Molecular Characterization of Two Point Mutants in the Chloroplast

Two point mutants of Chlamydomonas reinhardtii, previously found by recombination and complementation analysis to map in the chloroplast atpB gene encoding the /? subunit of the CFl/CFo ATP synthase, are here shown to be missense alterations near the 5’ end of that gene. One mutant (ac-u-c-2-9) has a change at amino acid position 47 of the /? subunit from leucine (CTA) to arginine (CGA). In the second mutant (ac-uc-2-29), the codon AAA (lysine) is changed to AAC (asparagine) at position 154. Spontaneous revertants of each mutant were isolated that restore the original wild type base pair. Northern analysis of total RNA and in vivo pulse labeling followed by immunoprecipitation reveals that both mutant atpB genes are transcribed and translated normally. However, immunoblots show that the amount of /? subunit associated with mutant thylakoids is only approximately 3% of that Seen in wild type and that the CF1 a and y subunits are missing entirely. The disruption of ATP synthase complex assembly in these mutants is much more severe than in Escherichia coli /? subunit gene point mutants, which retain significant amounts of a and /? subunits on their membranes (Noumi, T., Oka, N., Kanazawa, H., and Futai, M. (1986) J. Biol. Chem. 261, 70707075). These results support the hypothesis that there are differences in assembly of the ATP synthase between E. coli and chloroplasts. In particular they indicate that /? must be present for assembly of the a and y subunits of CF1 onto chloroplast membranes.