Cytochrome c6 from Cyanophora paradoxa

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Cytochrome c6 from Cyanophora paradoxa Characterization of the protein and the cDNA of the precursor and import into isolated cyanelles JuÈrgen M. Steiner1, Aurelio Serrano3, GuÈnter Allmaier2, Johannes Jakowitsch1 and Wolfgang LoÈffelhardt1 1

Institut fuÈr Biochemie und Molekulare Zellbiologie der UniversitaÈt Wien und Ludwig-Boltzmann-Forschungsstelle fuÈr Biochemie; Institut fuÈr Analytische Chemie der UniversitaÈt Wien, Vienna, Austria; 3Instituto de BioquõÂmica Vegetal y FotosõÂntesis, CSIC y Universidad de Sevilla, Spain

2

In the eukaryotic alga Cyanophora paradoxa, which does not contain plastocyanin, photosynthetic electron transport from the cytochrome b6 /f complex to photosystem I is mediated by cytochrome c6. Cytochrome c6 was purified to homogeneity by column chromatography and FPLC. The relative molecular mass of the holoprotein was determined by two different mass spectrometric methods (californium-252 plasma desorption and UV matrix-assisted laser desorption ionization) giving 9251 ^ 3.3 Da. N-terminal Edman microsequencing yielded information on approx. 30 amino acid residues. Based on these data and on highly conserved regions of cytochromes c6, degenerate oligonucleotides were designed and used for PCR to amplify the genomic DNA of C. paradoxa. Screening of a C. paradoxa cDNA library yielded several clones coding for preapo-cytochrome c6 . The deduced sequence of the mature protein was verified by plasma desorption mass spectrometric peptide mapping and shows high similarity to those of cytochromes c6 from cyanobacteria and algae. Cytochrome c6 appears to be encoded by a single nuclear gene ( petJ ) in C. paradoxa. As the mature protein is located in the lumen of the thylakoid membrane, it has to traverse three biological membranes as well as the unique peptidoglycan layer of the cyanelles before it reaches its final subcellular locale. Thus the transit sequence is composed of two different targeting signals: a stroma targeting peptide resembling those of higher plants with respect to hydropathy plots and amino acid composition and a hydrophobic signal peptide functioning as a thylakoid-traversing domain. There are indications for alternative sorting of part of the cyanelle cytochrome c6 pool to the periplasmic space. This is the first known bipartite transit sequence of a cyanelle precursor protein from C. paradoxa, a model organism concerning the endosymbiotic origin of plastids. Labeled precursor is efficiently imported into isolated cyanelles, then routed into thylakoids and processed to the mature protein. Hitherto, in vitro protein translocation was not reported for cyanobacterial-type thylakoids. Keywords: Cyanophora paradoxa; cyanelles; cytochrome c6; mass spectrometry; in vitro import. The cyanelles from Cyanophora paradoxa and other glaucocystophyceae are distinguished among plastids by the retention of a modified form of the peptidoglycan wall of their cyanobacterial ancestors [1]. The recently sequenced 136-kb cyanelle genome [2], though harboring a large number of genes, the protein products of which are nucleus-encoded in higher plants, nevertheless cannot account for all necessary polypeptides. Thus, like chloroplasts, cyanelles have to import a multitude of cytoplasmically synthesized precursor proteins [3]. These cyanelle precursors have to cross two membranes and the peptidoglycan layer in the case of stromal proteins, and their transit sequences resemble stroma-targeting peptides from higher plants as was shown for ferredoxin-NADP1 reductase Correspondence to Institut fuÈr Biochemie und Molekulare Zellbiologie, Biozentrum der UniversitaÈt Wien, Dr Bohrgasse 9, A-1030 Wien, Austria. Fax: 43 1 4277 9528. Tel.: 43 1 4277 52811. E-mail: [email protected] Abbreviations: FNR, ferredoxin-NADP1 reductase; PD, plasma desorption; MALDI, matrix-assisted laser desorption/ionization; CNBR, cyanogen bromide; endo, endoproteinase; DIG, digoxigenin; STP, stroma-targeting peptide. Dedication: Dedicated to professor Helmut Ruis on the occasion of his 60th birthday. (Received 8 February 2000, revised 12 April 2000, accepted 10 May 2000)

(FNR) [4] and for glyceraldehyde-3-phosphate dehydrogenase (H. Brinkmann, personal communication). Heterologous import of the FNR precursor from C. paradoxa into isolated pea chloroplasts occurred with high efficiency, and a higher plant pre-FNR was imported (to a lesser extent) into isolated cyanelles, pointing toward a similar organization and function of the cyanelle and chloroplast protein import machineries [5,6]. Our aim is to characterize more transit sequences of nucleus-encoded cyanelle proteins and, especially, to investigate the bipartite targeting signals of lumenal protein precursors [3] that, in addition, have to cross the thylakoid membrane. The soluble c-type cytochromes are ubiquitously distributed heme proteins which function in the electron transfer chains of mitochondria, bacteria, and the plastids of some algae [7]. These proteins vary in length from 79 to 129 amino acids and have redox potentials ranging from 100 to 400 mV. The major metabolic function of the soluble cytochromes c is in quinol oxidation via a membrane-associated complex of redox centers containing cytochromes b, a more hydrophobic class of cytochromes c (c1 or f ), and a high potential Fe-S center. This complex, common to most energy-transducing membranes, represents a phylogenetically ancient reaction pathway. In the chloroplasts of vascular plants, plastoquinol oxidation via the

