Title: HCF164 Receives Reducing Equivalents from Stromal Thioredoxin across the Thylakoid Membrane and Mediates Reduction of Target Proteins in the Thylakoid Lumen
Abstract: 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. 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 redox state of higher plant chloroplasts undergoes significant fluctuations in both light and dark conditions. In the light, photosynthetic electron transport via ferredoxin and ferredoxin-NADP+ reductase (FNR) 2The abbreviations used are: FNR, ferredoxin-NADP+ reductase; Trx, thioredoxin; PSI-N, the N subunit of photosystem I; HCF164sol, the soluble domain of HCF164; TCEP, tris-(2-carboxyethyl) phosphine; AMS, 4-acetamido-4′-maleimidylstilbene-2, 2′-disulfonic acid; PMF, peptide mass fingerprint; CcdA, cytochrome c defective A; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PIPES, 1,4-piperazinediethanesulfonic acid. results in the synthesis of NADPH, which is used as a source of reducing equivalents for carbon fixation. However, a portion of the electrons produced by the photosynthetic electron transport is transferred to thioredoxin (Trx) through ferredoxin and ferredoxin-thioredoxin reductase (1Buchanan B.B. Annu. Rev. Plant Physiol. 1980; 31: 341-374Crossref Google Scholar, 2Dai S. Schwendtmayer C. Schurmann P. 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A. 2001; 98: 4794-4799Crossref PubMed Scopus (205) Google Scholar, 12Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 13Balmer 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, 14Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (174) Google Scholar, 15Balmer 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, 16Balmer Y. Vensel W.H. Cai N. Manieri W. Schurmann P. Hurkman W.J. Buchanan B.B. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2988-2993Crossref PubMed Scopus (150) Google Scholar). This has allowed the characterization of a wide variety of previously unknown Trx-regulated enzymes in the chloroplast stroma, for example (6Meyer Y. Reichheld J.P. Vignols F. Photosynth. Res. 2005; 86: 419-433Crossref PubMed Scopus (187) Google Scholar, 7Buchanan B.B. Balmer Y. Annu. Rev. Plant Biol. 2005; 56: 187-220Crossref PubMed Scopus (701) Google Scholar, 8Gelhaye E. Rouhier N. Navrot N. Jacquot J.P. Cell. Mol. Life Sci. 2005; 62: 24-35Crossref PubMed Scopus (227) Google Scholar, 12Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 17Motohashi K. Koyama F. Nakanishi Y. Ueoka-Nakanishi H. Hisabori T. J. Biol. Chem. 2003; 278: 31848-31852Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 18Hisabori 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 contrast, our knowledge of the thiol-disulfide redox control system in the thylakoid lumen has been limited so far (19Hisabori T. Motohashi K. Hosoya-Matsuda N. Ueoka-Nakanishi H. Romano P.G. Photochem. Photobiol. 2007; (in press)PubMed Google Scholar). Meurer et al. identified mutants of Arabidopsis that displayed a high chlorophyll fluorescence phenotype (hcf mutants) under standard photosynthetic conditions (20Meurer J. Meierhoff K. Westhoff P. Planta. 1996; 198: 385-396Crossref PubMed Scopus (176) Google Scholar, 21Meurer J. Plucken H. Kowallik K.V. Westhoff P. EMBO J. 1998; 17: 5286-5297Crossref PubMed Scopus (178) Google Scholar), suggesting a defect in photosynthetic electron transport. Among these, the hcf164 mutant was found to be impaired in the stable assembly of the cytochrome b6 f complex within thylakoid membranes (22Lennartz K. Plucken H. Seidler A. Westhoff P. Bechtold N. Meierhoff K. Plant Cell. 2001; 13: 2539-2551Crossref PubMed Google Scholar). This mutant was shown to lack the Trx-like protein subsequently named HCF164, which was predicted to be localized in the thylakoid lumen. The HCF164 protein contains a membrane-spanning sequence and a thioredoxin-like CXXC motif in the N- and C-terminal regions, respectively (supplemental Fig. S1). The finding that the reduced form of the HCF164 protein possesses insulin reducing activity in vitro (22Lennartz K. Plucken H. Seidler A. Westhoff P. Bechtold N. Meierhoff K. Plant Cell. 2001; 