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• W2163269684 abstract "A strain of the cyanobacteriumSynechococcus elongatus was generated that expresses a hybrid version of the photosystem I subunit PsaF consisting of the first 83 amino acids of PsaF from the green alga Chlamydomonas reinhardtii fused to the C-terminal portion of PsaF from S. elongatus. The corresponding modified gene was introduced into the genome of the psaF-deletion strain FK2 by cointegration with an antibiotic resistance gene. The transformants express a new PsaF subunit similar in size to PsaF from C. reinhardtiithat is assembled into photosystem I (PSI). Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of magnitude in the rate of P700+ reduction by C. reinhardtii cytochrome c 6 or plastocyanin in 30% of the complexes as compared with wild type cyanobacterial PSI. The corresponding optimum second-order rate constants (k 2 = 4.0 and 1.7 × 107m1 s1 for cytochromec 6 and plastocyanin) are similar to those of PSI from C. reinhardtii. The remaining complexes are reduced at a slow rate similar to that observed with wild type PSI fromS. elongatus and the algal donors. At high concentrations of C. reinhardtii cytochrome c 6, a fast first-order kinetic component (t12=4 μs) is revealed, indicative of intramolecular electron transfer within a complex between the hybrid PSI and cytochromec 6. This first-order phase is characteristic for P700+ reduction by cytochromec 6 or plastocyanin in algae and higher plants. However, a similar fast phase is not detected for plastocyanin. Cross-linking studies show that, in contrast to PSI from wild typeS. elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c 6 or plastocyanin with a similar efficiency as PsaF from C. reinhardtii PSI. Our data indicate that development of a eukaryotic type of reaction mechanism for binding and electron transfer between PSI and its electron donors required structural changes in both PSI and cytochrome c 6 or plastocyanin. A strain of the cyanobacteriumSynechococcus elongatus was generated that expresses a hybrid version of the photosystem I subunit PsaF consisting of the first 83 amino acids of PsaF from the green alga Chlamydomonas reinhardtii fused to the C-terminal portion of PsaF from S. elongatus. The corresponding modified gene was introduced into the genome of the psaF-deletion strain FK2 by cointegration with an antibiotic resistance gene. The transformants express a new PsaF subunit similar in size to PsaF from C. reinhardtiithat is assembled into photosystem I (PSI). Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of magnitude in the rate of P700+ reduction by C. reinhardtii cytochrome c 6 or plastocyanin in 30% of the complexes as compared with wild type cyanobacterial PSI. The corresponding optimum second-order rate constants (k 2 = 4.0 and 1.7 × 107m1 s1 for cytochromec 6 and plastocyanin) are similar to those of PSI from C. reinhardtii. The remaining complexes are reduced at a slow rate similar to that observed with wild type PSI fromS. elongatus and the algal donors. At high concentrations of C. reinhardtii cytochrome c 6, a fast first-order kinetic component (t12=4 μs) is revealed, indicative of intramolecular electron transfer within a complex between the hybrid PSI and cytochromec 6. This first-order phase is characteristic for P700+ reduction by cytochromec 6 or plastocyanin in algae and higher plants. However, a similar fast phase is not detected for plastocyanin. Cross-linking studies show that, in contrast to PSI from wild typeS. elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c 6 or plastocyanin with a similar efficiency as PsaF from C. reinhardtii PSI. Our data indicate that development of a eukaryotic type of reaction mechanism for binding and electron transfer between PSI and its electron donors required structural changes in both PSI and cytochrome c 6 or plastocyanin. One of several minor differences found in the otherwise remarkably conserved electron transfer chains of oxygenic photosynthesis of cyanobacteria, algae, and land plants concerns the type of electron carrier proteins used to transfer electrons from the cytochromeb 6 /f complex to photosystem I and the way they interact with PSI 1The abbreviations used are: PSI, photosystem I; cyt c 6, cytochrome c 6; Pc, plastocyanin; PsaF, subunit III of photosystem I; psaF , gene of the PsaF subunit; P700, primary donor of photosystem I; PCR, polymerase chain reaction; Mops, 4-morpholinepropanesulfonic acid; kb, kilobase pairs. (1Reith M. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers Group, Drodrecht, Netherlands1996: 643-657Google Scholar, 2Morand L.Z. Cheng R.H. Krogman D.W. Ki Ho K. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers Group, Drodrecht, Netherlands1994: 381-407Crossref Google Scholar). All cyanobacteria investigated utilize a cytochromec 6 as a soluble periplasmic electron carrier. In several cases, e.g. Synechococcus elongatus,cytochrome c 6 is the only electron carrier in the periplasma and is constitutively expressed (1Reith M. Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers Group, Drodrecht, Netherlands1996: 643-657Google Scholar, 2Morand L.Z. Cheng R.H. Krogman D.W. Ki Ho K. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers Group, Drodrecht, Netherlands1994: 381-407Crossref Google Scholar, 3Hatakana H. Sonoike K. Hirano M. Katoh S. Biochim. Biophys. Acta. 1993; 1141: 45-51Crossref PubMed Scopus (45) Google Scholar). Other cyanobacteria like Anabaena sp. PCC 7119 andSynechocystis sp. PCC 6803 and most algae examined, however, use both cytochrome c 6 and the copper-containing plastocyanin as alternative periplasmic electron carrier proteins (4Sandman G. Böger P. Plant Sci. Lett. 1980; 17: 417-424Crossref Scopus (89) Google Scholar, 5Ho K.K. Krogmann D.W. Biochim. Biophys. Acta. 1984; 766: 310-316Crossref Scopus (112) Google Scholar, 6Merchant S. Bogorad L. Mol. Cell. Biol. 1986; 6: 462-469Crossref PubMed Scopus (139) Google Scholar, 7Briggs L.M. Pecorano V.L. McIntosh L. Plant Mol. Biol. 1990; 15: 633-642Crossref PubMed Scopus (68) Google Scholar, 8Nakamura M. Yamagishi M. Yoshizaki F. Sugimura Y. J. Biochem. (Tokyo). 1992; 111: 219-224Crossref PubMed Scopus (12) Google Scholar). In these organisms, they are differentially expressed depending mostly on the relative availability of copper and iron in the culture medium (5Ho K.K. Krogmann D.W. Biochim. Biophys. Acta. 1984; 766: 310-316Crossref Scopus (112) Google Scholar, 8Nakamura M. Yamagishi M. Yoshizaki F. Sugimura Y. J. Biochem. (Tokyo). 1992; 111: 219-224Crossref PubMed Scopus (12) Google Scholar). In contrast, plastocyanin is expressed constitutively in photosynthetic land plants that lack cytochromec 6. Thus, in the evolution of oxygen-evolving organisms a tendency to replace the originally used c-type cytochrome by plastocyanin is clearly discernible. The PSI complex functions as a light-driven oxidoreductase that transfers electrons from cytochrome c 6 or plastocyanin to ferredoxin or flavodoxin (see Refs. 9Golbeck J.H. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers Group, Drodrecht, Netherlands1994: 319-360Crossref Google Scholar and 10Nechushtai R. Eden A. Cohen Y. Klein J. Ort D.R. Yocum C.F. Advances in Photosynthesis, Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers Group, Drodrecht, Netherlands1996: 289-311Google Scholar for a review). According to the established atomic structure of PSI fromS. elongatus, the primary donor of PSI, P700, is located within the highly conserved reaction center core close to the periplasmic surface of the photosynthetic membrane (9Golbeck J.H. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers Group, Drodrecht, Netherlands1994: 319-360Crossref Google Scholar, 11Krauss N. Schubert W.D. Klukas O. Fromme P. Witt H.T. Saenger W. Nat. Struct. Biol. 1996; 3: 965-973Crossref PubMed Scopus (313) Google Scholar, 12Schubert W.D. Klukas O. Krauss N. Saenger W. Fromme P. Witt H.T. J. Mol. Biol. 1997; 272: 741-769Crossref PubMed Scopus (225) Google Scholar). Two horizontal helixes l and l′, attributed to the PSI core subunits PsaA and PsaB, are thought to form a recognition site for binding of the periplasmic electron carriers (11Krauss N. Schubert W.D. Klukas O. Fromme P. Witt H.T. Saenger W. Nat. Struct. Biol. 1996; 3: 965-973Crossref PubMed Scopus (313) Google Scholar, 12Schubert