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cytochrome b6 /f complex is catalyzed by plastocyanin instead of a c-type cytochrome, whereas most algae and cyanobacteria can use either of them. In C. paradoxa, photosynthetic electron transport from the cytochrome b6 /f complex to photosystem I is mediated exclusively by cytochrome c6 (previously named cytochrome c553). Plastocyanin is not present [8], nor does the corresponding gene exist [9]. Thus cytochrome c6 should be a relatively abundant protein of the thylakoid lumen that previously has already been detected in and partially purified from C. paradoxa [8±10]. Our goal was to purify the protein to homogeneity, characterize it by N-terminal Edman sequencing and mass spectrometry and to use the information thus obtained to isolate and characterize the cDNA encoding the respective precursor protein. An import experiment into isolated cyanelles using labeled precursor would be the first protein translocation observed in vitro across phycobilisome-bearing cyanobacterialike thylakoid membranes. Isolated cyanobacterial thylakoids are not suitable for such experiments as they do not form tight vesicles [6]. Among algae, the biosynthesis, translocation and assembly of cytochrome c6 has been thoroughly studied in Chlamydomonas reinhardtii [11]. Protein or gene (petJ) sequences are also available from the red algae Porphyra purpurea containing the only plastid-encoded member [12] and Porphyridium cruentum [13], from Euglena gracilis [14] and from several cyanobacteria [13±17] revealing highly conserved domains suitable for the design of PCR primers. Besides the well established role of cytochrome c6 in photosynthesis there is growing evidence for a function in respiratory electron transport. In cyanobacteria, cytochrome c6 can act as an electron donor to photosystem I as well as to cytochrome oxidase [7]. This is based on the following observations: (a) antibodies directed against cytochrome c6 from Anabaena sp. PCC 7119 inhibited not only photosynthetic, but also respiratory, electron transport in membrane preparations from this cyanobacterium [18]; (b) Anabaena variabilis cytochrome c6 effectively donates electrons to cytochrome oxidase in vitro [19], and (c) most of the cytochrome c6 in Anabaena sp. ATCC 29413 [20] and Nostoc sp. PCC 8009 [21] localizes to the periplasmic space. As cyanelles show many cyanobacterial features, a possible periplasmic localization of their cytochrome c6 should also be checked. SecY, a key compont of the bacterial preprotein translocase, is encoded in the cyanelle genome and was shown to function in Escherichia coli [22]. Nucleus-encoded SecY is supposed to play a role in thylakoid import of many but not all lumenal proteins in higher plants [3]. In cyanobacteria, SecA/Y dependent translocases were found in the thylakoid as well as the inner envelope membrane [23]. Here we report the isolation of the holoprotein and its mass spectrometric characterization as well as the cloning and sequencing of a cDNA for preapo-cytochrome c6 from C. paradoxa. A labeled precursor was imported in vitro into isolated cyanelles and correctly sorted to the thylakoid lumen. The mature protein resulting from two processing steps was shown to be protease-protected. Thylakoid lumen and periplasm are the subcompartments where cyanelle cytochrome c6 could be tentatively localized.

M AT E R I A L S A N D M E T H O D S Organisms C. paradoxa LB555UTEX was grown as previously described [4]. In general, cells were harvested in the exponential growth phase. Nucleic acids were isolated as described [4].