13: 2539-2551Crossref PubMed Google Scholar) leads to the possibility that, in a similar way to Trx, the HCF164 protein may act as a transducer of reducing equivalents in the thylakoid lumen. To understand the role of HCF164 in the thylakoid lumen, we have carried out a thorough investigation of the biochemical properties of the HCF164 protein. We found that m-type Trx is able to effectively transfer electrons to luminal HCF164 across the thylakoid membranes. A proteomic based screen then allowed us to identify putative thylakoid-localized HCF164 target proteins, one of which was confirmed to undergo HCF164-dependent reduction. The results presented here underline the physiological significance of HCF164 as a transducer of reducing equivalent within the thylakoid lumen. Cloning, Expression, and Purification of HCF164 and PSI-N— The genes for HCF164 and the N subunit of photosystem I (PSI-N) were obtained by PCR amplification from an Arabidopsis cDNA library, using the following oligonucleotides: for the soluble domain of HCF164 (HCF164sol, amino acid residues 116-261 (supplemental Fig. S1)) (At4g37200), 5′-aactgcagcatatggattttgggatttctttgaa-3′ (NdeI) and 5′-cggaattcttatccatggcttaagggat-3′ (EcoRI); for the mature form of PSI-N (amino acid residues 87-171) (At5g64040), 5′-aactgcagcatatgggcgtcattgacgaatacct-3′ (NdeI) and 5′-cggaattcaccatttccagaaaaca-3′ (EcoRI). The restriction sites for the enzyme shown in parentheses are underlined. The amplified DNA fragments were cloned into the NdeI and EcoRI sites of pET23c (Novagen), and the DNA sequences were confirmed. The recombinant HCF164sol was expressed in Escherichia coli BL21 (DE3) cells and purified as follows. E. coli cells were suspended in 25 mm Tris-HCl (pH 8.1) and 2 mm EDTA and disrupted by French press (5501-M, Ohtake Works, Tokyo, Japan) at 4 °C. The disrupted cells were centrifuged at 100,000 × g for 40 min, and the supernatant (crude extract) was applied to a QAE-Toyopearl 550c column (Tosoh, Japan). Proteins were then eluted with a 150-500 mm linear gradient of NaCl in 25 mm Tris-HCl (pH 8.1) and 2 mm EDTA. The peak fractions containing HCF164sol were collected, and solid ammonium sulfate was added to be a final concentration of 0.8 m. The solution was then applied to a Butyl-Toyopearl 650M column (Tosoh) and eluted with a 0.8-0 m inverse gradient of ammonium sulfate in 25 mm Tris-HCl (pH 8.1) and 2 mm EDTA. Recombinant PSI-N was sequestered in inclusion bodies in E. coli BL21 (DE3) cells and thus purified as follows. Cells were suspended in 25 mm Tris-HCl (pH 7.5) and 5 mm EDTA containing a protease inhibitor mixture tablet (Roche Applied Science), disrupted by sonication, and centrifuged at 11,000 × g for 10 min. The resulting inclusion bodies were washed and dissolved as described previously (17Motohashi K. Koyama F. Nakanishi Y. Ueoka-Nakanishi H. Hisabori T. J. Biol. Chem. 2003; 278: 31848-31852Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The supernatant was diluted 50-100-fold with 25 mm PIPES-NaOH (pH 6.9), 1 mm EDTA, 1 mm DTT, and 30% glycerol. The insoluble aggregate was removed by centrifugation, and the protein solution was applied to an SP-Toyopearl 650M column (Tosoh). The protein was eluted from the column with a 0-500 mm gradient of NaCl in 25 mm PIPES-NaOH (pH 6.9) and 1 mm EDTA. The peak fractions containing PSI-N were collected and stored at -80 °C. Preparation of Trx-f and Trx-m—Recombinant Trx-f and Trx-m were expressed in E. coli using the plasmids constructed for spinach Trxs (23Stumpp M.T. Motohashi K. Hisabori T. Biochem. J. 1999; 341: 157-163Crossref PubMed Scopus (38) Google Scholar). Trx-f was purified as follows. E. coli cells were suspended with 25 mm Tris-HCl (pH 8.1) and disrupted by French press at 4 °C. The disrupted cells were centrifuged at 100,000 × g for 40 min, and the supernatant was applied to a QAE-Toyopearl 550c column. The desired protein was then eluted with a 0-300 mm linear gradient of NaCl in 25 mm Tris-HCl (pH 8.1). The peak fraction containing Trx-f was dialyzed against 25 mm MES-NaOH (pH 6.1), applied to a SP-Toyopearl 650M column. and eluted