The Three Mitochondrial Encoded CcmF Proteins Form a Complex That Interacts with CCMH and c-Type Apocytochromes in Arabidopsis

Three reading frames called ccmFN1, ccmFN2, and ccmFc are found in the mitochondrial genome of Arabidopsis. These sequences are similar to regions of the bacterial gene ccmF involved in cytochrome c maturation. ccmF genes are always absent from animal and fungi genomes but are found in mitochondrial genomes of land plant and several evolutionary distant eukaryotes. In Arabidopsis, ccmFN2 despite the absence of a classical initiation codon is not a pseudo gene. The 3 ccmF genes of Arabidopsis are expressed at the protein level. Their products are integral proteins of the mitochondrial inner membrane with in total 11 to 13 predicted transmembrane helices. The conserved WWD domain of CcmFN2 is localized in the inter membrane space. The 3 CcmF proteins are all detected in a high molecular mass complex of 500 kDa by Blue Native PAGE. Direct interaction between CcmFN2 and both CcmFN1 and CcmFC is shown with the yeast two-hybrid split ubiquitin system, but no interaction is observed between CcmFN1 and CcmFC. Similarly, interaction is detected between CcmFN2 and apocytochrome c but also with apocytochrome c1. Finally, CcmFN1 and CcmFN2 both interact with CCMH previously shown to interact as well with cytochrome c. This strengthens the hypothesis that CcmF and CCMH make a complex that performs the assembly of heme with c-type apocytochromes in plant mitochondria.