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Cytochrome c6: purification and amino acid sequence analysis Preparation of total cell-free extracts and cyanelle extracts was performed as described [24]. The cyanelle extracts were subjected to salt fractionation. Solid ammonium sulphate was slowly added with stirring to the extract to make up to 40% saturation. After 20 min, the protein solution was centrifuged (40 000 g, 20 min) and the supernatant was brought to 90% ammonium sulphate saturation, before being treated as above and centrifuged. The pellet was resuspended in a minimum volume (less than 1 mL) of standard buffer (25 mm Tris/HCl pH 7.5, 1 mm EDTA, 2 mm dithiothreitol, 10% glycerol, 1 mm PhCH2SO2F), dialyzed against the same buffer, and eventually applied to a 1.6  18 cm DEAE-cellulose column (Whatman DE-52). When the column was washed with standard buffer to remove proteins not adsorbed, a pink band was slowly eluted. This fraction was pH-adjusted with a small amount of 3 m sodium acetate buffer pH 5.0, applied onto a 1  18 cm CM-cellulose column (Whatman) equilibrated with 60 mm sodium acetate buffer pH 5.0, 2 mm dithiothreitol, and then washed with the equilibration buffer. After concentration of the flow-through and buffer change to 25 mm Tris/HCl, pH 8.5 with an Amicon YM3 membrane, the pink-coloured fraction was applied to a second 1  5 cm DEAE-cellulose column equilibrated with the new buffer. In this case the protein remained bound to the bed and was eluted with buffer supplemented with 50 mm NaCl. The protein was further purified to homogeneity by gel filtration on a Superose 6HR 10/30 column (1  30 cm) using an automated Pharmacia FPLC system. Isocratic elution was carried out at 25 8C with standard buffer at a flow rate of 0.5 mL´min21. Fractions containing a single polypeptide were pooled, concentrated on a Microcon 3 device (Amicon) and sequenced on an Applied Biosystems 476A gas phase sequenator at the Vienna Biocenter. Cytochrome spectra were recorded on a Hitachi U-3000 photometer. Antibody production and Western blotting Antibodies were raised in rabbits against purified cytochrome c6 from C. paradoxa and used at a dilution of 1 : 2000 on semidry blotted nylon membranes containing proteins from the pellet fraction of cyanelles of C. paradoxa subjected to various treatments, and from the corresponding supernatants. As secondary antibodies, goat anti-(rabbit IgG) conjugated to alkaline phosphatase (Promega) were used according to the instructions of the manufacturer. Mass spectrometry Plasma desorption (PD) time-of-flight mass spectrometry. Positive and negative ion mass spectra were obtained on a short linear californium-252 PD time-of-flight instrument (BioIon AB, Uppsala, Sweden). Acceleration voltage was between 15 and 20 kV and mass spectra were accumulated for 1±30 million fission events. Calibration of the mass spectra was done in the positive ion mode with H1 and Na1 ions and in the negative ion mode with H2 and CN2 ions. Average mass values are reported throughout the whole paper for measured and calculated values. For the sample preparation of the intact protein (approx. 9 mg of protein deposited) and unseparated peptide mixtures, the nitrocellulose adsorption technique was applied [25]. After sample deposition, all targets were rinsed three times with 20 mL of cold water containing 0.05%

4234 J. M. Steiner et al. (Eur. J. Biochem. 267)

trifluoroacetic acid to remove alkali contamination prior to PD mass spectrometric analysis. Matrix-assisted laser desorption/ionization (MALDI) time-offlight mass spectrometry. Mass spectrometric measurements were performed in the reflector mode on a Kompact MALDI IV (Shimadzu Kratos Analytical, Manchester, UK) laser desorption time-of-flight instrument equipped with a type VSL-337ND (Laser Science, Newton, MA, USA) nitrogen laser (l ˆ 337 nm, 3 ns pulse width). Positive ions were accelerated from the target in the continuous mode to a final potential of 24 kV and laser power density was kept near threshold levels. Mass spectra were obtained by signal averaging of 10 laser shots. Calibration of the instrument was done externally using the [M 1 H]1 and [M 1 2H]21 ions of the standard protein cytochrome c (horse heart; Sigma, St Louis, MO, USA), and average mass values are reported. The lyophilized protein sample (50 mg) was redissolved in 100 mL of water/acetonitrile (1 : 1) containing 0.1% trifluoroacetic acid. Sinapinic acid was applied as matrix (Sigma). Matrix solution (0.8 mL, saturated solution in water/acetonitrile 1 : 1) was placed in the center of the sample well of the disposable stainless-steel 20-well target and mixed with 0.2 mL of the sample solution on the target at room temperature. After the droplet had dried, insertion into the ion source followed. Chemical and enzymatic cleavage reactions Cyanogen bromide (CNBr) cleavage. The lyophilized, purified cytochrome c6 (50 mg) was dissolved in 150 mL of water followed by 350 mL of formic acid and incubated with CNBr (methionine/CNBr molar ratio 1 : 200) in the dark under nitrogen at room temperature for 24 h. The reaction was stopped by flash-freezing and lyophilization. The sample was redissolved in 100 mL of water and 10 mL aliquots were used for direct PD mass spectrometric analysis or subsequent enzymatic cleavage. Endoproteinase Lys-C cleavage. A 10-mL aliquot of the unseparated CNBr peptide mixture was diluted with 90 mL of 0.1 m ammonium bicarbonate buffer (pH 8.6) and degraded with 0.5 mg of the endoproteinase Lys-C (endo Lys-C; Boehringer Mannheim) for 18 h at 37 8C. A 10-mL aliquot was used for subsequent PD mass spectrometric analysis. Endoproteinase Glu-C cleavage. A 10-mL aliquot of the unseparated CNBr peptide mixture was diluted with 90 mL of 0.1 m ammonium bicarbonate buffer (pH 7.8) and degraded with 4 mg of the endoproteinase Glu-C (endo Glu-C; Boehringer Mannheim) for 16 h at 37 8C. A 10-mL aliquot was used for direct mass spectrometric analysis. PCR amplification of cytochrome c6-encoding genomic DNA The degenerate nucleotide sequence determined via reverse translation of the partial amino acid sequence and of a highly conserved region of cytochromes c6 required for heme attachment were used to design a 768-fold degenerate, forward PCR primer (PR1 ˆ 5 0 -AACTGCAGAYGGNGCNGCNATHTTYAC-3 0 ) and a 256-fold degenerate reverse PCR primer (PR2 ˆ 5 0 -AAGAATTCNGGCATNGCRTTYTTNCC-3 0 ). The PCR reaction mixture (50 mL) included 100 ng of DNA from C. paradoxa, 0.1 mm concentration of each primer species, 10 mm Tris/HCl (pH 8.3), 50 mm KCl, 1 mm MgCl2, 0.2 mm dNTPs, and 1 U of Taq DNA polymerase (Dynazyme, Finnzymes Oy). The following thermal cycle was