with a 0-250 mm linear gradient of NaCl in 25 mm MES-NaOH (pH 6.1). Trx-m was purified as described previously without DTT (17Motohashi K. Koyama F. Nakanishi Y. Ueoka-Nakanishi H. Hisabori T. J. Biol. Chem. 2003; 278: 31848-31852Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Protease Protection Assay—“Intact thylakoids” from Arabidopsis were prepared as described (24Casazza A.P. Tarantino D. Soave C. Photosynth. Res. 2001; 68: 175-180Crossref PubMed Scopus (60) Google Scholar). “Sonicated thylakoids” were prepared by sonicating thylakoid membranes for 3 min at 0 °C, by microtip of the sonifier (Branson Sonifier 250, output 2, duty cycle 30%). Both thylakoid preparations (chlorophyll concentration, 50 μg/ml) were incubated with 0.1 mg/ml thermolysin at 0 °C in 0.1 m sorbitol, 5 mm MgCl2, 10 mm NaCl, 20 mm KCl, 30 mm Tricine-KOH (pH 8.0), and 5 mm CaCl2 (21Meurer J. Plucken H. Kowallik K.V. Westhoff P. EMBO J. 1998; 17: 5286-5297Crossref PubMed Scopus (178) Google Scholar). Protease reactions were terminated at the times indicated by addition of EDTA (pH 8.0, final 50 mm). Thylakoids were precipitated with trichloroacetic acid (final 5%) and washed with ice-cold acetone. The protein samples were analyzed by SDS-PAGE and Western blotting. Polyclonal anti-HCF164 and anti-PSI-N serum were raised in rabbits against purified HCF164sol or PSI-N protein. Polyclonal anti-maize L-FNR serum was a gift from Toshiharu Hase (Osaka University, Osaka, Japan) (25Onda Y. Matsumura T. Kimata-Ariga Y. Sakakibara H. Sugiyama T. Hase T. Plant Physiol. 2000; 123: 1037-1045Crossref PubMed Scopus (122) Google Scholar). Orientation of Active Cysteines of HCF164 on Thylakoid Membranes—Intact thylakoids or sonicated thylakoids from Arabidopsis (chlorophyll concentration, 200 μg/ml) were incubated with 5 mm tris-(2-carboxyethyl) phosphine (TCEP), 0.1 m sorbitol, 5 mm MgCl2, 10 mm NaCl, 20 mm KCl, and 30 mm HEPES-KOH (pH 7.1) for 30 min at 25 °C. After incubation, both thylakoid samples were washed twice with the same solution without TCEP, precipitated with trichloroacetic acid (final 5%), washed with ice-cold acetone, and finally dissolved in 50 mm Tris-HCl (pH 6.8), 1% SDS, and 10 mm 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) (12Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 17Motohashi K. Koyama F. Nakanishi Y. Ueoka-Nakanishi H. Hisabori T. J. Biol. Chem. 2003; 278: 31848-31852Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The protein samples were then analyzed by 13% nonreducing SDS-PAGE and Western blotting using anti-HCF164 serum. Trx-dependent Reduction Assay of HCF164 and PSI-N in Thylakoids—Arabidopsis (ecotype Columbia) was grown on MS medium (1× MS salts, 3 μg/ml thiamine hydrochloride, 5 μg/ml nicotinic acid, 0.5 μg/ml pyridoxine hydrochloride, and 0.2% gellan gum), in a 16-h light/8-h dark cycle at 22 °C. Intact thylakoids (chlorophyll concentration, 80 μg/ml), were incubated with or without Trx-f (final 5 μm) or Trx-m (final 5 μm)in the presence or absence of DTT (final 10 μm) in 0.1 m sorbitol, 5 mm MgCl2, 10 mm NaCl, 20 mm KCl, 30 mm Tricine-KOH (pH 8.0) for 60 min at 25 °C. Experiments to measure the DTT concentration- and time-dependent reduction of HCF164 in intact thylakoids were performed under conditions described in the legend of Fig. 2. Following completion of the reaction, the samples were precipitated with trichloroacetic acid (final 5%), washed with ice-cold acetone, and finally dissolved in buffer containing 50 mm Tris-HCl (pH 6.8), 1% SDS, and 10 mm AMS (12Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 17Motohashi K. Koyama F. Nakanishi Y. Ueoka-Nakanishi H. Hisabori T. J. Biol. Chem. 2003; 278: 31848-31852Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Reduced and oxidized proteins were separated by non-reducing SDS-PAGE and detected by Western blotting. Insulin Reduction Assay—To check the disulfide reduction activity of the recombinant soluble domain of HCF164 and to compare with that of stromal Trx proteins, we measured the change in turbidity of the insulin solution because of precipitation of free insulin