Biochemical Characterization of Thylakoids and CF0CF1 Isolated from Chlamydomonas Reinhardtii CW15 and from atpA and atpB Deletion Mutants

Fiedler, Heike R.; Leu, Stefan; Shavit, Noun; Strotmann, Heinrich

doi:10.1007/978-94-009-0173-5_463

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“…Active thylakoid membranes were prepared as described (15,16). Cells were grown at 20°C in high salt medium containing 0.2% acetate at a light intensity of 900 lux in a 14-h light to 10-h dark cycle, in 400-ml cultures bubbled with sterile air.…”

Section: Methodsmentioning

confidence: 99%

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Catalytic Properties and Sensitivity to Tentoxin of Chlamydomonas reinhardtii ATP Synthases Changed in Codon 83 of atpB by Site-directed Mutagenesis

Fiedler

Golan

et al. 1997

Journal of Biological Chemistry

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The participation of the amino acid ␤83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by sitedirected mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid ␤83 was changed from Glu to Asp (␤E83D) and to Lys (␤E83K), and the highly conserved tetrapeptide ␤T82-E83-G84-L85 (⌬TEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15⌬atpB and FUD50 with the mutated atpB genes. The transformants containing the ␤E83D and ␤E83K mutant genes grew well photoautotrophically. The ⌬TEGL transformant did not grow photoautotrophically, and no CF 1 subunits were detected by immunostaining of Western blots using CF 1 specific antibodies. The rates of ATP synthesis at clamped ⌬pH with thylakoids isolated from cw15 and the two mutants, ␤E83D and ␤E83K, were similar. However, only the phosphorylation activity of the mutant ␤E83D was inhibited by tentoxin with 50% inhibition attained at 4 M. These results confirm that amino acid ␤83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF 1 s isolated from cw15, ␤E83D, and ␤E83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF 1 from the ␤E83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the ␤E83K-CF 1 than that of CF 1 from the other two strains. Moreover, the optimal activity of the ␤E83K-CF 1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF 1 -ATPase.The eukaryotic unicellular green algae Chlamydomonas reinhardtii constitutes a powerful experimental model system for the study of the photosynthetic machinery. It is accessible to genetic analysis and grows photoautotrophically on minimal medium or heterotrophically with acetate as the sole carbon source. These properties have been used to isolate numerous photosynthetic mutants which have helped to examine the function of the photosynthetic apparatus (1).The chloroplast of C. reinhardtii contains approximately 80 copies of its 196-kb 1 circular genome (2). Due to recent progress in the molecular genetics of C. reinhardtii, chloroplast proteins can be altered by site-directed mutagenesis of the corresponding genes followed by transformation into the chloroplast (3-7). Chloroplast transformation was first demonstrated in 1988 by Boynton and co-workers (3), by complementation of an atpB deletion mutant with the cloned wild type gene. The transforming DNA integrates into the recipient chloroplast DNA by homologous recombination. Goldschmidt-Clermont (5) has constructed a chimeric selectable marker using ...

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Section: Methodsmentioning

confidence: 99%

“…The atpB deletion mutant cw15⌬atpB was prepared from the cell wall deficient strain cw15 by replacing the atpB open reading frame by the aadA cassette (14). The use of the cell wall deficient strain cw15 allowed us to isolate well preserved thylakoids and to characterize electron transport and photophosphorylation activities of these membranes (15,16).…”

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confidence: 99%