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used: Step 1, 96 8C for 5 min; Step 2, 94 8C for 1 min; Step 3, 45 8C for 2 min; Step 4, 72 8C for 3 min; Step 5, repeat steps 2±4 35 times; Step 6, 72 8C for 7 min. The predominant PCR product was digested with EcoRI and PstI and subcloned into the phagemid pBluescript KS1 (Stratagene) that had been digested with those enzymes. Gel electrophoresis Proteins were separated on SDS-containing polyacrylamide gels [26], RNA on 1.2% agarose gels containing formaldehyde [27], and DNA on 0.8±1.5% agarose gels in 89 mm Tris, 89 mm boric acid, 2 mm Na2EDTA or in 50 mm Tris, 20 mm sodium acetate, 27 mm acetic acid, 2 mm Na2EDTA [27]. cDNA library screening A C. paradoxa cDNA library in the vector lNM1149 was obtained from Dr H. Brinkmann (Braunschweig, Germany). Plaque hybridization screening using the PCR-generated probe, labeled with the digoxigenin Labeling/Detection System (Boehringer Mannheim), was performed under high stringency conditions [27]. Southern and Northern blot analysis Total DNA (10 mg per digest) from C. paradoxa was incubated for 20 h at 37 8C with 40 U of various restriction enzymes, single or in combination. Agarose gel electrophoresis and blotting, as well as the prehybridization, hybridization, and washing steps were carried out under standard conditions [27]. Total RNA (15 mg per lane) from light or dark grown cultures of C. paradoxa was separated electrophoretically, blotted and hybridized to the same DIG-labeled petJ cDNA probe [27]. DNA sequencing Plasmid DNA was purified and both strands were sequenced by the method of Kraft et al. [28]. In parallel, the sequence was determined in an automatic sequencer (LONGREADIR 4200, LI-COR). The petJ cDNA sequence of C. paradoxa was deposited in the EMBL Nucleotide Sequence Database under the accession no. AJ271743. Isolation of import-competent cyanelles Cyanophora cells suspended in 25 mm Hepes buffer, pH 7.6, 0.35 m sucrose, 2 mm EDTA were broken in a Waring Blendor: 5  1 min full speed, in between 1 min cooling in ice water [6]. Inspection under the light microscope should reveal more than 90% of broken cells. Cyanelles were pelleted at 1500 g (2 min in a Sorvall centrifuge, GSA rotor, with the brake off). The pellet was carefully suspended in 8 mL of SRM buffer (50 mm Hepes, 0.33 m sorbitol, pH 8.0) using a fine brush. Cyanelle suspension (4 mL) was layered on top of a cushion of 40% Percoll (in SRM buffer, 4 mL). After centrifugation for 7.5 min in a Sorvall SS34 rotor at 2000 g, the blue-green pellet was gently dissolved in 1 mL of SRM buffer, diluted to 40 mL with SRM buffer and pelleted at 1000 g for 2 min. This washing procedure was repeated twice. Import assay The radiolabeled precytochrome c6 was synthesized by in vitro transcription/translation of the respective cDNA cloned into the pBAT vector [29], as described previously [5]. A cyanelle

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Cyanelle cytochrome c6 (Eur. J. Biochem. 267) 4235

suspension in SRM buffer, equivalent to 100 mg of chlorophyll was incubated with the translation mixture for 15 min at 25 8C in a total volume of 240 mL. Prebinding of precursor proteins to cyanelles was done under conditions identical to those described above except that all steps were carried out at 0 8C. Cyanelles were then isolated again by centrifugation for 2 min at 800 g. Thermolysin treatment. Thermolysin (100 mg/mL) was added to the resuspended cyanelles containing 10 mm CaCl2 and incubation was continued for 30 min at 4 8C. Reaction products were separated by SDS/PAGE. Alternatively, thermolysintreated cyanelles were lysed by lysozyme treatment (0.3 mg´mL21, 15 min, 4 8C) followed by gentle homogenization, and separated into thylakoid and stroma fractions by centrifugation at 10 000 g for 5 min. Import data were analyzed using a PhosphoImager and the Molecular Dynamics image quant version 3.3 program, such that all the signals remained in the linear detection range.