W.D. Klukas O. Krauss N. Saenger W. Fromme P. Witt H.T. J. Mol. Biol. 1997; 272: 741-769Crossref PubMed Scopus (225) Google Scholar). However, despite the high degree of structural conservation of the PSI core subunits in all oxygen-evolving organisms, the mechanism of interaction between plastocyanin or cytochrome and PSI varies in different species. In higher plants, electron transfer from plastocyanin to P700+ is a biphasic process that includes a first-order kinetic component with a half-life of about 12 μs which is attributed to electron transfer from plastocyanin to P700+ within a stable complex between plastocyanin and PSI formed prior to the photooxidation of P700 (13Bottin H. Mathis P. Biochemistry. 1985; 24: 6453-6460Crossref Scopus (90) Google Scholar, 14Haehnel W. Ratajczak R. Robenek H. J. Cell Biol. 1989; 108: 1397-1405Crossref PubMed Scopus (57) Google Scholar, 15Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (116) Google Scholar). Biochemical studies indicate that the PsaF subunit of PSI is involved in the formation of this complex (16Wynn R.M. Malkin R. Biochemistry. 1988; 27: 5863-5869Crossref PubMed Scopus (109) Google Scholar, 17Hippler M. Ratajczak R. Haehnel W. FEBS Lett. 1989; 250: 280-284Crossref Scopus (91) Google Scholar, 18Bengis C. Nelson N. J. Biol. Chem. 1977; 252: 4564-4569Abstract Full Text PDF PubMed Google Scholar). Similar first-order kinetic components with half-lives of about 3 μs are observed for the reduction of P700+ by both plastocyanin and cytochrome c 6 in the green alga Chlamydomonas reinhardtii (19Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar). Electron transfer from both donors to PSI from a psaF-deficient mutant of C. reinhardtii was shown to be drastically slower indicating that PsaF is essential for efficient electron transfer from both plastocyanin and cytochrome c 6 to PSI (19Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar, 20Farah J. Rappaport F. Choquet Y. Joliot P. Rochaix J.-D. EMBO J. 1995; 14: 4976-4984Crossref PubMed Scopus (109) Google Scholar). Similar first-order kinetics have also been observed for PSI reduction by plastocyanin or cytochrome c in the green algaeChlorella and Monoraphidium braunii (21Delosme R. Photosynth. Res. 1991; 29: 45-54PubMed Google Scholar, 22Diaz A. Hervás M. Navarro J.A. De la Rosa M.A. Tollin G. Eur. J. Biochem. 1994; 222: 1001-1007Crossref PubMed Scopus (28) Google Scholar). This suggests that the formation of complexes between PSI and these electron donors which involve the PsaF subunit is likely to occur generally in algae and plants. In contrast, in the cyanobacteriumSynechocystis sp. PCC 6803, the reduction of P700+ by cytochrome c 6 or plastocyanin is a second-order process and fast phases which can be attributed to electron transfer within a stable preformed complex could not be detected (3Hatakana H. Sonoike K. Hirano M. Katoh S. Biochim. Biophys. Acta. 1993; 1141: 45-51Crossref PubMed Scopus (45) Google Scholar, 23Hervás M. Ortega J.M. Navarro J.A. De la Rosa M.A. Bottin H. Biochim. Biophys. Acta. 1994; 1184: 235-241Crossref Scopus (64) Google Scholar, 24Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Crossref PubMed Scopus (130) Google Scholar, 25Xu Q. Xu L. Chitnis V. Chitnis P. J. Biol. Chem. 1994; 269: 3205-3211Abstract Full Text PDF PubMed Google Scholar). In addition, spectroscopic investigations of PSI complexes from S. elongatus andSynechocystis sp. PCC 6803 lacking the PsaF subunit show that the absence of PsaF does not affect the rates of P700+reduction by cytochrome c 6 (3Hatakana H. Sonoike K. Hirano M. Katoh S. Biochim. Biophys. Acta. 1993; 1141: 45-51Crossref PubMed Scopus (45) Google Scholar, 25Xu Q. Xu L. Chitnis V. Chitnis P. J. Biol. Chem. 1994; 269: 3205-3211Abstract Full Text PDF PubMed Google Scholar). It has been shown that the PsaF subunit of PSI from spinach cross-links at one of its N-terminal lysines between residues 10–23 or 24–51 to the conserved acidic amino acids 42–44 and 59–61 of plastocyanin, respectively (26Hippler M. Reichert J. Sutter M. Zak E. Altschmied L. Schröer U. Herrmann R.G. Haehnel W. EMBO J. 1996; 15: 6374-6384Crossref PubMed Scopus (127) Google Scholar). This region