Complementation of aChlamydomonas reinhardtii mutant defective in the nuclear gene encoding the chloroplast coupling factor 1 (CF1) ?-subunit (atpC)

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Abstract
Chlamydomonas reinhardtii strainatpC1 is a mutant defective in the nuclear gene that encodes the CF1 ATP synthase γ-subunit polypeptide. Photoautotrophic growth was restored toatpC1 after it was transformed with wild-type DNA. Transformed strains were acetate-independent and arsenate-sensitive, similar in phenotype to the progenitor wild-type strain from whichatpC1 was generated. Three transformed strains were examined in detail. Southern blot analyses demonstrated that the transformants were complements and not revertants. The transforming DNA integrated into the nuclear genome in a nonhomologous manner and at a low copy number. Northern blot analyses showed that the γ-subunit mRNA in the complemented strains was expressed at the same relative level as that of wild-type. Western blots of total protein showed that whereasatpC1 was unable to synthesize any CF1 γ-subunit, all three complemented strains could. Furthermore, the Western blot analyses demonstrated that the mutation inatpC1 had a pleiotropic effect on the accumulation of the CF1 β-subunit which was relieved upon complementation. Cell extracts fromatpC1 did not have any CF1-dependent catalytic activity, whereas extracts from all of the complemented strains and the wild-type strain had identical activities.