R E S U LT S Purification of cytochrome c6 from C. paradoxa As a relatively abundant, soluble, cyanelle hemoprotein, the purification of cytochrome c6 could easily be followed during ion-exchange column chromatography on two gel matrices and under different buffer conditions. The separation from bulk protein already occurred during ammonium sulphate fractionation and the first DEAE-cellulose column step. Spectrophotometric analysis (not shown) of the resulting pink fraction indicated that it was composed mainly of the electron-transfer protein cytochrome c6 with the a, b, and g peaks at 553 nm, 523 nm, and 417 nm, respectively. The presence of dithiothreitol in all the buffers allowed us to purify this chromoprotein in the reduced state. Although cytochrome c6 eluted during washing in the next (CM-cellulose) step, being only slightly retained by the column bed, it was further enriched. This was indicated by the ratio A275/A553, a typical purity index for this protein, which decreased by a factor of two. Finally, a second DEAE-cellulose column equilibrated with a more basic buffer was adopted. In this case the protein remained bound to the bed and was eluted with buffer supplemented with 50 mm NaCl. The behaviour of the protein during the ion-exchange chomatographic steps underlines its rather acidic nature. The isoelectric point calculated from the amino acid sequence is 5.0; that reported by Burnap and Trench was 4.2 [10]. The cytochrome c6 preparation was pure, as indicated by the absorbance spectrum which showed a ratio A275/A553 of about 1. One single band of approximately 11 kDa was observed after SDS/PAGE upon staining with Coomasssie Blue or silver stain (not shown). The homogeneity of the protein was also demonstrated by FPLC gel filtration. Only one protein with an apparent molecular mass of about 11 kDa was found, the single A280 peak exactly coinciding with the A553 maximum of the collected fractions. The high purity of the preparation allowed the determination of the N-terminal amino acid sequence from a sample of approximately 0.5 mg of cytochrome c6: A-D-G-A-I-F-T-N-N-X-A-AX-H-A-G-G-N-N-V-I-A-A-E-K-T-L-K-K. No amino acids were detected at positions 10 and 13 (the protein was not subjected to cysteine derivatization with 4-vinylpyridine prior to analysis), pointing toward two conserved cysteine residues for heme attachment.

Fig. 1. Positive ion reflectron MALDI mass spectrum of the intact (including the heme moiety) cytochrome c6 from C. paradoxa.

Molecular mass determination of C. paradoxa cytochrome c6 The relative molecular mass of the purified cytochrome c6 was determined by the two soft ionization techniques MALDI and PD time-of-flight mass spectrometry. The positive ion MALDI mass spectrum (Fig. 1) exhibits one main component corresponding to the protonated ([M 1 H]1) and double-protonated ([M 1 2H]21) molecular ions at m/z 9250.1 and m/z 4626.5, respectively, from which a relative molecular mass of 9250.0 ^ 1.9 Da was calculated. Furthermore, the positive and negative ion PD mass spectra were obtained from the same protein sample, showing abundant [M 1 H]1, [M 2 H]2, [M 1 2H]21and [M 1 3H]31 ions. Based on positive and negative ion data a relative molecular mass of 9251.9 ^ 4.6 Da was calculated. Mass spectrometric peptide mapping of C. paradoxa cytochrome c6 The purified protein was cleaved with CNBr in formic acid and the unfractionated mixture was analyzed by PD mass spectrometry. The high specifity of CNBr and the generation of few but large peptide fragments compared to most enzymatic cleavages, were the main reason for choosing CNBr for the initial protein cleavage. Based on cDNA data, cytochrome c6 contains one methionine residue, hence two peptides would be expected from CNBr cleavage of the protein. PD mass spectrometric analysis of the mixture gave strong signals ([M 1 H]1 ions) assignable to both peptides C-1, amino acids 1±57 (observed m/z 6398.6 as homoserine, calculated m/z 6399.0) and C-2, amino acids 58±84 (observed m/z 2846.8, calculated m/z 2843.1). The mass values of both signals agreed well with the theoretical mass values of the CNBr peptide fragments predicted from the DNA-derived amino acid sequence of cytochrome c6. In order to obtain a more detailed PD mass spectrometric peptide mapping of the protein, proteolytic cleavage with either endo Lys-C or endo Glu-C protease was performed on the unseparated CNBr peptide mixture. The PD mass spectrometric analysis of the endo Lys-C-generated peptides produced protonated molecules diagnostic for three (T-1, T-4 and T-4/5/6 ± incomplete cleavage) of the seven cleavage peptides (T-1 to T-7) expected at m/z 3121.1 (amino acids 1±26), m/z 1841.0 (amino acids 31±47) and m/z 2861.6 (amino acids 31±57, detected as double-formylated homoserine lactone), respectively. The mass accuracy obtained was better than ^ 0.4 Da. Fragments of only one or up to three residues in

4236 J. M. Steiner et al. (Eur. J. Biochem. 267)

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Fig. 2. Amino acid sequence of cytochrome c6 from C. paradoxa. Open arrows indicate N-terminal Edman sequence data; cyanogen bromide cleaved peptides (CNBr); endo Lys-C cleaved peptides (Endo Lys-C); endo Glu-C cleaved peptides (Endo Glu-C), all peptides are indicated with a bar. The nucleotide sequence of the coding region of the mature protein is superimposed. Forward and reverse internal primers used for sequencing are given in bold letters.

