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Chloroplast Cyclophilin Is a Target Protein of Thioredoxin

2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês

10.1074/jbc.m304258200

1083-351X

Ken Motohashi, Fumie Koyama, Yoichi Nakanishi, Hanayo Ueoka‐Nakanishi, Toru Hisabori,

Peptidase Inhibition and Analysis

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 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. Cyclophilin (CyP) 1The abbreviations used are: CyP, cyclophilin; CyPox, oxidized cyclophilin; CyPred, reduced cyclophilin; CyPNoCys, cyclophilin containing no cysteines; DTT, dithiothreitol; PPIase, peptidyl-prolyl cis-trans isomerase; Trx, thioredoxin; AMS, 4-acetamido-4′-maleimidyl-stilbene-2,2′-disulfonate; HPLC, high pressure liquid chromatography. is a member of immunophilin superfamily and a target of the immunosuppressive drug cyclosporin A (1Dolinski K. Heitman J. Gething M.-J. Guidebook to Molecular Chaperones and Protein-folding Catalysts. Oxford University Press, 1997: 359-369Google Scholar). CyP shows peptidyl-prolyl cis-trans isomerase (PPIase) activity and functions as a catalyst in protein folding, facilitating the slow isomerization around Xaa-Pro peptide bonds, which is a general rate-limiting step of protein folding (2Takahashi N. Hayano T. Suzuki M. Nature. 1989; 337: 473-475Crossref PubMed Scopus (941) Google Scholar, 3Fischer G. Wittmann-Liebold B. Lang K. Kiefhaber T. Schmid F.X. Nature. 1989; 337: 476-478Crossref PubMed Scopus (1212) Google Scholar). The second major role of CyP is its function as a component in cellular signaling. CyP is essential for the regulation of immunosuppression in mammalian T-cells (4Schreiber S.L. Cell. 1992; 70: 365-368Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 5Walsh C.T. Zydowsky L.D. McKeon F.D. J. Biol. Chem. 1992; 267: 13115-13118Abstract Full Text PDF PubMed Google Scholar). In plants CyP was first reported by Gasser et al. (6Gasser C.S. Gunning D.A. Budelier K.A. Brown S.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9519-9523Crossref PubMed Scopus (146) Google Scholar). Breiman et al. (7Breiman A. Fawcett T.W. Ghirardi M.L. Mattoo A.K. J. Biol. Chem. 1992; 267: 21293-21296Abstract Full Text PDF PubMed Google Scholar) found cyclosporin A-sensitive PPIase in chloroplasts. The corresponding gene for this enzyme was cloned in the nuclear genome of Arabidopsis thaliana as a single gene targeted to chloroplast (8Lippuner V. Chou I.T. Scott S.V. Ettinger W.F. Theg S.M. Gasser C.S. J. Biol. Chem. 1994; 269: 7863-7868Abstract Full Text PDF PubMed Google Scholar). Members of the CyP family were identified in various organelles of plants (8Lippuner V. Chou I.T. Scott S.V. Ettinger W.F. Theg S.M. Gasser C.S. J. Biol. Chem. 1994; 269: 7863-7868Abstract Full Text PDF PubMed Google Scholar, 9Luan S. Lane W.S. Schreiber S.L. Plant Cell. 1994; 6: 885-892Crossref PubMed Scopus (75) Google Scholar, 10Hayman G.T. Miernyk J.A. Biochim. Biophys. Acta. 1994; 1219: 536-538Crossref PubMed Scopus (16) Google Scholar, 11Saito T. Ishiguro S. Ashida H. Kawamukai M. Matsuda H. Ochiai H. Nakagawa T. Plant Cell Physiol. 1995; 36: 377-382Crossref PubMed Scopus (20) Google Scholar, 12Saito T. Niwa Y. Ashida H. Tanaka K. Kawamukai M. Matsuda H. Nakagawa T. Plant Cell Physiol. 1999; 40: 77-87Crossref PubMed Scopus (35) Google Scholar), and the A. thaliana genome project identified at least 10 isoforms of typical cyclophilins (13Kaul S. Koo H.L. Jenkins J. Rizzo M. Rooney T. Tallon L.J. Feldblyum T. Nierman W. Benito M.-I. Lin X. Town C.D. Venter J.C. Fraser C.M. Tabata S. Nakamura Y. et al.Nature. 2000; 408: 796-815Crossref PubMed Scopus (7166) Google Scholar). However, the physiological significance of CyP in plant cytoplasm and in chloroplasts has been obscure thus far (9Luan S. Lane W.S. Schreiber S.L. Plant Cell. 1994; 6: 885-892Crossref PubMed Scopus (75) Google Scholar, 14Luan S. Albers M.W. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 984-988Crossref PubMed Scopus (66) Google Scholar, 15Sheldon P.S. Venis M.A. Biochem. J. 1996; 315: 965-970Crossref PubMed Scopus (20) Google Scholar). The important function of CyP in the process of T-cell activation for the regulation of immunosuppression in mammalian cells suggests that CyP also has an important physiological role in the plant cell, e.g. in signal transduction. Although CyPs of some eukaryotic organisms have several conserved cysteines, there is still no information available on the role of these cysteines. However, we found that CyP is the potential target protein of chloroplast thioredoxin (Trx) (16Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar). Trx is a small, ubiquitous, disulfide oxidoreductase with two redox active cysteines at the active center (17Buchanan B.B. Annu. Rev. Plant Physiol. 1980; 31: 341-374Crossref Google Scholar, 18Schürmann P. Jacquot J.P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 371-400Crossref PubMed Scopus (326) Google Scholar, 19Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). The active site sequence (-Trp-Cys-Gly-Pro-Cys-) of Trx is well conserved regardless of the lower overall sequence homology. Trx induces a conformational change in the target protein via exchange of the disulfide bond and thereby modulates the activity of these enzymes. The mechanism for reduction of target proteins by Trx has been studied in vitro (20Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 21Holmgren A. Structure. 1995; 3: 239-243Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). The first Cys in the active site sequence of Trx probably forms a mixed disulfide intermediate with the target protein. Then the established disulfide bond between the two redox partners is attacked by another cysteine of Trx. Consequently the reduced form of the target protein is released from Trx, and Trx itself is oxidized. In the chloroplasts of higher plants two Trx isoforms, designated f-type (Trx-f) and m-type (Trx-m) based on their first identified target proteins, are well known (18Schürmann P. Jacquot J.P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 371-400Crossref PubMed Scopus (326) Google Scholar, 22Jacquot J.P. Lancelin J.M. Meyer Y. New Phytol. 1997; 136: 543-570Crossref Scopus (160) Google Scholar, 23Ruelland E. Miginiac-Maslow M. Trends Plant Sci. 1999; 4: 136-141Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The various chloroplast enzymes are regulated by reduction of their internal disulfide or reoxidation of thiols. Calvin cycle enzymes, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and phosphoribulokinase, are regulated by their redox states, and their activities are enhanced in the reduced enzymes (24Baumann U. Juttner J. Cell Mol. Life Sci. 2002; 59: 1042-1057Crossref PubMed Scopus (41) Google Scholar). Recently several approaches to identify the target proteins of Trx have been challenged (16Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 25Yano 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, 26Balmer 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). Within the captured candidate proteins, we identified CyP as an unreported Trx target in chloroplasts (16Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar). In the present study, we revealed that CyP in chloroplasts is an actual target protein of Trx and that the PPIase activity of CyP is regulated by the reduction of the internal disulfide bond by Trx-m. In addition, cysteine residues involved in this redox regulation were identified. Preparation of Trx-m—The recombinant Trx-m was expressed in Escherichia coli (27Stumpp M.T. Motohashi K. Hisabori T. Biochem. J. 1999; 341: 