close to the N-terminal end of PsaF could form an amphipathic α-helix, whose positively charged face may interact with plastocyanin (26Hippler M. Reichert J. Sutter M. Zak E. Altschmied L. Schröer U. Herrmann R.G. Haehnel W. EMBO J. 1996; 15: 6374-6384Crossref PubMed Scopus (127) Google Scholar). Amino acid sequence comparison, however, shows that a 27-amino acid domain of this well conserved N terminus of PsaF from plants and algae is missing in cyanobacteria (9Golbeck J.H. Bryant D.A. The Molecular Biology of Cyanobacteria. Kluwer Academic Publishers Group, Drodrecht, Netherlands1994: 319-360Crossref Google Scholar,27Chitnis P.R. Purvis D. Nelson N. J. Biol. Chem. 1991; 266: 20146-20151Abstract Full Text PDF PubMed Google Scholar). It was therefore suggested that the introduction of this new N-terminal domain into algal and plant-type PsaF may be responsible for the formation of the complex between PSI and cytochromec 6 or plastocyanin that is characteristic for PSI from algae and plants (26Hippler M. Reichert J. Sutter M. Zak E. Altschmied L. Schröer U. Herrmann R.G. Haehnel W. EMBO J. 1996; 15: 6374-6384Crossref PubMed Scopus (127) Google Scholar). In order to test this hypothesis we have introduced a chimeric psaF gene containing the N-terminal coding region from the green alga C. reinhardtiiinto the genome of a psaF-deletion strain of S. elongatus and report the functional characterization of a cyanobacterial photosystem I complex carrying an algal type PsaF subunit. DNA manipulations were performed in Escherichia coli strain XL1 blue according to standard protocols (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Integrative cartridge vectors were constructed by modifications of a genomic 2.45-kb BamHI/XhoI fragment from S. elongatus carrying the psaF/psaJ operon subcloned into pBSC M13+ (29Mühlenhoff U. Haehnel W. Witt H.T. Herrmann R.G. Gene (Amst.). 1993; 127: 71-78Crossref PubMed Scopus (59) Google Scholar) (Fig. 1). A DNA construct, pCRSEF/2, was constructed in which the region of the psaF gene fromS. elongatus encoding the mature PsaF subunit was substituted completely by the psaF region from C. reinhardtii (30Franzén L.G. Frank G. Zuber H. Rochaix J.D. Plant Mol. Biol. 1989; 12: 463-474Crossref PubMed Scopus (52) Google Scholar). First, an EcoRV site was introduced at codons 24 and 25 of psaF (i.e. codons 1 and 2 of the mature PsaF from S. elongatus), and the termination codon was modified to create an XbaI site. These modifications were introduced by inverse PCR in the presence of oligonucleotides SEF1 (5′-TGCGATATCAGCGGAGGCTAGG-3′) and SEF2 (5′-TCTCTAGATTTGCTGTTTGTTG-3′) to create the vector pSEF1. Second, apsaF cDNA clone from C. reinhardtii (30Franzén L.G. Frank G. Zuber H. Rochaix J.D. Plant Mol. Biol. 1989; 12: 463-474Crossref PubMed Scopus (52) Google Scholar) was modified by PCR in the presence of oligonucleotides CRF1 (5′-CCGATATCGCGGGCCTGACC-3′) and CRF2 (5′-CAGCTAGCGGGGAGACACGG-3′) to create an EcoRV site at codons 63 and 64 (i.e.codons 1 and 2 of the mature PsaF) and an NheI site at the termination codon. This fragment was inserted into the EcoRV and XbaI sites of pSEF1 to produce the plasmid pCRSEF/2 which carries the psaF/psaJ operon from S. elongatus in which the part of the C. reinhardtii psaFgene encoding the entire mature PsaF is fused in frame to the presequence of psaF from S. elongatus. In addition, a second plasmid was constructed in which the codons for amino acids Ile64 to His145 of PsaF from C. reinhardtii were inserted in frame between codon Asp24 and codon Ala83 ofpsaF from S. elongatus. First, the genomicClaI/XhoI fragment from S. elongatuswas modified to create a BalI site at codons 81 and 82 by PCR in the presence of oligonucleotide FBal (5′-CTTGGCCATGCCGGTGATTTTC-3′) and the M13 reverse primer. This PCR fragment was inserted into the BalI and XhoI sites of vector pCRSEF/2 to create vector FBalCRF/2. The fusion part of the DNA construct was sequenced. Finally, the SmaI fragment of pHP45Ω (31Prentki P. Krisch H.M. Gene (Amst.). 