length are not normally found by this mass spectrometric technique, due to the removal of small hydrophilic peptides by the washing step during the sample preparation (nitrocellulose adsorption). The peptide C-2, which exhibits no tryptic cleavage site, was not detected in the unseparated peptide mixture anymore. In additition, an endo Glu-C cleavage of the CNBr peptide mixture was performed to cover especially the C-terminal end of the protein in more detail and to obtain overlapping amino acid sequences. Four endo Glu-C peptides (G-1, G-2, G-7 and G-4/5 ± incomplete cleavage) were determined by positive ion PD mass spectrometry at m/z 2991.4 (calculated m/z 2993.1, amino acids 1±25), m/z 1002.7 (calculated m/z 1002.3, amino acids 26±34), m/z 1144.2 (calculated m/z 1145.2, amino acids 75±84) and m/z 1162.1 (calculated m/z 1162.3, amino acids 58±68) spanning almost the entire protein sequence and the C-terminal end. The only peptides unaccounted for were the peptides G-3 and G-6. In summary (Fig. 2), the PD mass spectrometry, in combination with the two enzymatic and one chemical cleavage reaction without any separation step, has allowed the rapid and convenient confirmation (100%) of the DNA-deduced primary sequence of cytochrome c6 isolated from C. paradoxa. The calculated relative molecular mass of this sequence (taking into acount that the two cysteine residues are linked covalently to the heme-iron system) is 9252.1 Da, which is in excellent agreement with the mass spectrometric (MALDI and PD) determination (9251.0 Da).

PCR amplification of cytochrome c6 gene fragment A partial amino acid sequence was determined from the N-terminus of holo-cytochrome c6 from C. paradoxa. This sequence was quite similar to sequences of cytochromes c6 from C. reinhardtii, the red alga P. purpurea and cyanobacteria and served to design a degenerate oligonucleotide (PR1, see Materials and methods). Another degenerate oligonucleotide was designed, corresponding to a highly conserved domain required for heme attachment (PR2, see Materials and methods). Using these primers, a 190-bp fragment was amplified from C. paradoxa nuclear DNA. The deduced amino acid sequence of this fragment exhibited 64% identity with the corresponding sequence of P. purpurea, thereby confirming that a C. paradoxa cytochrome c6 gene fragment had been amplified. Isolation of the full-length cytochrome c6 gene When this study was started, the sequence of the cyanelle genome was not yet completed. Therefore we used heterologous gene probes derived from the corresponding genes from C. reinhardtii [11], P. purpurea [12], and Synechocystis sp. PCC 6803 [17] for Southern blot experiments. Positive signals were obtained with nuclear but not with cyanelle DNA from C. paradoxa. Homologous hybridizations performed later confirmed these results and localized petJ to two adjacent

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Cyanelle cytochrome c6 (Eur. J. Biochem. 267) 4237

Fig. 3. Sequence comparison of precytochromes c6 from C. paradoxa (C.p.) and C. reinhardtii (C.r.). Arrows indicate the putative cleavage sites of stromal processing protease. The starting amino acids of the mature proteins are given in bold. Conserved stretches are boxed.

XhoI fragments (single cleavage site within the gene) of 600 and 300 bp, pointing toward a single copy gene (data not shown). In order to isolate the full-length cytochrome c6 gene, a cDNA library in Lambda NM1149 was screened with a DIG-labeled DNA probe produced from the subcloned 190-bp PCR product. Four independent clones were isolated in this manner, and restriction analysis indicated that all four clones contained common sequences. One of these clones was digested with EcoRI, and the resulting 552-bp restriction fragment was subcloned into pBluescript KS1. This subclone was sequenced using a combination of universal and genespecific primers. This analysis indicated the presence of one single open reading frame of 435 bp (Fig. 2). Comparison of the deduced amino acid sequence with the sequence of cyanobacterial and algal cytochromes c6 showed high identity scores with respect to the apoprotein, while the complete targeting signal could only be compared with that of the homologous precursor from C. reinhardtii (Fig. 3). The 60-amino-acid bipartite presequence consists of a 38-amino-acid transit sequence with the putative cleavage site for stroma processing protease VRM#S [30] and a 22 amino acid signal sequence with the cleavage site for thylakoid processing protease VFA#A. The plastid-encoded precytochrome c6 from P. purpurea contains a signal sequence only; thus this protein can serve as a paradigm for the concept of `conservative sorting' [31] in a primitive primary plastid. In the case of E. gracilis (the complex plastid of which is thought to originate from a secondary endosymbiotic event), precytochrome c6 shows a tripartite presequence containing an additional N-terminal signal sequence [32].

mature form of 9.2 kDa, comigrating with the holoprotein (Fig. 5). The time-course showed that most of the import was completed already after 10 min. Prolonged incubation resulted in eventual degradation of the mature protein, presumably through lumenal proteinases [33]. No intermediate was observed under these assay conditions, indicating that envelope import is the rate-determining step. However, two-step processing, as reported for the homologue from C. reinhardtii [11], could be demonstrated when intermediate accumulated due to the addition of thylakoid translocase inhibitors or during incubation at lower temperatures (J. M. Steiner, J. BerghoÈfer, R. B. KloÈsgen & W. LoÈffelhardt unpublished data). Fractionation of cyanelles after the import experiment showed that the

petJ expression and transcript size PetJ from C. paradoxa gave rise to an abundant 0.7-kb transcript that was considerably enhanced by light (Fig. 4). The signal was stronger than that observed for FNR [4] and approximately corresponded in intensity to that for cyanelle transketolase (Y. Ma, unpublished data). Accordingly, petJ cDNA clones were well represented and easy to isolate upon screening of different C. paradoxa libraries. Import of cytochrome c6 in vitro Using a modified cyanelle isolation procedure, the efficiency of homologous import could be greatly increased. The choice of the osmolytes appeared to be less important than previously thought [5]. The crucial point is to avoid any osmotic shock and to work as fast as possible. Import-competent isolated cyanelles (tested with pre-FNR) from C. paradoxa efficiently took up the 15-kDa preapocytochrome c6 and converted it into the protease-protected

