CCME, a Nuclear-encoded Heme-binding Protein Involved in Cytochrome c Maturation in Plant Mitochondria

Nathalie Spielewoy,Henk Schulz,Jean Michel Grienenberger,Linda Thöny-Meyer,Géraldine Bonnard

doi:10.1074/jbc.m008853200

Abstract

The maturation of c-type cytochromes requires the covalent attachment of the heme cofactor to the apoprotein. For this process, plant mitochondria follow a pathway distinct from that of animal or yeast mitochondria, closer to that found in alpha- and gamma-proteobacteria. We report the first characterization of a nuclear-encoded component, namely AtCCME, the Arabidopsis thaliana orthologue of CcmE, a periplasmic heme chaperone in bacteria. AtCCME is targeted to mitochondria, and its N-terminal signal peptide is cleaved upon import. AtCCME is a peripheral protein of the mitochondrial inner membrane, and its major hydrophilic domain is oriented toward the intermembrane space. Although a AtCCME (Met(79)-Ser(256)) is not fully able to complement an Escherichia coli CcmE mutant strain for bacterial holocytochrome c production, it is able to bind heme covalently through a conserved histidine, a feature previously shown for E. coli CcmE. Our results suggest that AtCCME is important for cytochrome c maturation in A. thaliana mitochondria and that its heme-binding function has been conserved evolutionary between land plant mitochondria and alpha-proteobacteria.

Highlights

Electron transport constituents of respiratory chains in prokaryotes and eukaryotes are composed mainly of proteins containing a variety of cofactors including metal ions, iron-sulfur clusters, nucleotides, or hemes
We report the first characterization of a nuclear-encoded component, namely AtCCME, the Arabidopsis thaliana orthologue of CcmE, a periplasmic heme chaperone in bacteria
Our results suggest that AtCCME is important for cytochrome c maturation in A. thaliana mitochondria and that its heme-binding function has been conserved evolutionary between land plant mitochondria and ␣-proteobacteria

Summary

EXPERIMENTAL PROCEDURES

Strains and Plasmids—AtCCME cDNA clone (GenBankTM accession number ATU72502) was obtained from F. Mitoplast and outer membrane fractions were isolated after centrifugation through a bovine serum albumin-free discontinuous gradient of 22, 33, and 47% sucrose. Intact mitoplasts were collected from the 33/47% interface, washed and resuspended in 20 mM MOPS, pH 7.2, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride at a protein concentration of 3 mg/ml, and broken by three freeze/thaw cycles followed by sonication (5ϫ 10 s, 300 W, Sonic Vibra Cells). The membrane (P) and soluble (S) fraction of mitoplasts were separated by a 30-min centrifugation at 100,000 ϫ g in a Beckman TLA-100 rotor. A 30-min centrifugation at 100,000 ϫ g in a Beckman TLA-100 rotor allows the separation of soluble (peripheral) from insoluble (integral) protein fractions. Import assays were carried out as described [38]

RESULTS

TABLE I Strains and plasmids used in this work

This work This work

DISCUSSION