B chain by reduction (14Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (174) Google Scholar, 26Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). The assay mixture contained 100 mm potassium phosphate (pH 7.0), 2 mm EDTA, and 130 μm bovine insulin. The reaction was initiated by adding 330 μm DTT into the assay mixture, and the change in turbidity was monitored at 650 nm at 25 °C. Determination of the Redox Potential of HCF164sol Protein— Wild type HCF164sol (1 μm) was incubated at 25 °C for 3 or 16 h in 50 mm potassium phosphate buffer (pH 7.0), 100 mm oxidized DTT, and various concentrations of reduced DTT (0.5 μm to 2 mm), under nitrogen atmosphere. To minimize oxidation by air, buffer solutions were thoroughly degassed. After incubation, samples were treated with trichloroacetic acid (final 5%). The protein precipitates were washed with ice-cold acetone and then dissolved in buffer containing 50 mm Tris-HCl (pH 6.8), 1% SDS, and 2 mm AMS. Reduced (AMS derivative) and oxidized (nonderivative) forms of HCF164sol were separated by 15% nonreducing SDS-PAGE, stained with Coomassie Brilliant Blue R-250, and quantified by using LAS-3000mini CCD-imaging (Fujifilm, Tokyo, Japan) (27Inaba K. Takahashi Y.H. Ito K. J. Biol. Chem. 2005; 280: 33035-33044Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The equilibrium constant and the standard redox potential were calculated as described (28Huber-Wunderlich M. Glockshuber R. Folding Des. 1998; 3: 161-171Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), using a value of -330 mV for the standard redox potential of DTT as a reference (29Singh R. Lamoureux G.V. Lees W.J. Whitesides G.M. Methods Enzymol. 1995; 251: 167-173Crossref PubMed Scopus (46) Google Scholar). When Trx-f (1 μm) and Trx-m (1 μm) were incubated in the redox equilibrium buffer, the reduced DTT concentrations were varied from 20 μm to 100 mm against 100 mm oxidized DTT. Large Scale Preparation of Arabidopsis Thylakoids for Screening of HCF164 Target Proteins—Arabidopsis (ecotype Columbia) rosette leaves from 4- to 5-week-old plants were harvested and then rapidly homogenized in a Waring blender in ice-cold 330 mm sorbitol, 5 mm sodium ascorbate, 0.05% BSA, 2 mm EDTA, 1 mm MgCl2, 1 mm MnCl2, and 50 mm HEPES-NaOH (pH 7.6), filtered through Miracloth (Calbiochem), and centrifuged at 2,000 × g for 5 min. The resulting chloroplasts were resuspended in 330 mm sorbitol, 5 mm sodium ascorbate, 1 mm MgCl2, and 50 mm HEPES-NaOH (pH 7.6) and centrifuged at 1,300 × g for 5 min. They were then resuspended with 330 mm sorbitol, 2 mm sodium ascorbate, 1 mm MgCl2, 1 mm MnCl2, 2 mm EDTA, 2 mm NaNO3, 5 mm NaHCO3, 0.5 mm K2HPO4, 5 mm sodium diphosphate, and 50 mm HEPES-NaOH (pH 7.6) and centrifuged at 1,300 × g for 5 min. The precipitated chloroplasts were resuspended with 50 mm Tricine-KOH (pH 8.0), 2 mm EDTA, 1% protease inhibitor mixture for plant cell extracts (Sigma) at a final concentration of 0.5 mg/ml chlorophyll. The broken chloroplast suspension was then centrifuged at 7,500 × g for 10 min at 4 °C and washed twice with 10 mm sodium dihydrogen pyrophosphate-NaOH (pH 7.8) to remove peripheral proteins (30Kieselbach T. Hagman A. Andersson B. Schroder W.P. J. Biol. Chem. 1998; 273: 6710-6716Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Arabidopsis thylakoids were obtained as a precipitate of this suspension following centrifugation for 10 min at 7,500 × g, for screening of HCF164 target proteins. Screening of HCF164 Target Proteins in Arabidopsis Thylakoids—HCF164sol (CS)-mutant (149WCEVC153 to 149WCEVS153) was immobilized on CNBr-activated Sepharose 4B (GE Healthcare) according to the manufacturer's instructions. Arabidopsis thylakoids were solubilized in 50 mm Tricine-KOH (pH 8.0), 2 mm EDTA, 1% protease inhibitor mixture for plant cell extracts (Sigma) and 1% n-octyl-β-d-glucoside for 60 min at 4 °C with gentle mixing and centrifuged at 140,000 × g for 30 min. The solubilized thylakoid protein fraction was obtained as a supernatant. The detergent-solubilized fraction was then incubated with the HCF164sol (CS)-mutant immobilized resin