GENETIC-ANALYSIS OF CHLOROPLAST C-TYPE CYTOCHROME ASSEMBLY IN CHLAMYDOMONAS-REINHARDTII - ONE CHLOROPLAST LOCUS AND AT LEAST 4 NUCLEAR LOCIARE REQUIRED FOR HEME ATTACHMENT

Chloroplasts contain up to two c-type cytochromes, membrane-anchored c ytochrome f and soluble cytochrome c(6). To elucidate the post-transla tional events required for their assembly, acetate-requiring mutants o f Chlamydomonas reinhardtii that have combined deficiencies in both pl astid-encoded cytochrome f and nucleus-encoded cytochrome c(6) have be en identified and analyzed. For strains ct34 and ct59, where the pheno type displays uniparental inheritance, the mutations were localized to the chloroplast ccsA gene, which was shown previously to be required for heme attachment to chloroplast apocytochromes, The mutations in an other eight strains were localized to the nuclear genome. Complementat ion tests of these strains plus three previously identified strains of the same phenotype (ac206, F18, and F2D8) indicate that the 11 ccs st rains define four nuclear loci, CCS1-CCS4. We conclude that the produc ts of the CCS1-CCS4 loci are not required for translocation or process ing of the preproteins but, like CcsA, they are required for the heme attachment step during assembly of both holocytochrome f and holocytoc hrome c(6). The ccsA gene is transcribed in each of the nuclear mutant s, but its protein product is absent in ccs1 mutants, and it appears t o be degradation susceptible in ccs3 and ccs4 strains. We suggest that Ccs1 may be associated with CcsA in a multisubunit ''holocytochrome c assembly complex,'' and we hypothesize that the products of the other CCS loci may correspond to other subunits.