Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.
Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.
de Vitry
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Physiologie Membranaire et Moléculaire du Chloroplaste, Centre National de la Recherche Scientifique UPR1261, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France.
Finazzi
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Baymann
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Kallas
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics*; D002736:Chloroplasts / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism; D045346:Cytochrome b6f Complex; D017931:DNA Primers; D019281:Dimerization; D004579:Electron Transport; D014450:Electron Transport Complex III; D007506:Iron-Sulfur Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017384:Sequence Deletion
D002135:Calcium-Binding Proteins; D003573:Cytochrome b Group; D017931:DNA Primers; D007506:Iron-Sulfur Proteins; C037989:Rieske iron-sulfur protein; D045346:Cytochrome b6f Complex; D014450:Electron Transport Complex III