH, we were able to determine that Cys330 is the cysteine required to form the disulfide bond the Cys330 have been to be at of the in reported of MDH proteins from other K. S. M. Lemaire-Chamley M. Issakidis-Bourguet E. Miginiac-Maslow M. H. PubMed Scopus Google Scholar, D. Full Text Full Text PDF PubMed Scopus Google Scholar). This the of whether recombinant cytMDH is able to the reported conditions. the oxidized form of cytMDH to further the of the reported disulfide dimer described mapping analysis disulfide bond between and that is not involved in to the enzyme activity of Moreover, the reported of the MDH G. 1989; PubMed Scopus Google Scholar), the of the of these cysteine may not be to form a disulfide the molecular of the cytMDH of A. thaliana is to be from the reported of analysis of dimer formation by we the and this was not to the suggested disulfide bond both and also this the reducing the mutant not the We found that the of these the reduced protein was with to not We that the is the result of formation of the disulfide bond between the Cys330 and the other in the cytMDH which must be in the by during To characterize the oxidized form cytMDH in we the molecular using chromatography and analysis in the oxidized cytMDH in both the molecular was the and the for the reduced form of cytMDH was identified as dimer the the for oxidized form has the of this must be a dimer The presence of may significant into the molecular the the analysis a for very that the with the of to the reported of cytMDH, a change is to occur disulfide bond formation between the Cys330 the This change may the molecular and the Moreover, this change may and the formation of The in the between the oxidized and reduced form of cytMDH may be to the change in of the analysis of the oxidized form cytMDH the of In plant cytosol, the reducing equivalents transferred from are to NADH by The resulting NADH is then used as reducing equivalents in the cytosol or transferred into mitochondria by malate system where is used for In cytosolic NADPH levels in plant are maintained by the irrespective of the light/dark (21Igamberdiev A.U. Gardestrom P. Biochim. Biophys. Acta. 2003; 1606: 117-125Crossref PubMed Scopus (198) Google Scholar) cytosolic NADPH is an important reducing equivalent required for acid and acid In the reduction level of cytosolic Trx-h isoforms is to be by the cytosolic NADPH/NADP+ the cytosolic Trx-h protein is efficiently reduced by Trx-h is to be in a high ratio of cytMDH physiological conditions. is the for the reduction of oxidized cytMDH by the physiological importance of cytMDH that its activity must be Trx-h may a mechanism required to of cytMDH by disulfide bond the of cytosolic Trx isoforms has been and is known the roles of these The metabolic enzymes captured by Trx affinity chromatography (30Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (174) Google Scholar) are to be of significant physiological of these enzymes is to result in a in To this plant are with a and reduction system of Trx-h which to reduce redox-sensitive disulfide bonds these important into the between Trx-h isoforms and their target proteins and the of the metabolic enzymes in the cytosol into the and functions of the cytosolic Trx-h We for to M. for and with