A Pathogenic Cytochrome b Mutation Reveals New Interactions between Subunits of the Mitochondrial bc1Complex

Abstract

Energy transduction in mitochondria involves five oligomeric complexes embedded within the inner membrane. They are composed of catalytic and noncatalytic subunits, the role of these latter proteins often being difficult to assign. One of these complexes, the bc1 complex, is composed of three catalytic subunits including cytochrome b and seven or eight noncatalytic subunits. Recently, several mutations in the human cytochrome b gene have been linked to various diseases. We have studied in detail the effects of a cardiomyopathy generating mutation G252D in yeast. This mutation disturbs the biogenesis of the bc1 complex at 36°C and decreases the steady-state level of the noncatalytic subunit Qcr9p. In addition, the G252D mutation and the deletion of QCR9 show synergetic defects that can be partially bypassed by suppressor mutations at position 252 and by a new cytochrome b mutation, P174T. Altogether, our results suggest that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between the catalytic subunits, this role being essential at high temperature.Numerous cellular functions are performed by oligomeric complexes composed of catalytic and noncatalytic subunits and the role of these latter proteins is often difficult to assign. Energy transduction in mitochondria involves five oligomeric complexes that are embedded within the inner membrane. One of these complexes, the bc1 complex is composed of three catalytic and seven or eight noncatalytic subunits (for review, see Ref. 1). The three catalytic subunits, cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein (Rieske) are conserved in the bacterial equivalents of this complex. However, several noncatalytic subunits are present in the mitochondrial bc1 complexes and are often referred to as supernumerary subunits although some are required for the enzymatic activity. In Saccharomyces cerevisiae the bc1 complex contains seven supernumerary subunits that are conserved in the mammalian enzyme. The mammalian bc1 complex presents an additional subunit that has no equivalent in yeast. The mitochondrial bc1 complexes from bovine, chicken, and yeast have been crystallized (2-5) and an analysis of these structures reveals that the general shape, size, and topology of the complexes are similar in the three organisms.

DMR1 (CCM1/YGR150C) of Saccharomyces cerevisiae Encodes an RNA-Binding Protein from the Pentatricopeptide Repeat Family Required for the Maintenance of the Mitochondrial 15S Ribosomal RNA

“…We constructed strains bearing the deletion of DMR1 in the background of the strain W303, which has been extensively used in the study of nucleomitochondrial interactions. To test the possible involvement of Dmr1p in the splicing of mitochondrial introns, we constructed dmr1D strains bearing either wild-type long mtDNA (13 introns), or the intronless variant (Seraphin et al 1987;Saint-Georges et al 2002). The phenotype of tight respiratory deficiency ( Figure 1A) was identical for both strains, which rules out the involvement in the splicing of mitochondrial introns as the primary defect in dmr1D mutants.…”

Mitochondria-associated Yeast mRNAs and the Biogenesis of Molecular Complexes

“…The Saccharomyces cerevisiae strain CW252 has an intronless mitochondrial genome and is nuclearly isogenic to W303 (Saint-Georges et al, 2002). Cells were grown at 30°C in galactose medium (1% bactopeptone, 1% yeast extract, Address correspondence to: Claude Jacq (jacq@biologie.ens.fr).…”

“…The Saccharomyces cerevisiae strain CW252 has an intronless mitochondrial genome and is nuclearly isogenic to W303 (Saint-Georges et al, 2002 Quantitative PCR analysis of asymmetric mRNA localization. Schematically, mitochondrion-bound polysomes were purified as in Kellems et al (1975), and RNA was extracted using the hot-phenol method.…”

Human Disease-related Mutations in Cytochrome b Studied in Yeast

“…The analyses were performed as described in Ref. 10. The mitochondrial membrane preparations (40 g of total protein/sample) were electrophoresed on SDS-polyacrylamide gels (4 -20% linear gradient polyacryamide gel) prior to transfer to polyvinylidene difluoride membrane by semi-dry electroblotting.…”