THE PLASTID-ENCODED CCSA GENE IS REQUIRED FOR HEME ATTACHMENT TO CHLOROPLAST C-TYPE CYTOCHROMES

A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cyto chromes, was tested for its function in chloroplast cytochrome biogene sis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 ge ne results in a non-photosynthetic phenotype attributable to the absen ce of c-type cytochromes. The cloned ycf5 gene also complements the ph ototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is una ble to synthesize functional forms of cytochromes f and c(6) owing to a chloroplast genome mutation that prevents heme attachment. The selec ted (phototrophic growth) as well as the unselected (holocytochrome c( 6) accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), i s expressed in wild-type and B6 cells but is non-functional in B6 owin g to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revert ant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein require d for heme attachment to c-type cytochromes.