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

Abstract

Three reading frames called ccmF N1 , ccmF N2 , and ccmF c 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, ccmF N2 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 CcmF N2 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 CcmF N2 and both CcmF N1 and CcmF C is shown with the yeast two-hybrid split ubiquitin system, but no interaction is observed between CcmF N1 and CcmF C . Similarly, interaction is detected between CcmF N2 and apocytochrome c but also with apocytochrome c 1 . Finally, CcmF N1 and CcmF N2 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.

Mutations in an Arabidopsis Mitochondrial Transcription Termination Factor–Related Protein Enhance Thermotolerance in the Absence of the Major Molecular Chaperone HSP101

“…There were almost no changes in the transcripts of atp4, atp6-1, atp6-2, atp9, and nad4L. The only downregulated transcript in the shot1-2 mutant was the ccmF C transcript, which encodes a subunit of a protein complex reported to be involved in cytochrome c maturation (Rayapuram et al, 2008).…”

The Heme Chaperone ApoCcmE Forms a Ternary Complex with CcmI and Apocytochrome c

“…C, shown is an elution profile (absorbance at 280 nm as a function of the volume) of a mixture containing His-apoCcmE, FLAGCcmI, and Strep-apocytochrome c 2 from the Sephacryl S200 column in buffer containing 0.01% DDM. Estimation of the molecular masses (kDa) was done according to the calibration of the column using bovine serum albumin (67 kDa), carbonic anhydrase (39 kDa), and horse heart cytochrome c (12. yeast two hybrid screens, revealed that in Arabidopsis thaliana CcmFN2 and CcmH interacted with apocytochrome c (32,33). E. coli CcmH could reduce the disulfide bond of a apocytochrome c-like peptide in vitro (16), and R. capsulatus Cys-less CcmG could co-purify with apocytochrome c 2 (34).…”

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

“…We believe that multiple functions described here provide the rationale for these conservations and observations. Interestingly, in plant mitochondrial genomes, CcmF is separated as two (72) and sometimes three individual ORFs, called CcmF N1 , CcmF N2 , and CcmF C , which form a complex with CcmH (70,121). Has genomic reorganization of the mitochondrial endosymbiont naturally separated multiple functions of the CcmF protein, albeit while still forming a complex?…”