The Cbp3–Cbp6 complex, which has been shown previously to promote cytochrome b synthesis and assembly, plays a key role in adjusting cytochrome b expression to the efficiency of assembly of the respiratory chain bc1 complex.

A complex specifically required for the biogenesis of the respiratory chain component cytochrome b binds to the tunnel exit of yeast mitochondrial ribosomes to coordinate protein synthesis and assembly.

Edited by Linda SpremulliThe mitochondrial electron transport chain consists of individual protein complexes arranged into large macromolecular structures, termed respiratory chain supercomplexes or respirasomes. In the yeast Saccharomyces cerevisiae, respiratory chain supercomplexes form by association of the bc 1 complex with the cytochrome c oxidase. Formation and maintenance of these assemblies are promoted by specific respiratory supercomplex factors, the Rcf proteins. For these proteins a regulatory function in bridging the electron transfer within supercomplexes has been proposed. Here we report on the maturation of Rcf2 into an N-and C-terminal peptide. We show that the previously uncharacterized Rcf3 (YBR255c-A) is a homolog of the N-terminal Rcf2 peptide, whereas Rcf1 is homologous to the C-terminal portion. Both Rcf3 and the C-terminal fragment of Rcf2 associate with monomeric cytochrome c oxidase and respiratory chain supercomplexes. A lack of Rcf2 and Rcf3 increases oxygen flux through the respiratory chain by up-regulation of the cytochrome c oxidase activity. A double gene deletion of RCF2 and RCF3 affects cellular survival under non-fermentable growth conditions, suggesting an overlapping role for both proteins in the regulation of the OXPHOS activity. Furthermore, our data suggest an association of all three Rcf proteins with the bc 1 complex in the absence of a functional cytochrome c oxidase and identify a supercomplex independent interaction network of the Rcf proteins.Eukaryotic cells cover their energy demands mainly with ATP generated by oxidative phosphorylation in mitochondria.

The cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain. The complex accepts electrons from cytochrome c and passes them onto molecular oxygen. This process contributes to energy capture in the form of a membrane potential across the inner membrane. The enzyme complex assembles in a stepwise process from the three mitochondria-encoded core subunits Cox1, Cox2 and Cox3, which associate with nuclear-encoded subunits and cofactors. In the yeast Saccharomyces cerevisiae, the cytochrome c oxidase associates with the bc1-complex into supercomplexes, allowing efficient energy transduction. Here we report on Cox26 as a protein found in respiratory chain supercomplexes containing cytochrome c oxidase. Our analyses reveal Cox26 as a novel stoichiometric structural subunit of the cytochrome c oxidase. A loss of Cox26 affects cytochrome c oxidase activity and respirasome organization.

a b s t r a c tThe mitochondrial respiratory chain is essential for the conversion of energy derived from the oxidation of metabolites into the membrane potential, which drives the synthesis of ATP. The electron transporting complexes bc 1 complex and the cytochrome c oxidase assemble into large supercomplexes, allowing efficient energy transduction. Currently, we have only limited information about what determines the structure of the supercomplex. Here, we characterize Aim24 in baker's yeast as a protein, which is integrated in the mitochondrial inner membrane and is required for the structural integrity of the supercomplex. Deletion of AIM24 strongly affects activity of the respiratory chain and induces a growth defect on non-fermentable medium. Our data indicate that Aim24 has a function in stabilizing the respiratory chain supercomplexes.

The authors have noted an error to a sentence in the abstract. The correct sentence appears below.The html and pdf versions of this article were corrected on June 20, 2011. The error remains only in the print version.On the one hand, the interaction of Cbp3 and Cbp6 with mitochondrial ribosomes is necessary for efficient translation of cytochrome b transcript. JCB: Correction

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The respiratory chain in the inner membrane of the yeast mitochondrion is organized as a network of individual complexes and large supercomplex structures.These supercomplexes are composed of dimeric complex III and one or two copies of complex IV (III2IV and III2IV2). Even though the existence of respiratory supercomplexes has been shown for a variety of organisms, it is not fully understood which purpose they serve and how they are assembled as well as regulated. Lipids, protein complexes and single proteins were proposed to take part in these intricate processes. To the latter group of potential supercomplex regulators belongs the Rcf protein family which is composed of three related proteins: Rcf1, Rcf2 and the so far uncharacterized YBR255C-A/ Rcf3. Rcf1 was shown to be essential for the formation of III2IV2. To obtain a deeper understanding of the role of the Rcf protein family in supercomplex formation and stability, this study aimed at an in-depth investigation of Rcf2 and Rcf3.Like Rcf1 and Rcf2, Rcf3 proved to be a constituent of supercomplexes via its association with complex IV*, an Rcf-specific version of complex IV. All three Rcfs furthermore revealed the ability to interact with complex III in the absence of complex IV, positioning them at the interface of both complexes. In contrast to Rcf1, Rcf3 and Rcf2 are dispensable for supercomplex formation. However, despite unchanged supercomplex organization, simultaneous deletion of RCF2 and RCF3 leads to severely reduced respiratory growth. This indicates a functional overlap, which is further supported by the sequence similarities of Rcf3 with the N-terminus of Rcf2 and the observed processing of Rcf2. This study revealed that Rcf2 is subjected to limited proteolysis after import into mitochondria. The resulting N-terminal fragment, Rcf2 N , was neither observed in individual complexes nor in supercomplexes. Whether it is degraded or preserved to fulfill a regulatory function within the respiratory chain could not be clarified on the basis of the present data. In contrast, the C-terminal fragment, Rcf2 C , is assembled into complex IV* along with the remaining full-length Rcf2. It was hence found in supercomplexes. Further investigations will elucidate its function and the significance of the processing event in regard to supercomplex organization.