for 60 min at room temperature. The resin was washed with 50 mm Tricine-KOH (pH 8.0), 2 mm EDTA, and 1% n-octyl-β-d-glucoside (Buffer A) to remove any non-specifically bound proteins. The resin was then washed with Buffer A plus 500 mm NaCl and with Buffer A containing 500 mm NaCl and 0.1% SDS. Each washing step was repeated until the absorbance of the washed solution at 280 nm approached zero. The resin was finally suspended in Buffer A plus 0.1% SDS and 20 mm DTT for 60 min, and the eluted proteins were analyzed by SDS-PAGE (15% (w/v)) and stained with Coomassie Brilliant Blue R-250. Stained protein bands were identified by N-terminal amino acid analysis using a peptide sequencer and by peptide mass fingerprint (PMF) analysis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (12Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 14Yamazaki D. Motohashi K. Kasama T. Hara Y. Hisabori T. Plant Cell Physiol. 2004; 45: 18-27Crossref PubMed Scopus (174) Google Scholar, 31Hosoya-Matsuda N. Motohashi K. Yoshimura H. Nozaki A. Inoue K. Ohmori M. Hisabori T. J. Biol. Chem. 2005; 280: 840-846Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). HCF164-dependent Reduction of the Recombinant PSI-N in Vitro—PSI-N (8 μm) in 25 mm Tris-HCl (pH 7.5) was incubated for 60 min at 25 °C with 50 μm CuCl2 or various concentrations of DTT in the presence or absence of HCF164sol (5 μm). The redox states of PSI-N were assessed by the AMS labeling as described (12Motohashi K. Kondoh A. Stumpp M.T. Hisabori T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11224-11229Crossref PubMed Scopus (333) Google Scholar, 17Motohashi K. Koyama F. Nakanishi Y. Ueoka-Nakanishi H. Hisabori T. J. Biol. Chem. 2003; 278: 31848-31852Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). PSI-N bands were visualized by Western blotting using anti-PSI-N antibody because the mobility of reduced form of PSI-N was almost same as that of HCF164sol. A Disulfide Bond in the Thioredoxin Domain of HCF164 Faces the Thylakoid Lumen—To probe the redox-related function of HCF164, we initially sought to determine the source of the reducing equivalents supplied to the luminal side of the thylakoid membranes. To achieve this, we first prepared Arabidopsis thaliana (Arabidopsis) thylakoid membranes and determined their degree of intactness; incubation of isolated thylakoids with the protease thermolysin resulted in the rapid degradation of FNR, which is located on the stroma-exposed surface of thylakoid membranes, whereas the amount of the extrinsic luminal PSI-N protein (32Nielsen V.S. Mant A. Knoetzel J. Moller B.L. Robinson C. J. Biol. Chem. 1994; 269: 3762-3766Abstract Full Text PDF PubMed Google Scholar, 33Scheller H.V. Jensen P.E. Haldrup A. Lunde C. Knoetzel J. Biochim. Biophys. Acta. 2001; 1507: 41-60Crossref PubMed Scopus (170) Google Scholar) was unaffected (Fig. 1A, left panels). In contrast, following sonication of intact thylakoid membrane samples, both FNR and PSI-N were rapidly degraded by thermolysin (Fig. 1A, right panels), clearly indicating that our thylakoid preparations were intact. Using these thylakoid preparations, we carried out a protease protection assay of HCF164. Although the N-terminal region of HCF164, which is predicted to extend into the stroma, was degraded by protease within 2 min (Fig. 1A, left upper panel), the 19-kDa fragment of HCF164 corresponding to the hydrophilic C-terminal domain, which contains the active cysteine residues, was protected for at least 30 min. In contrast, the largest component of the HCF164 protein was immediately degraded in sonicated thylakoid membranes by thermolysin treatment. Moreover, we determined the orientation of the active cysteines of the thioredoxin domain of HCF164 using the membrane-impermeable reducing reagent, TCEP (34Pelkmans L. Puntener D. Helenius A. Science. 2002; 296: 535-539Crossref PubMed Scopus (595) Google Scholar, 35Koriazova L.K. Montal M. Nat. Struct. Biol. 2003; 10: 13-18Crossref PubMed Scopus (271) Google Scholar, 36Hsu M.F. Sun S.P. Chen Y.S. Tsai C.R. Huang L.J. Tsao L.T. Kuo S.C. Wang J.P. Biochem. Pharmacol. 