Overlapping Role of Respiratory Supercomplex Factor Rcf2 and Its N-terminal Homolog Rcf3 in Saccharomyces cerevisiae

Abstract

The 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 bc₁ 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 bc₁ complex in the absence of a functional cytochrome c oxidase and identify a supercomplex independent interaction network of the Rcf proteins.

Keywords: cytochrome c oxidase (complex IV); membrane protein; mitochondria; protein assembly; respiratory chain.

FIGURE 1.

After import a part of Rcf2 is processed into Rcf2^C and an unstable Rcf2^N fragment. A, radiolabeled Rcf2 was imported into isolated mitochondria for 15 min in the presence or absence of membrane potential (Δψ). Samples were treated with proteinase K where indicated and analyzed by SDS-PAGE and digital autoradiography. For comparison with endogenous Rcf2^C, mitochondria were analyzed by SDS-PAGE followed by Western blotting and immunodecoration. B, radiolabeled Rcf2 and N- and C-terminally truncated constructs (ΔN and ΔC) were analyzed by SDS-PAGE and compared with endogenous Rcf2^C. C, subcellular fractionation of wild-type and rcf2Δ cells expressing ^FLAGRcf2. Samples of homogenized cells (T), post mitochondrial supernatant (S), and mitochondrial pellet (P) were analyzed by SDS-PAGE and Western blotting. Cytosolic proteins are represented by Pgk1, and mitochondrial proteins are represented by Aco1. D, Isolated mitochondria of the strains used in C were analyzed by SDS-PAGE and Western blotting. Rcf2 variants were detected using FLAG and Rcf2 antibodies. E, model for Rcf2 processing in the inner mitochondrial membrane. The data shown in A–D represent the results of at least two individual experiments.

FIGURE 2.

Rcf2^C is low abundant in the oxidase, whereas Rcf2^N is free and not assembled into respiratory supercomplexes. A, radiolabeled N- and C-terminally truncated Rcf2 constructs (ΔN and ΔC) were imported into isolated mitochondria for 45 min in the presence or absence of membrane potential (Δψ). Proteinase K-treated samples were split for solubilization in 1% digitonin buffer and SDS sample buffer. The samples were analyzed by BN-PAGE or SDS-PAGE and digital autoradiography. B, isolated wild-type mitochondria were solubilized in 1% digitonin and analyzed by BN-PAGE followed by a second dimension SDS-PAGE and Western blotting. Cox1 and Rip1 indicate the positions of respiratory supercomplexes (III₂IV₂ and III₂IV), complex III dimer (III₂) and complex IV. Rcf2 and Rcf2^C were detected using Rcf2 antibody. C, isolated mitochondria of ^FLAGRcf2-expressing rcf2Δ were analyzed as in B. Qcr8 was used as marker for complex III. Rcf2 variants were detected using a FLAG and Rcf2 antibodies. The data shown in A–C represent the results of at least two individual experiments.

FIGURE 3.

Rcf3 (YBR255C-A) is an integral inner mitochondrial membrane protein. A, alignment of Rcf1, Rcf2, and Rcf3 (YBR255C-A). Dark green and cyan indicate transmembrane spans (TM); light green indicates a possible transmembrane span or hydrophobic patch. B, live cell fluorescence microscopy of Rcf3^GFP expressing cells with stained mitochondria by MitoTracker treatment. Scale bar, 5 μm. C, mitochondria of Rcf3^SF expressing cells were subjected to carbonate extraction or lysed with 1% Triton X-100 and were separated by ultracentrifugation into pellet (P), supernatant (S), and total (T). Samples were analyzed by SDS-PAGE and Western blot analysis. D, wild-type mitochondria were left untreated (M), swollen (MP), or lysed with 1% Triton X-100, treated with proteinase K where indicated, and subjected to SDS-PAGE and Western blotting. The data shown in B–D represent the results of at least two individual experiments.

FIGURE 4.

Deletion of RCF3 and RCF2 influence mitochondrial respiration via complex IV. A, rcf3Δ cells were tested for growth on minimal media supplemented with glucose or lactate at indicated temperatures in comparison to wild-type and respiratory deficient cox5aΔ. B, oxygen flux rates of mitochondria derived from wild-type and rcf3Δ cells were analyzed in an Oxygraph 2 k (Oroboros) at 30 °C (n = 4). C, rcf2Δ/rcf3Δ cells with additional expression of Rcf2 derivates or Rcf3 were tested for growth on minimal medium supplemented with glucose or glycerol at the indicated temperatures in comparison to wild-type and respiratory deficient shy1Δ. D, oxygen flux rates were measured in isolated mitochondria of wild type and rcf2Δ/rcf3Δ in an Oxygraph 2 k (Oroboros) at 30 °C (n = 4). The data shown in A–D represent the results of at least three individual experiments.

FIGURE 5.

Rcf3 associates with complex III and IV and assembles into respiratory supercomplexes. A, mitochondria isolated from wild-type, Cox4^ZZ, and Cor1^ZZ strains were solubilized in 1% digitonin or 0.6% DDM and subjected to IgG chromatography. Upon TEV protease cleavage, eluates were analyzed by SDS-PAGE and Western blotting. Cox4* marks Cox4 after removal of ZZ by TEV cleavage. B, radiolabeled Rcf3 or Rcf1 were imported into isolated rcf3Δ mitochondria for indicated times in the presence or absence of membrane potential (ΔΨ). Proteinase K-treated samples were solubilized in 1% digitonin or 0.6% DDM and analyzed by BN-PAGE and digital autoradiography. IV_Dig and IV_DDM indicate the size of the Rcf1-containing pool of monomeric complex IV when solubilized in the respective detergent. C, radiolabeled Rcf3 or Rcf1 were imported into isolated rcf3Δ mitochondria for 45 min and analyzed as specified in B. For comparison, wild-type mitochondria were solubilized in 1% digitonin or 0.6% DDM and analyzed by BN-PAGE and Western blotting. Complex III and IV containing assemblies were visualized by immunodetection of Rip1 and Cox1, respectively. D, digitonin-solubilized mitochondria of wild type, cox4Δ, cyt1Δ, or rcf3Δ were used for immunoprecipitation of Rcf3. Totals and glycine eluates were analyzed by SDS-PAGE and Western blotting. E, digitonin-solubilized mitochondria of wild type, cox4Δ, or cyt1Δ were used for immunoprecipitation of Rcf1, Rcf2, or Rcf3. Totals and glycine eluates were analyzed by SDS-PAGE and Western blotting. The data shown in A–E represent the results of at least two individual experiments.