157-163Crossref PubMed Scopus (38) Google Scholar) and was purified as follows. The E. coli cells were suspended in 25 mm Tris-HCl (pH 7.5) containing 0.5 mm dithiothreitol (DTT), disrupted by a French pressure cell (5501-M, Ohtake Works, Tokyo), and centrifuged at 100,000 × g for 40 min at 4 °C. The supernatant was applied to a DEAE-Toyopearl 650 m column (Tosoh, Tokyo) and then eluted with a 0–150 mm linear gradient of NaCl in 25 mm Tris-HCl (pH 7.5) and 0.5 mm DTT. The peak fraction containing Trx-m was collected, and solid ammonium sulfate was added to be the final concentration of 1.6 m. The solution was then applied to a butyl-Toyopearl 650 m column, and eluted with a 1.6 to 0 m inverse gradient of ammonium sulfate. The protein concentration was calculated from the A 278 using the published molar absorption coefficient value for Trx-m, 20,500 m–1·cm–1 (28Schürmann P. Methods Enzymol. 1995; 252: 274-283Crossref PubMed Scopus (53) Google Scholar). Preparation of CyP—E. coli BL21(DE3) cell carrying CyP-pET23c, the plasmid for the wild-type CyP (CyPWT), was cultured at 37 °C, and the desired protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (0.5 mm) at 25 °C. CyPWT was obtained as soluble protein and purified as described previously (16Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar). Preparation of the Mutant CyP—To prepare the mutant CyPs, CyPC53S/C170S, CyPC128S/C175S, and CyPC53S/C128S/C170S/C175S (CyPNoCys), site-directed mutagenesis was performed with mega-primer PCR method (29Landt O. Grunert H.P. Hahn U. Gene. 1990; 96: 125-128Crossref PubMed Scopus (639) Google Scholar) using KOD DNA polymerase (Toyobo, Osaka). The sequences of the resultant plasmids were confirmed by DNA sequencing (Prism 310, Applied Biosystems). When the desired proteins were expressed in E. coli BL21(DE3), CyPC128S/C175S was obtained as soluble protein and could be purified by the method for CyPWT. However, CyPC53S/C170S and CyPNoCys were insoluble proteins and were purified as follows. After induction, the cells were suspended in 25 mm Tris-HCl (pH 7.5) and 5 mm EDTA, disrupted by sonication, and centrifuged at 11,000 × g for 10 min. The inclusion bodies obtained were suspended in 25 mm Tris-HCl (pH 7.5), 5 mm EDTA, and 2% (v/v) Triton X-100, incubated for 30 min at room temperature, and centrifuged at 11,000 × g for 10 min. This washing step was repeated twice. Then the precipitate was washed twice with 25 mm Tris-HCl (pH 7.5) and 1 mm EDTA and dissolved finally in 25 mm Tris-HCl (pH 7.5), 1 mm EDTA, 8 m urea, and 0.5 mm DTT. The insoluble fraction was removed by centrifugation, and the supernatant was diluted 50–100-fold with 25 mm Tris-HCl (pH 7.5), 1 mm EDTA, and 0.5 mm DTT. Solid ammonium sulfate was added to the diluted solution up to 1.3 m and the solution was applied to a butyl-Toyopearl 650 m column, which was equilibrated with 25 mm Tris-HCl (pH 7.5) containing 1.3 m ammonium sulfate. The protein was eluted from the column with a 1.3–0 m inverse gradient of ammonium sulfate. The peak fraction containing CyP was collected and stored at –80 °C. The protein concentration of CyP was determined by the method described previously (30Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). In Vitro Reduction of CyP Assisted by Trx—The recombinant CyPs (both wild-type and mutants) were obtained in reduced form, and the oxidized form of CyP (CyPox) was prepared as described (16Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar). Oxidized CyP (3.0 μm) was incubated with DTT (50 μm) in the presence or absence of Trx-m (3 μm) for 1 h at 25 °C. To assess the redox state of CyP, 4-acetamido-4′-maleimidyl-stilbene-2,2′-disulfonate (AMS) (Molecular Probes), a maleimidyl reagent that specifically modifies cysteine residues, was used. AMS labeling was carried out as described previously (16Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar), and CyPox and reduced CyP (CyPred) were separated by 15% (w/v) SDS-PAGE. PPIase Activity of the Recombinant CyP—The PPIase activity of CyP was measured as described (31Liu J. Albers M.W. Chen C.M. Schreiber S.L. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2304-2308Crossref PubMed Scopus (231) Google Scholar, 32Fischer G. Bang H. Berger E. Schellenberger A. Biochim. Biophys. Acta. 1984; 791: 87-97Crossref PubMed Scopus (134) Google Scholar, 33Hayano T. Takahashi N. Kato S. Maki N. Suzuki M. Biochemistry. 1991; 30: 3041-3048Crossref PubMed Scopus (124) Google Scholar) with minor modifications. An assay mixture containing 35 mm HEPES-NaOH (pH 8.0), 50 μm N-succinyl-Ala-Ala-Pro-Phe-4-methylcoumaryl-7-amide (Peptide Institute) was incubated at 10 °C. α-Chymotrypsin (final concentration 20 μm) was added to initiate the reaction. CyP was added after 15 s. Absorbance at 360 nm was monitored by UV spectrophotometry. First-order rate constants (k obs) were derived by a curve fit to a first-order rate equation (A 360 = A 1+ A 0 e –kt, where k is the rate constant). The k cat/K m values were calculated according to the equation k cat/K m = (k obs – k 0)/[PPIase], where k 0 is the first-order rate constant for spontaneous cis-trans isomerization (31Liu J. Albers M.W. Chen C.M. Schreiber S.L. Walsh C.T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2304-2308Crossref PubMed Scopus (231) Google Scholar). Peptide Mapping and Analysis—To determine the internal disulfide partner cysteines, CyPox was digested with sequencing grade modified trypsin (catalog no. V5111, Promega) for 20 h at 37 °C in 25 mm (NH4)HCO3, pH 7.0, with a protein-to-protease ratio of 90:1 (w/w), or sequencing grade chymotrypsin (catalog no. 1-418-467, Roche Diagnostics) for 15 h at 37 °C in 25 mm (NH4)HCO3, pH 7.8, containing 2 mm CaCl2, with a protein-to-protease ratio of 50:1 (w/w). The proteolytic peptides obtained were analyzed directly by reversed-phase HPLC or reduced before analysis. For reduction of the fragments obtained from CyPox, the mixture of proteolytic peptides was incubated with 10 mm DTT for 1 h at 37 °C in 25 mm Tris-HCl (pH 7.5). Then, the peptides were separated by reversed-phase HPLC column (Cosmosil 5C18 AR-300, 4.6 × 150 mm, Nacalai tesque, Kyoto, Japan) at a flow rate of 0.5 ml/min with solvent A (0.1% (v/v) trifluoroacetic acid) and solvent B (90% (v/v) acetonitrile and 0.1% trifluoroacetic acid) using the gradient elution (2% solvent B at 0–5 min, 2–50% solvent B at 5–50 min, 50–80% solvent B at 50–60 min, 80% solvent B at 60–70 min). The peptide fragments were monitored at 220 nm. N-terminal amino acid sequences of the separated fragments were analyzed by N-terminal peptide sequencing (PPSQ-21, Shimadzu, Kyoto, Japan) and their molecular masses were determined by using a matrix-assisted laser desorption ionization/time-of-flight mass spectrometer (Axima-CFR, Shimadzu). The Quantitative Analysis of Sulfhydryl Groups—The number of free sulfhydryl groups in CyP molecule was determined using 5,5′-dithio-bis(2-nitrobenzoic acid) (34Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21474) Google Scholar). 5,5′-Dithio-bis(2-nitrobenzoic acid) (final concentration of 0.4 mm) was added to CyP in 100 mm Tris-HCl (pH 8.0), 10 mm EDTA and 6 m guanidine HCl. The absorbance at 412 nm was measured, and the amounts of reactive sulfhydryls were determined from ϵ412 = 13,380 m–1·cm–1 (35Glazer A.N. Delange R.J. Sigman D.S. Delange R.J. Sigman D.S. Chemical Modification of Proteins. Elsevier, Amsterdam1975: 21-25Google Scholar). The Oxidized CyP Is Inactive as PPIase—In the previous study, we reported that chloroplast CyP is a possible target protein for Trx-m and that CyPox could be reduced by Trx-m in vitro (16Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar). CyP, known as a member of the immunophilin superfamily, promotes the isomerization from cis-form to trans-form in peptidylproline. Therefore we examined whether the PPIase activity of CyP is affected by reduction or oxidation. We measured the PPIase activity using the model substrate N-succinyl-Ala-Ala-Pro-Phe-4-methylcoumaryl-7-amide and monitored the change of the absorbance at 360 nm derived by the release of the methylcoumaryl moiety from the artificial model peptide when the trans-form peptide was digested by α-chymotrypsin. A slow increase in absorbance was observed even in the absence of the catalyzing enzyme, indicating that the model substrate was gradually transferred from the cis-form to the trans-form irrespective of PPIase (Fig. 1A, none). The addition of CyPox did not change the rate of increase in absorbance at 360 nm (Fig. 1A, CyPox). Thus, PPIase activity of CyP must be suppressed when CyP is present in the oxidized form. In contrast, the model substrate was rapidly shifted to the trans-form and digested by α-chymotrypsin when CyPred was added to the assay mixture (Fig. 1A, CyPred). The K obs value of PPIase activity was measured in the presence of various concentrations of CyPred or CyPox, and the k cat/K m values were calculated as described under "Experimental Procedures" (Fig. 1B and Table I). The activity of CyPox was less than 1% of the CyPred activity, suggesting that CyP is inactive in the oxidized form.Table IPPIase activity of oxidized or reduced CyPConditionkcat/Kms-1 μm-1CyPred8.32CyPox + Trx-m + DTT4.57CyPox + DTT1.14CyPox + Trx-m0.04CyPox0.07 Open table in a new tab CyP ox Can Be Reduced, and the PPIase Activity Is Reactivated by Trx-m—We examined whether CyPox could be reduced and the PPIase activity reactivated by Trx-m. When CyPox was incubated with Trx-m only, PPIase activity was not observed (Fig. 1B). Weak PPIase activity was detected when 50 μm DTT was used instead of Trx-m. In contrast, PPIase activity recovered to 55% of the rate observed in the reduced state when Trx-m was added together with 50 μm DTT (Fig. 1B and Table I). Inactivation and partial recovery of PPIase activity was dependent on the redox conditions following the redox state of CyP, which was visualized by AMS labeling (Fig. 2). Identification of Cysteine Pairs Involved in Disulfide Bond Formation in CyP ox —CyP from A. thaliana contains four cysteine residues allowing six different combinations of disulfide bonds. To specify the cysteine pairs involved in disulfide formation that are responsible for the regulation of the PPIase activity, CyPox was digested by proteases, and the resultant peptide fragments were separated by reversed-phase HPLC after incubation under non-reduced or reduced conditions (Fig. 3). We identified a single redox-responsive peptide fragment in trypsin-digested and in chymotrypsin-digested fragments of CyPox, respectively (Fig. 3A, TO1, and Fig. 3C, CTO1). Under reduced conditions, TO1 emerged with two specific peaks (Fig. 3B, TR1 and TR2). In the case of the reduced fragments after chymotrypsin digestion, CTO1 disappeared and three specific peaks emerged (Fig. 3D, CTR1, CTR2, and CTR3). The elution profiles of the peptide fragments from CyPred were very similar to those of CyPox proteolytic fragments after reduction (data not shown). These redox-specific peptide fragments were analyzed by N-terminal peptide sequencing and mass spectrometry (Table II). TO1 obtained by trypsin digestion of CyPox was composed of two peptides, which were recovered as two peaks, TR1 and TR2, under reduced conditions. The TR1 peptide contains Cys175, whereas TR2 contains Cys128. Therefore these two cysteines should form the disulfide bond. In the case of chymotrypsin cleavage, CTO1 was composed of two peptides containing Cys53 and Cys170, respectively. The