2005; 70: 1320-1329Crossref PubMed Scopus (17) Google Scholar). In intact thylakoids, HCF164 was found to be present in the oxidized form and was not reduced by TCEP (Fig. 1B, Intact). In contrast, HCF164 was easily reduced when thylakoids were disrupted by sonication (Fig. 1B, Sonicated). These results substantiate existing evidence that the large hydrophilic domain of HCF164, which includes the active site Cys motif, is located in the thylakoid lumen, as suggested previously by Lennartz et al. (22Lennartz K. Plucken H. Seidler A. Westhoff P. Bechtold N. Meierhoff K. Plant Cell. 2001; 13: 2539-2551Crossref PubMed Google Scholar). HCF164 Receives Electrons from Trx Located on the Stromal Side—Having characterized the orientation of HCF164 within the membrane, we proceeded to determine the source of the reducing equivalents for reduction of the luminal portion of the HCF164 protein. In chloroplasts, electrons produced by photosynthetic electron transport ultimately accumulate within the stroma, the site of the chloroplast Trxs. In prokaryotic bacteria, the polytopic membrane protein cytochrome c defective A (CcdA) is known to act as a transducer of reducing equivalents, bridging the transfer of electrons from the cytoplasmic reduced form Trx to substrates within the periplasm across the membrane (37Katzen F. Deshmukh M. Daldal F. Beckwith J. EMBO J. 2002; 21: 3960-3969Crossref PubMed Scopus (77) Google Scholar); an ortholog of the bacterial CcdA protein is also present in Arabidopsis (38Nakamoto S.S. Hamel P. Merchant S. Biochimie (Paris). 2000; 82: 603-614Crossref PubMed Scopus (29) Google Scholar). We therefore sought to investigate the possibility of a related pathway in thylakoid membranes. To determine the source of reducing equivalents, which could reduce the active cysteines of HCF164 on the luminal side, we monitored the redox state of HCF164 in intact thylakoids treated with an exogenous supply of reductant. The HCF164 protein was found to occur in the oxidized form in freshly prepared intact Arabidopsis leaf thylakoids (Fig. 2A, -Trx without DTT). This oxidized form of HCF164 could be partially reduced by the reduced form of Trx-m (Fig. 2A, Trx-m with DTT) but not by oxidized Trx-m (Fig. 2A, Trx-m without DTT). The reduced forms of Trx-f or 10 μm DTT without Trx were ineffective in reducing HCF164 (Fig. 2A, Trx-f with DTT and -Trx with DTT). Reduction of HCF164 was accomplished by Trx-m even in the presence of low concentrations of DTT but not by Trx-f (Fig. 2B). The reduction rate of HCF164 by Trx-m was fast (t1/2 <10 min) reaching a maximum within 30 min (Fig. 2C). These results clearly indicate that the stromal reduced form Trx-m is able to transfer reducing equivalents across the membrane to the luminal HCF164 protein. Disulfide Reduction Activity and Redox Potential Determination of the HCF164 Protein—To further characterize the biochemical properties of HCF164, we constructed an expression system consisting of the soluble C-terminal domain of the HCF164 protein containing the Trx motif alone (hereafter designated as HCF164sol), lacking both the N-terminal chloroplast signal sequence and the membrane spanning domain (supplemental Fig. S1) (22Lennartz K. Plucken H. Seidler A. Westhoff P. Bechtold N. Meierhoff K. Plant Cell. 2001; 13: 2539-2551Crossref PubMed Google Scholar). The insulin reduction activity displayed by purified HCF164sol was found to be lower than that measured for the well characterized stromal Trxs Trx-f and Trx-m (Fig. 3). To evaluate the feasibility of a putative electron cascade from stromal Trx to HCF164 in the thylakoid lumen, the redox potential values of HCF164sol and Trxs were determined using a DTTred/DTTox redox buffer. Following equilibration of HCF164 and Trxs with increasing ratios of DTTred/DTTox at 25 °C, the proteins were acid-denatured and treated with the thiol modifier AMS (27Inaba K. Takahashi Y.H. Ito K. J. Biol. Chem. 2005; 280: 33035-33044Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 39Kobayashi T. Kishigami S. Sone M. Inokuchi H. Mogi T. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11857-118