Fig. 4. Northern hybridization of RNA isolated from light (lane L) or dark (lane D) grown C. paradoxa to a DIG-labeled petJ cDNA probe.

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Fig. 5. Import of 35S-labeled preapocytochrome c6 into isolated cyanelles. T, translation mix; ±/1, without/with addition of thermolysin; p, precursor; m, mature protein. Lanes 4±9: time course of import at 25 8C. Lanes 2 and 3: incubation at 0 8C (precursor binding only).

mature protein localized to the thylakoid fraction (Fig. 6). Due to the leaky nature of cyanelle (and cyanobacterial) thylakoid vesicles [6], only partial protease protection could be observed. During cyanelle lysis and fractionation, cyanelle thylakoid vesicles lose part of the internalized mature cytochrome c6 to the stroma fraction. Upon protease treatment of the thylakoid preparation, further leakage and digestion of cytochrome c6 occurs. Suborganellar localization of cytochrome c6 C. paradoxa cells were broken in a blendor under standard conditions. Aliquots of the cyanelle pellet were suspended in sucrose solutions of various osmolarities and were (a) kept on ice for 30 min, (b) subjected to freeze-thawing, or (c) ultrasonicated. After centrifugation (3000 g, 5 min) the corresponding postcyanelle supernatants and cyanelle pellets were subjected to SDS/PAGE and Western blotting. Blots were treated with an antiserum raised against pure cytochrome c6 from C. paradoxa. It appeared that almost all of this protein had leaked out of the cyanelles upon freeze-thawing (Fig. 7). As under these conditions the rather delicate outer membrane [34] is expected to rupture, a periplasmic localization of a significant part of cyanelle cytochrome c6 has to be envisaged. Cytoplasmic proteins like Rubisco and thylakoid-associated proteins like phycocyanin (stromal face) are retained in the cyanelle pellet upon freeze-thawing (data not shown). Sonication led to complete destruction of the cyanelles, as evidenced by the intense blue phycobiliprotein coloration of

Fig. 6. Fractionation of isolated cyanelles after import (3 min incubation) of 35S-labeled preapocytochrome c6. C, intact cyanelles; S, stroma; T, thylakoids; 1 , thermolysin-treated; p, precursor; m, mature protein.

Fig. 7. Localization of cytochrome c6 within cyanelles by Western blotting. C. paradoxa cells were lysed in a blendor under standard conditions (0.35 m sucrose). Cyanelle pellets were suspended in buffered sucrose solutions of different osmolarity from 0.7 m to 0.01 m (lanes labeled .7 to .01). Two separate assays were subjected to freeze-thawing (fr), or ultrasonication (us), under the osmotic conditions indicated. Afterwards, cyanelles were spun down again and the pellets and corresponding supernatants analyzed with respect to their content in cytochrome c6. 1, SDS extract from an aliquot of the untreated original pellet (positive control).

the supernatant which contained approximately the same amount of cytochrome c6 as the supernatant from freezethawing. Strongly hypotonic conditions (0.01 m sucrose) resulted in only low amounts of cytochrome c6 in the supernatant (Fig. 7).

DISCUSSION A common origin of all primary plastids (surrounded by two membranes: chloroplasts of higher plants and green algae, rhodoplasts and cyanelles) resulting from a singular primary endosymbiotic event is suggested by the comparative analysis of completely sequenced plastid genomes [2,35]. Consequently, the envelope import machinery must have evolved at the stage of the universal precursor organelle, the `protoplastid' [6]. The import apparatus of cyanelles is expected to show the general features of the corresponding machinery in chloroplasts but doubtless is closest to the prototype that evolved in the protoplastid that was also still surrounded by a peptidoglycan wall. The third known transit sequence for a nucleus-encoded cyanelle preprotein again resembles stroma-targeting peptides for higher plant chloroplasts in the hydrophobic N-terminus and the positive net charge, although the content of hydroxylated amino acids is unusually low (Fig. 3). This transit sequence shows a conserved amino terminus M(A)AFVA/V as with the other two stroma-targeting peptides (STPs) from C. paradoxa, but is considerably shorter than that of FNR (65 amino acids) and GAPDH (78 amino acids), and shows a slightly higher net charge (14) than previously found for FNR (13) or GAPDH (13). There is but one negatively charged amino acid present, whereas (unusual for STPs) five and six such residues are found