1984; 29: 303-313Crossref PubMed Scopus (1343) Google Scholar) carrying the streptomycin/spectinomycin resistance genes was inserted into the HpaI site of pCRSEF/2 and FBalCRF/2, generating plasmids pCRSEF/3 and FBalCRF/3 which served as integrative vectors for the genetic manipulation of S. elongatus. Wild type and mutant strains from S. elongatus were cultivated as described (32Mühlenhoff U. Chauvat F. Mol. Gen. Genet. 1996; 252: 93-100Crossref PubMed Scopus (61) Google Scholar). Genetic manipulations of the psaF/psaJ locus from S. elongatus were carried out using thepsaF −/Kmr strain FK2, which carries a psaF gene disrupted by a kanamycin resistance marker (32Mühlenhoff U. Chauvat F. Mol. Gen. Genet. 1996; 252: 93-100Crossref PubMed Scopus (61) Google Scholar). Prior to transformation, FK2 was grown in medium D supplemented with 40 μg/ml kanamycin. Cells were transformed by electroporation in the presence of plasmids pCRSEF/3 or FBalCRF/3 essentially as described previously, selected for streptomycin resistance in liquid cultures, and colony-purified on solid media (32Mühlenhoff U. Chauvat F. Mol. Gen. Genet. 1996; 252: 93-100Crossref PubMed Scopus (61) Google Scholar). From these initial clones, strains with the desiredKms/Smr phenotype were selected by replica plating on solid media containing either 2 μg/ml streptomycin or 25 μg/ml kanamycin. Cells carrying the C. reinhardtii psaFgene were identified by either immunoblot analysis of photosynthetic membranes isolated from small scale cultures using anti-C. reinhardtii (20Farah J. Rappaport F. Choquet Y. Joliot P. Rochaix J.-D. EMBO J. 1995; 14: 4976-4984Crossref PubMed Scopus (109) Google Scholar) or anti-S. elongatus PsaF antibodies 2U. Mühlenhoff, unpublished. or by Southern blot analysis of genomic DNA as described (32Mühlenhoff U. Chauvat F. Mol. Gen. Genet. 1996; 252: 93-100Crossref PubMed Scopus (61) Google Scholar). Photosystem I complexes were extracted from PSII-depleted membranes by 0.6% w/v β-dodecyl maltoside and purified by centrifugation in sucrose gradients as described (33Rögner M. Mühlenhoff U. Boekema E. Witt H.T. Biochim. Biophys. Acta. 1990; 1015: 415-424Crossref Scopus (87) Google Scholar, 34Rögner M. Nixon P.J. Diner B.A. J. Biol. Chem. 1990; 265: 6189-6196Abstract Full Text PDF PubMed Google Scholar). PSI fromC. reinhardtii was isolated as described (19Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar), and plastocyanin and cytochrome c 6 were isolated from C. reinhardtii following the protocol of Ref. 6Merchant S. Bogorad L. Mol. Cell. Biol. 1986; 6: 462-469Crossref PubMed Scopus (139) Google Scholar with modifications described in Ref. 19Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar. Cytochromec 6 from S. elongatus was isolated essentially according to Ref. 35Koike H. Katoh S. Plant Cell Physiol. 1979; 20: 1157-1161Google Scholar. Plastocyanin and cytochromec 6 concentrations were determined spectroscopically using absorption coefficients of ε597 nm = 4.9 mm1cm1 and ε552 nm= 20 mm1 cm1, respectively (19Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar). For the fast immunoblot analysis of photosynthetic membranes, 3.5-ml cultures of S. elongatus were grown to OD750 = 1, harvested by centrifugation, washed once in 1 ml of HMCM buffer (20 mm Hepes, pH 7.8, 10 mmCaCl2, 5 mm MgCl2, 0.5m mannitol), resuspended in 50 μl of HMCM buffer, and frozen. The thawed suspension was incubated with 2 mg/ml lysozyme for 30 min at 48 °C and frozen. Cells were then lysed by the addition of 10 volumes of MCM (MMCM minus mannitol), and the photosynthetic membranes were recovered by centrifugation in a microcentrifuge at 4 °C for 10 min at maximum speed. Membranes were washed once in 500 μl of MCM supplemented with 0.1% sulfobetain 10, pelleted by centrifugation, and resuspended in SDS loading buffer at a concentration of approximately 0.25 mg of chlorophyll/ml. Cytochrome c 6 and plastocyanin were chemically cross-linked to photosystem I essentially as described (19Hippler M. Drepper F. Farah J. Rochaix J.-D. Biochemistry. 