peptide containing Cys53 of CyP was recovered as CTR1 in the reduced fragments. The peptide containing Cys170 was not identified in the fragments under reduced conditions. In contrast, CTR2, which possibly contains Cys53, and CTR3, containing Cys175, were obtained only from the reduced fragments. The failure to detect additional peaks corresponding to cysteine containing fragments under reduced and oxidized conditions might be attributed to an incomplete digestion by chymotrypsin or the insufficient separation of the peptides by the reversed-phase HPLC. As a consequence, we were able to determine two disulfide bond pairs, Cys53–Cys170 and Cys128–Cys175, in CyPox (Fig. 4A). To confirm the formation of these two disulfide bonds in CyP, the number of free sulfhydryl groups in oxidized or reduced CyP was quantified with 5,5′-dithio-bis(2-nitrobenzoic acid). According to these measurements CyPox contained 0.2 mol of thiol/mol of CyP, and CyPred contained 4.0 mol of thiol/mol of CyP.Table IIPeptide mapping analysis of oxidized CyP using N-terminal peptide sequencer and MALDI-TOF mass spectrometryPeptide fractionPeptide fragmentaResidue numbers denote the amino acid positions in the sequence of the mature protein. The underlines indicate the amino acid sequences determined by N-terminal peptide sequencer.Fragment mass (Obs.)bObs., observed mass (m/z) estimated by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry.Fragment mass (Calc.)cCalc., calculated monoisotopic mass of a single-charged molecule ([M+H]+) based on the assigned peptide sequence.TO1dThese fractions were analyzed by MALDI-TOF mass spectrometry after reduction with DTT.105HTGPGILSMAN...ICTVK131Not detectedeBecause of signal suppression by matrix molecules in matrix-assisted laser desorption ionization, the signals corresponding to the peptides were not detected.2762.3172IYACGELPLDA1821164.71164.6TR1172IYACGELPLDA1821164.81164.6TR2105HTGPGILSMAN...ICTVK1312762.92762.3CTO1dThese fractions were analyzed by MALDI-TOF mass spectrometry after reduction with DTT.53CTGEKKY59Not detectedeBecause of signal suppression by matrix molecules in matrix-assisted laser desorption ionization, the signals corresponding to the peptides were not detected.828.4164DVPKKGCRIY1731178.81178.6CTR153CTGEKKY59fPeptide sequencing of this fraction indicated that this fragment was digested by chymotrypsin just after Tyr59.Not determined828.4CTR239GEVVPKTVENF49Not detectedeBecause of signal suppression by matrix molecules in matrix-assisted laser desorption ionization, the signals corresponding to the peptides were not detected.1218.639GEVVPKTVENFRAL52Not detectedeBecause of signal suppression by matrix molecules in matrix-assisted laser desorption ionization, the signals corresponding to the peptides were not detected.1558.939GEVVPKTVEN...CTGEKKY59Not detectedeBecause of signal suppression by matrix molecules in matrix-assisted laser desorption ionization, the signals corresponding to the peptides were not detected.2368.2CTR3174ACGELPLDA182888.5888.4a Residue numbers denote the amino acid positions in the sequence of the mature protein. The underlines indicate the amino acid sequences determined by N-terminal peptide sequencer.b Obs., observed mass (m/z) estimated by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry.c Calc., calculated monoisotopic mass of a single-charged molecule ([M+H]+) based on the assigned peptide sequence.d These fractions were analyzed by MALDI-TOF mass spectrometry after reduction with DTT.e Because of signal suppression by matrix molecules in matrix-assisted laser desorption ionization, the signals corresponding to the peptides were not detected.f Peptide sequencing of this fraction indicated that this fragment was digested by chymotrypsin just after Tyr59. Open table in a new tab Fig. 4The suggested disulfide bonds in CyP and the redox state of Cys mutants. A, the identified disulfide bonds in CyP are presented schematically. B, the oxidized (ox) or reduced (red) form of CyP mutants (3 μm) was incubated with Trx-m (3 μm) or DTT (50 μm) or both. The redox states of CyP were monitored by the AMS labeling method. In lane "red," original CyPs were AMS-labeled; lane "ox," CyPs were oxidized before AMS-labeling; lane "ox + DTT + M," oxidized CyPs were incubated with Trx-m and DTT. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Both Disulfide Bonds in CyP ox Inactivate the PPIase Activity and Are Reduced by Trx-m—A CyPNoCys mutant, containing no cysteines, and two cysteine mutants, CyPC53S/C170S and CyPC128S/C175S, which cannot form either of the internal disulfide bonds identified above, were constructed. The PPIase activities of these mutants were examined under oxidizing and reducing conditions to determine the redox-responsive disulfide bonds in the molecule. Although the PPIase activities of three mutants were affected by the number of deleted cysteines, redox sensitivity of the PPIase activity was clearly maintained in the case of CyPC53S/C170S and CyPC128S/C175S (Table III). Redox sensitivity of the internal disulfide bonds in these mutants was confirmed by AMS-labeled CyP mobility on SDS-PAGE (Fig. 4B). The PPIase activity of CyPNoCys was remarkably lower than that of other mutants and was not affected by the reduction or oxidation (Table III). This insensitivity was also confirmed by AMS labeling (Fig. 4B). As observed in Fig. 4B, CyPC128S/C175S was greatly reduced by Trx-m and DTT, although activity was not recovered very much by reduction (Table III). This apparent discrepancy might be due to the instability of the mutant protein after the redox treatment. Taken together, both Cys53–Cys170 and Cys128–Cys175 disulfide bonds were redox-sensitive and involved in redox regulation of the PPIase activity of CyP.Table IIIPPIase activity change of CyP mutants under redox conditionsCypkcat/KmCyPredCyPoxCyPox + Trx-m + DTTs-1 μm-1WT8.210.135.45C53S/C170S7.120.123.02C128S/C175S5.740.071.83No Cys1.361.331.13 Open table in a new tab CyP Is a Trx-Regulated Enzyme—In the present study, we observed that the PPIase activity of CyP is modulated by the redox state of the molecule and revealed that CyPox is an inactive PPIase, whereas CyPred is an active one (Fig. 1). A disulfide bond in CyPox was definitely reduced by Trx-m, and the PPIase activity of CyP was recovered by the reduction of disulfide bonds in CyP. We identified two critical disulfide bonds involved in the thiol modulation of PPIase activity, Cys53–Cys170 and Cys128–Cys175. Cyclophilin 3 from Caenorhabditis elegans has four cysteine residues, which correspond to the residues in A. thaliana. Their amino acid sequence homology is very high; the identity of the amino acids was 65%. The crystal structure of cyclophilin 3 has been reported (36Dornan J. Page A.P. Taylor P. Wu S. Winter A.D. Husi H. Walkinshaw M.D. J. Biol. Chem. 1999; 274: 34877-34883Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and the distance between Cα–Cα in each disulfide bond pair of CyPox in A. thaliana could be estimated based on the coordinates of the three-dimensional structure of cyclophilin 3. The distances between Cα–Cα in Cys53–Cys170 and Cys128–Cys175 was estimated as 16.49 and 18.91 Å, respectively. When Dornan et al. (36Dornan J. Page A.P. Taylor P. Wu S. Winter A.D. Husi H. Walkinshaw M.D. J. Biol. Chem. 1999; 274: 34877-34883Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) reported the structure, they suggested the possible disulfide bond formation in cyclophilin 3 because the distance between the sulfur atoms of Cys40 and Cys168 (corresponding to Cys53 and Cys175 of CyP from A. thaliana) was close enough (5.38 Å) to form the