AtCCMH, an essential component of the c-type cytochrome maturation pathway in Arabidopsis mitochondria, interacts with apocytochrome c.
The maturation of c-type cytochromes requires the covalent ligation of the heme cofactor to reduced cysteines of the CXXCH motif of apocytochromes. In contrast to mitochondria of other eukaryotes, plant mitochondria follow a pathway close to that found in alpha- and gamma-proteobacteria. We identified a nuclear-encoded protein, AtCCMH, the Arabidopsis thaliana ortholog of bacterial CcmH/CycL proteins. In bacteria, CcmH and the thioredoxin CcmG are components of a periplasmic thio-reduction pathway proposed to maintain the apocytochrome c cysteines in a reduced state. AtCCMH is located exclusively in mitochondria. AtCCMH is an integral protein of the inner membrane with the conserved RCXXC motif facing the intermembrane space. Reduction assays show that the cysteine thiols in the RCXXC motif of AtCCMH can form a disulfide bond that can be reduced by enzymatic thiol reductants. A reduced form of AtCCMH can reduce the intra-disulfide bridge of a model peptide of apocytochrome c. When expressed in Escherichia coli, AtCCMH coimmunoprecipitates with the bacterial CcmF, a proposed component of the heme lyase. Blue-native PAGE of mitochondrial membrane complexes reveals the colocalization of AtCCMH and AtCcmF(N2) in a 500-kDa complex. Yeast two-hybrid assays show an interaction between the AtCCMH intermembrane space domain and A. thaliana apocytochrome c. A. thaliana ccmh/ccmh knockout plants show lethality at the torpedo stage of embryogenesis. Our results show that AtCCMH is an essential mitochondrial protein with characteristics consistent with its proposed apocytochrome c-reducing and heme lyase function.
Meyer
Etienne H
EH
Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, 12 Rue du Général Zimmer, 67084 Strasbourg Cedex, France.
Giegé
Philippe
P
Gelhaye
Eric
E
Rayapuram
Naganand
N
Ahuja
Umesh
U
Thöny-Meyer
Linda
L
Grienenberger
Jean-Michel
JM
Bonnard
Géraldine
G
D020816:Amino Acid Motifs; D029681:Arabidopsis Proteins / Q000378:metabolism*; D003545:Cysteine; D003574:Cytochrome c Group / Q000378:metabolism* / Q000502:physiology; D045304:Cytochromes c / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D029681:Arabidopsis Proteins; D003574:Cytochrome c Group; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D045304:Cytochromes c; C506270:CCMH protein, Arabidopsis; D050862:Oxidoreductases Acting on Sulfur Group Donors; D003545:Cysteine