1997; 36: 6343-6349Crossref PubMed Scopus (92) Google Scholar); PSI particles at a concentration of 0.1 mg of chlorophyll/ml in 30 mm Hepes, pH 7.5, 3 mm MgCl2, and 1 mm ascorbate were incubated in the presence of 20 μm plastocyanin or cytochrome c 6with 5 mm N-ethyl-3-(3-diaminopropyl)carbodiimide and 10 mm N-hydroxysulfosuccinimide for 45 min in darkness. The reactions were terminated by addition of ammonium acetate to a final concentration of 0.2 m and diluted 4-fold. PSI complexes were sedimented by centrifugation at 200,000 ×g for 45 min and resuspended in 20 mm Hepes, pH 7.5, 0.05% Triton X-100. Analytical SDS-polyacrylamide gel electrophoresis was carried out using 15 or 16.5% (w/v) polyacrylamide gels (36Harlow E. Lane D. Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). For immunoblot analysis, PSI complexes equivalent to 4 μg of chlorophyll and membrane preparations equivalent to 10 μg of chlorophyll were analyzed. Western blots and antibody incubations were carried out essentially as described (36Harlow E. Lane D. Antibodies, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Immunodetection reactions were performed using anti-rabbit IgG antibodies linked to horseradish peroxidase followed by enhanced chemiluminescence detection (ECL, Amersham Pharmacia Biotech). Flash-induced absorption changes at 817 nm were measured at 296 K on a single beam spectrophotometer essentially as described (15Drepper F. Hippler M. Nitschke W. Haehnel W. Biochemistry. 1996; 35: 1282-1295Crossref PubMed Scopus (116) Google Scholar). Flash excitation was performed using a frequency-doubled Nd:YAG laser (5 ns full width at half maximum). The measuring light was provided by a luminescence diode (Hitachi HE8404SG, 40 milliwatts, 30 nm full width at half maximum), filtered through an 817-nm interference filter (9 nm full width at half maximum), and passed through a cuvette with an optical path length of 1 cm that contained 200 μl of sample. For flash-absorption experiments, PSI reaction centers were suspended in the presence or absence of cytochrome c 6 or plastocyanin at a standard concentration of 50 μm chlorophyll in 30 mmMops, pH 7.0, supplemented with 0.05% β-dodecyl maltoside, 0.2 mm methyl viologen, 0.1 mm diaminodurene, 1 mm sodium ascorbate, and MgCl2 as indicated in the figure legends. For measurements at high donor concentrations (Fig.6), a cuvette with 3-mm optical path length was used that contained 30 μl of sample. Four individual signals were averaged, and the resulting kinetic traces were fitted to a sum of one or two exponential components and a constant offset with the program GNUPLOT (Unix version 3.5, Williams), performing nonlinear least squares fitting using the Marquart-Levenberg algorithm. To generate an S. elongatusstrain carrying the psaF gene from C. reinhardtii, integrative vectors were introduced into the S. elongatus strain FK2 that carries a psaF gene interrupted by a kanamycin resistance cassette and thus served as apsaF-free background ((32) see Fig.1, middle part). Vector constructions were performed using the genomic fragment from S. elongatus carrying the psaF/psaJ locus ((29) seetop of Fig. 1) as follows. First, we took into account that the PsaF subunit is expressed as a precursor protein carrying an N-terminal export sequence required for the translocation of the N terminus of the protein into the periplasma. In order to be efficiently recognized by the cyanobacterial export apparatus, the hybridpsaF gene should therefore contain the entire cyanobacterial export sequence and the ASA-D cleavage site of the export protease. Second, in cyanobacteria, psaF and psaJ are arranged in an operon. In order to avoid any interference with the expression of the PsaJ subunit which is likely to be involved in the binding of PsaF to PSI, the organization of the psaF/psaJoperon should be maintained (25Xu Q. Xu L. Chitnis V. Chitnis P. J. Biol. Chem. 1994; 269: 3205-3211Abstract Full Text PDF PubMed Google Scholar) (see Fig. 1). As shown in Fig. 1 (bottom) and Fig.2, the integrative vector FBalCRF/3 carries a gene encoding a hybrid PsaF protein that contains the cyanobacterial signal sequence (up to codon Asp-24 of S. elongatus psaF), the N-terminal domain of PsaF from C. reinhardtii (30Franzén L.G. Frank G. Zuber H. Rochaix J.D. Plant Mol. Biol. 1989; 12: 463-474Crossref PubMed Scopus (52) Google Scholar) (i.e. between codons Asp-63/Ile-64 to codon His-145), and the hydrophobic C-terminal part of the cyanobacterial