bond. However, disulfide bond formation between Cys53 and Cys175 has not been found in our case (Table II and Fig. 4). As the calculated distances between Cα–Cα in Cys53–Cys170 and Cys128–Cys175 were too great to form the intramolecular disulfides, a remarkable conformational change may occur when CyP is oxidized, which may be the cause of the loss of PPIase activity. Physiological Role of Redox Regulation of CyP—In the thylakoid lumen, a 40-kDa cyclophilin-like protein named TLP40 had been reported (37Fulgosi H. Vener A.V. Altschmied L. Herrmann R.G. Andersson B. EMBO J. 1998; 17: 1577-1587Crossref PubMed Scopus (110) Google Scholar). TLP40 shows PPIase activity and affects the dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain (38Vener A.V. Rokka A. Fulgosi H. Andersson B. Herrmann R.G. Biochemistry. 1999; 38: 14955-14965Crossref PubMed Scopus (87) Google Scholar). Another cyclophilin is CyP40, which has a cyclophilin-like PPIase domain in the N terminus and has been identified as a regulatory factor in the vegetative phase of A. thaliana (39Berardini T.Z. Bollman K. Sun H. Poethig R.S. Science. 2001; 291: 2405-2407Crossref PubMed Scopus (133) Google Scholar). Thus cyclophilin-related molecules seem to have significant roles in various compartments of the plant cell and in chloroplasts. So far, the role of CyP in chloroplasts is uncertain. Here we found that CyP in chloroplasts is redox-sensitive and its PPIase activity is modulated by the redox states via Trx-m. Light initiates many physiological phenomena in chloroplasts including biogenesis of proteins. Therefore, the accurate activation of the PPIase activity of CyP under reducing conditions must be physiologically meaningful, as CyP is a member of the protein folding catalyst proteins. Recently, Lee et al. (40Lee S.P. Hwang Y.S. Kim Y.J. Kwon K.S. Kim H.J. Kim K. Chae H.Z. J. Biol. Chem. 2001; 276: 29826-29832Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) have reported that a mammalian cyclophilin, CyP-A, binds to 1-Cys peroxiredoxin and supports the peroxidase activity as an electron donor. This finding allowed proposing the cascade from Trx to 1-Cys peroxiredoxin via cyclophilin in the cell. However we could not yet identify the counterpart proteins of CyP in chloroplasts under the physiological conditions, and further studies will be required to understand the physiological significance of the redox transmission network involving CyP in chloroplast. Perspective—It appears that the reduction/oxidation cascade via Trx in chloroplasts must be an important regulation network for the metabolic pathways in chloroplasts. Actually various metabolic enzymes are suggested to be involved in this network (16Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 26Balmer 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). In the present study, we have clearly shown the redox regulation of the PPIase activity of CyP. Although we do not have information on the physiological contribution of CyP in chloroplasts at the moment, and we do not know why CyP-PPIase activity was affected by their redox states, our finding should be important in revealing the role of CyP in chloroplasts. Further studies in vivo are necessary to clarify the physiological significance of redox regulation of CyP together with structure analysis to understand the redox regulation of PPIase activity. We thank E. Muneyuki for helpful discussion on the kinetic analysis of the PPIase activity and H. Taguchi and A. Koike for technical assistance and instruction of peptide sequencing and mass spectrometry. We also thank D. Yamazaki for helpful discussions and Georg Groth (Heinrich Heine University, Düsseldorf, Germany) for critically reading the manuscript. We extend special thanks to Masasuke Yoshida for continuous encouragement and valuable suggestions.