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 ...

We have generated the mutation T168S in the L L subunit of the chloroplast ATP synthase complex of Chlamydomonas reinhardtii by site directed mutagenesis and chloroplast transformation. CF I and the K K Q L L Q Q Q complex of this mutant strain were isolated and their enzymatic activities were characterized and compared to those of the corresponding wild type complexes. Without activation the mutant CF I exhibits MgATPase activity with at least 10 times higher rates than the wild type enzyme. The MgATPase activity could be stimulated to some extent by methanol, but less by ethanol and octylglucoside. The K K Q L L Q Q Q complex had an even higher MgATPase activity, which was only slightly enhanced by ethanol or methanol. The ATPase activities of the mutant complexes, like those of the wild type complexes, displayed a sharp concentration optimum for Mg P+ . Free ADP inhibited neither the mutant nor the wild type ATPase significantly. Azide, which strongly inhibited the ATPase activity of the wild type enzyme, inhibited the mutant enzyme only at an about 30 times higher concentration suggesting that the mutation T168S prevents trapping of a tightly bound MgADP by a catalytic site that regulates chloroplast ATPase activity. The mutant cells grew photoautotrophically at a growth rate of about 50%. Similar to the wild type the cells survived on minimal medium in the dark. Under heterotrophic conditions with acetate as energy and carbon source the mutant cells grew much faster than the wild type cells, but the chlorophyll content per cell decreased dramatically.z 1998 Federation of European Biochemical Societies.

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