subunit (starting with codon Ala-60 of S. elongatus psaF) which is assumed to anchor the protein to the hydrophobic core of PSI. A streptomycin/spectinomycin resistance gene cassette was inserted at the HpaI site located 165 base pairs downstream of psaJ, well downstream of the transcribed region of the psaF/psaJ operon (see “Experimental Procedures” for details of the construction). In addition to FBalCRF/3, a vector, pCRSEF/3, was constructed in which the complete region of the psaF gene from S. elongatusencoding the entire mature PsaF subunit was replaced by the corresponding part of psaF from C. reinhardtii(see “Experimental Procedures”). However, following the introduction of this vector into S. elongatus, C. reinhardtii PsaF was not recovered in PSI although it was verified by Southern blot analysis that the gene was cointegrated together with the antibiotic resistance marker and inserted correctly into the genome (not shown). Following the electroporation of thepsaF-deletion strain FK2 from S. elongatus in the presence of the plasmid pFBalCRF/3, streptomycin-resistant transformants with the desired Kms/Smrphenotype were selected by replica plating on solid media, and those expressing the chimeric C. reinhardtii-S. elongatus PsaF protein were screened by immunoblot analysis using anti-C. reinhardtii-PsaF antibodies (not shown, see “Experimental Procedures”). The organization of the psaF/psaJ locus of one of these strains, H53, was investigated by Southern blot analysis shown in Fig. 3. First, upon hybridization with a 500-base pair fragment from a C. reinhardtii psaF cDNA clone carrying part of the gene encoding the mature PsaF, a single 6.3-kb genomic EcoRV restriction fragment is detected that is not present in S. elongatus wild type nor in strain FK2 (Fig. 3 A). The same DNA fragment is detected in H53 DNA after hybridization with a probe carrying theSm r /Sp r genes inserted into pFBalCRF/3 (Fig. 3 B). Taken together, these blots indicate that the region of the psaF gene of C. reinhardtii has been inserted with the antibiotic marker gene into the genome of S. elongatus. Furthermore, when the blots were probed with the ClaI/XhoI fragment carrying thepsaF, psaJ, and rpl9 genes, only single EcoRV fragments of ∼13 and 14.5 kb are observed in DNA from wild type and strain FK2. The size difference is due to the presence of the Km r marker gene in FK2 (Fig.3 C, see Fig. 1). For strain H53, however, twoEcoRV fragments of ∼8.0 and 6.3 kb are detected, indicating that a new EcoRV site has been introduced into the psaF/psaJ locus of H53. Since theSm r /Sp r marker genes do not contain an EcoRV site, the existence of a newEcoRV restriction site together with theSm r /Sp r genes at thepsaF/psaJ gene locus indicates that the entire part of thepsaF gene that originates from C. reinhardtii has been introduced into strain H53, because C. reinhardtii psaFis flanked by a new EcoRV site and theSm r /Sp r genes in the integrative vector pFBalCRF/3 (Fig. 1). Finally, when the blots were probed with a fragment carrying the Kmr marker present in strain FK2, no hybridization signal was observed in DNA from strain H53 (Fig.3 D). This result indicates that H53 represents a fully segregated mutant in which the original psaF/psaJ gene locus of strain FK2 that carried a kanamycin resistance gene has been completely replaced by the new psaF gene at thepsaF/psaJ gene locus in all copies of the polyploid genome. Essentially the same conclusion was obtained by a similar Southern blot analysis that was carried out using EcoRI-restricted DNA (not shown). The expression pattern of the psaF genes in S. elongatus wild type and strains FK2 and H53 was monitored by Western blot analysis of photosynthetic membran" @default.
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• W2163269684 title "Insertion of the N-terminal Part of PsaF from Chlamydomonas reinhardtii into Photosystem I from Synechococcus elongatus Enables Efficient Binding of Algal Plastocyanin and Cytochrome c 6" @default.
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• W2163269684 doi "https://doi.org/10.1074/jbc.274.7.4180" @default.
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