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. 2016 Jul 8;44(12):5785-97.
doi: 10.1093/nar/gkw490. Epub 2016 Jun 1.

Ribosome recycling defects modify the balance between the synthesis and assembly of specific subunits of the oxidative phosphorylation complexes in yeast mitochondria

Jelena Ostojić  1 , Cristina Panozzo  1 , Alexa Bourand-Plantefol  1 , Christopher J Herbert  1 , Geneviève Dujardin  2 , Nathalie Bonnefoy  3
Affiliations

Ribosome recycling defects modify the balance between the synthesis and assembly of specific subunits of the oxidative phosphorylation complexes in yeast mitochondria

Jelena Ostojić et al. Nucleic Acids Res. .

Abstract

Mitochondria have their own translation machinery that produces key subunits of the OXPHOS complexes. This machinery relies on the coordinated action of nuclear-encoded factors of bacterial origin that are well conserved between humans and yeast. In humans, mutations in these factors can cause diseases; in yeast, mutations abolishing mitochondrial translation destabilize the mitochondrial DNA. We show that when the mitochondrial genome contains no introns, the loss of the yeast factors Mif3 and Rrf1 involved in ribosome recycling neither blocks translation nor destabilizes mitochondrial DNA. Rather, the absence of these factors increases the synthesis of the mitochondrially-encoded subunits Cox1, Cytb and Atp9, while strongly impairing the assembly of OXPHOS complexes IV and V. We further show that in the absence of Rrf1, the COX1 specific translation activator Mss51 accumulates in low molecular weight forms, thought to be the source of the translationally-active form, explaining the increased synthesis of Cox1. We propose that Rrf1 takes part in the coordination between translation and OXPHOS assembly in yeast mitochondria. These interactions between general and specific translation factors might reveal an evolutionary adaptation of the bacterial translation machinery to the set of integral membrane proteins that are translated within mitochondria.

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Figures

Figure 1.
Figure 1.
MtDNA stability and respiratory growth in mitochondrial translation mutants. (A) The structure of the M. tuberculosis RRF and the predicted structure of the last 183 residues of S. cerevisiae Rrf1 are shown together with the superposition. (B) The names of bacterial translation factors are indicated on a schematic representation of the main steps of translation; corresponding yeast mitochondrial factors are given in parentheses. LSU: large ribosomal subunit, SSU: small ribosomal subunit. (C) Dilution series of cells from wild type (wt) and a series of mitochondrial translation factor mutants were spotted onto fermentable (glucose) and respiratory (glycerol) media and incubated for four days at 28°C and 36°C.
Figure 2.
Figure 2.
In vivo mitochondrial translation and mRNA accumulation in mutants devoid of ribosome recycling (Rrf1) or translation initiation factors (Ifm1 or Mif3). (A) Products of mitochondrial translation were labeled in vivo for 10 min with [35S]-methionine and cysteine. Cox1, Cox2, Cox3, Cytb, Atp6, Atp8, Atp9 are the mitochondrially encoded subunits of respiratory complexes IV (Cox), III (Cytb) and V (Atp). Var1 is a mitochondrially-encoded small ribosomal subunit. Upper panel: autoradiography of the gel. Lower panel: Coomassie staining. The total incorporation, obtained by quantifying with the Image J. software and summing up all bands of the autoradiography corresponding to mitochondrial proteins, is given as a percentage of the wild type labeling normalized using the Coomassie staining. (B) The labeling of individual proteins was also quantified with the Image J. software and normalized to the Coomassie staining. (C) Total RNAs (10 μg) were analyzed by Northern blotting with probes specific for the protein-encoding mitochondrial genes COX1, COX2, CYTB and ATP9, and the mitochondrial rRNAs 21S and 15S; actin (ACT1) was used as a loading control.
Figure 3.
Figure 3.
Assembly of the OXPHOS complexes in mutants devoid of ribosome recycling (Rrf1) or translation initiation factors (Ifm1 or Mif3). (A) Total proteins from [35S]-labeling experiments were analyzed by Western blotting with antibodies against Cox1, Cytb, Atp9, Cox2; Porin was used as a loading control (upper panel). Quantification was done using the Image J software (lower panel). (B) Cytochrome absorption spectra were recorded on whole cells. The position of the absorption maxima of cytochromes a + a3 (complex IV), cytochrome b and c1 (complex III) and c are indicated. (C and D) Mitochondrial proteins were solubilized with either dodecylmaltoside (DM, panel C) or digitonin (DG, panel D), separated on BN-PAGE 4–16% (left) or 3–12% (right) and immunoblotted with antibodies against Cyt1, Cox2 and Atp2. Positions of respiratory complexes III and IV, of supercomplexes III2 + IV2, III2 + IV as well as those of multimers (Vn), monomers (V) and F1-part of complex V are indicated. The protein molecular mass markers are also indicated (kDa).
Figure 4.
Figure 4.
Pulse-chase analysis of mitochondrial translation in the wile type and Δrrf1 strains. (A) Products of mitochondrial translation were labeled in vivo for 2.5, 5, 7.5 or 10 min with [35S]-methionine and cysteine and then chased for 60, 120 or 240 min in presence of an excess of cold methionine and cysteine. Total incorporation was calculated as described in Figure 2A. (B) Histograms showing the quantification of individual proteins calculated as described in Figure 2B.
Figure 5.
Figure 5.
Genetic interactions and localization of Rrf1 and the translational activators. (A) In vivo [35S] incorporation as in Figure 2A. (B) Mitochondria were purified from cells producing Mss51-c-Myc. Mitochondrial proteins (M) were either sonicated (left) or alkali treated (right) and after centrifugation the soluble proteins were recovered in the supernatant (S) whereas the membrane-associated or membrane-spanning proteins remained in the pellet (P). Proteins were analyzed by Western blotting with antibodies recognizing Rrf1 or the epitope c-Myc. The soluble matrix protein Hsp60 and the integral mitochondrial membrane protein Cox2 were used as controls.
Figure 6.
Figure 6.
Functional interaction between Rrf1 and Mss51. (A) Mitochondrial proteins from RRF1-HA or Δrrf1 cells producing Mss51-c-Myc were immunoprecipitated using anti-HA or anti-c-Myc-agarose beads. T: total; S: supernatant; W: wash; IP: immunoprecipitate. Proteins were analyzed by Western blotting with antibodies recognizing Rrf1, Ssc1, Cox1, Mrp20, HA or c-Myc. The ribosomal protein Mrp20 undergoes some degradation during these immunoprecipitation experiments unless a cocktail of protease inhibitors is added. (B) Mitochondrial proteins of wt or Δrrf1 cells producing Mss51-c-Myc were analyzed by SDS-PAGE (left panel) or were lysed in digitonin, separated on BN-PAGE 3–12% (right panel) and immunoblotted with antibodies recognizing c-Myc, Cox1, Ssc1, Tom40. The protein molecular mass markers are indicated. Mss51A: high molecular weight assembly-competent complexes; Mss51T: low molecular weight complex thought to be the source of translationally-active complex. (C) Model of the effect of an absence of Rrf1 on Cox1 synthesis and complex IV assembly. We hypothesize that in the absence of Rrf1, the dissociation of the ribosomal subunits is slowed down, reducing the accessibility of Mss51 to its binding site on the C-terminus of Cox1 and concomitantly preventing the formation of the high molecular weight complex MSS51A. This would displace the equilibrium between Mss51T and Mss51A toward Mss51T, thus reducing the assembly of complex IV.
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