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in the two other published transit sequences from C. paradoxa [6]. Based on the (in general) higher positive net charge of higher plant STPs we speculated that cyanelle transit sequences are subjected to a constraint in this respect, in order to minimize electrostatic interactions with the negatively charged peptidoglycan wall [36]. The transit sequence of cytochrome c6 seems to contradict our hypothesis. It remains to be seen if additional identified STPs from C. paradoxa will show a trend toward lower positive net charges or not. As pre-FNR [5], precytochrome c6 from C. paradoxa is readily imported and processed by isolated chloroplasts from spinach or pea (J.M. Steiner et al. unpublished). From a mere comparison of STPs we cannot explain the difficulties we meet with inverse heterologous import, i.e. of higher plant precursors into isolated cyanelles [36]. There might be a feature of chloroplast STPs that is incompatible with a peptidoglycan barrier. The respective constraint could have been released in the course of STP evolution after loss of the organelle wall. The parallels between the STPs of C. paradoxa and C. reinhardtii are not surprising (Fig. 3) as in general STPs for the same gene in different organisms are more related than STPs for different proteins in the same organism. This holds true also for the respective promoters, and can be envisaged as a consequence of independent gene transfer during protoplastid evolution [37]. An amphiphilic helix structure has been proposed for the central part of STPs from C. reinhardtii [38] and of the presequence of the FNR from C. paradoxa [39]. However, such a structure does not seem to be favored in the corresponding region of the cytochrome c6 transit peptide of C. paradoxa where the spacing of the charged residues is not sufficiently regular. The PetJ signal peptide from C. paradoxa allows us to test a hypothesis on the relative hydrophilicity of the hydrophobic core (H-) domains of nuclear-encoded thylakoid signal peptides as compared to the plastid-encoded ones [40]. This is thought to prevent undesired cytosolic signal recognition particle binding. Indeed, the respective H-domain of precytochrome c6 is enriched in alanine (Fig. 3), whereas the respective domains of the cyanelle-encoded precursors to the lumenal or lumenalexposed thylakoid proteins cytochrome f, cytochrome c550, and PsaF contain phenylalanine, leucine, and isoleucine, instead [41], which also applies for plastid-encoded pre-PetJ from P. purpurea [12]. Progress was made during the pre-PetJ import experiments in two directions. (a) Optimization of the cyanelle isolation procedure resulted in efficiencies of homologous in vitro import comparable to that of heterologous import into pea chloroplasts. It turned out that even mild osmotic shock turned cyanelles into plastid counterparts of mitoplasts (which cannot be obtained with chloroplasts). The organelle wall stabilized such cyanelle preparations that looked intact in the phase contrast microscope but showed a poor performance in import, in organello protein biosynthesis, etc. [41]. (b) For the first time protein translocation across cyanobacterial-type phycobilisome-bearing thylakoid membranes could be demonstrated in vitro. Cyanobacterial and cyanelle thylakoids do not form tight vesicles that provide protease protection to internalized proteins [6]. This problem can be circumvented by conducting the import experiments in cyanello. The interenvelope membrane space is not very well defined for chloroplasts. There is but one protein, Tic 22, an extrinsic component of the inner membrane translocon, that has been reported to localize to this subcompartment [42]. The cyanelle periplasm contains the wall and more than 10 enzymes involved in its biosynthesis [43,44]. Electron transfer chains, redox reactive components, etc. have been invoked for the chloroplast inner envelope membrane [45]. A primitive plastid like the

Cyanelle cytochrome c6 (Eur. J. Biochem. 267) 4239

cyanelle with its numerous cyanobacterial features is even more likely to possess such properties. Preliminary data indicate cytochrome oxidase activity in isolated cyanelles and crossreaction of cyanelle protein blots with heterologous antibodies directed against cytochrome oxidase subunits (G.A. Peschek, personal communication). In this scenario, the additional role for (periplasmic) cytochrome c6 would be that of an electron carrier in a (respiratory?) electron transport chain in the inner envelope membrane. This would be compatible with the large amount of cytochrome c6 found in cyanelles but does not explain why almost all of this protein is released to the supernatant upon freeze-thawing whereas phycobiliproteins and Rubisco remain inside. Connections/continuities between the cytoplasmic membrane and the thylakoid membranes of cyanobacteria have been reported [46,47]. An artifactual permeabilization for small proteins of the thylakoid [48] and inner envelope membrane cannot be ruled out at present. However, then one would expect that the only slightly larger phycocyanin subunits (16 kDa) would also be found in the supernatant, which is not the case. In the absence of a reliable marker for the cyanelle periplasmic compartment, the dual localization of cytochrome c6 is tentative. A decision might be possible after immunoelectron microscope localization of cyanelle cytochrome c6.

ACKNOWLEDGMENTS This work was made possible through grants (P12876-MOB, to W. L. and P11183-CHE, to G. A.) from the Austrian `Fonds zur FoÈrderung der Wissenschaftlichen Forschung'. We thank Drs Sabeeha Merchant, Himadri Pakrasi and Michael Reith for providing petJ gene probes and Drs Henner Brinkmann and RuÈdiger Cerff for a C. paradoxa cDNA library in lNM1149. Travel money was made available by the Scientific-Technical Cooperation Austria-Spain (Acciones Integradas).

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