Insertion of a "chloroplast-like" regulatory segment responsible for thiol modulation into gamma-subunit of F0F1-ATPase of the cyanobacterium Synechocystis 6803 by mutagenesis of atpC.
A regulatory sequence in the gamma subunit of the F0F1-ATPase complex of higher plant chloroplasts, responsible for so-called thiol modulation, is absent in the corresponding polypeptides of the cyanobacterial complexes analysed so far. We have modified the atpC gene encoding this gamma subunit in Synechocystis 6803 by site-directed mutagenesis. A segment was introduced coding for nine additional amino acids, including the two functional cysteines, which constitutes the sequence of the respective element in the chloroplast gamma subunit. The growth rate as well as the rate of photosynthesis of the transformant was comparable to that of the wild-type, but the transitory increase in respiration observed immediately after a period of illumination was significantly lower in the mutant than in the wild-type. The F1 subcomplex solubilized from thylakoid membranes of both the wild-type and the transformant can be activated by trypsin to yield Ca(2+)-dependent ATPase activity, but only the F1 from the transformant can be activated by the thiol reagent dithiothreitol.
Werner-Grüne
S
S
Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Germany.
Gunkel
D
D
Schumann
J
J
Strotmann
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002736:Chloroplasts / Q000201:enzymology*; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D003624:Darkness; D005798:Genes, Bacterial; D005947:Glucose / Q000494:pharmacology; D008027:Light; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010101:Oxygen Consumption / Q000187:drug effects; D016133:Polymerase Chain Reaction; D012045:Regulatory Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014675:Vegetables / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D005947:Glucose