Oxidative phosphorylation (OXPHOS) is vital for the regeneration of the vast majority of ATP in eukaryotic cells1. OXPHOS is carried out by large multi-subunit protein complexes in the cristae membranes, which are invaginations of the mitochondrial inner membrane. The OXPHOS complexes are a mix of subunits encoded in the nuclear and mitochondrial genomes. Thus, the assembly of these dual-origin complexes is an enormous logistical challenge for the cell. Using super-resolution microscopy (nanoscopy) and quantitative cryo-immunogold electron microscopy, we determined where specific transcripts are translated and where distinct assembly steps of the dual-origin complexes in the yeast Saccharomyces cerevisiae occur. Our data indicate that the mitochondrially encoded proteins of complex III and complex IV are preferentially inserted in different sites of the inner membrane than those of complex V. We further demonstrate that the early, but not the late, assembly steps of complex III and complex IV occur preferentially in the inner boundary membrane. By contrast, all steps of complex V assembly occur mainly in the cristae membranes. Thus, OXPHOS complex assembly is spatially well orchestrated, probably representing an unappreciated regulatory layer in mitochondrial biogenesis.
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We thank A. Kremer and S. Lippens of the VIB BioImaging Core in Ghent, Belgium, for acquisition of the FIB-SEM data. We thank J. Herrmann, TU Kaiserslautern, for providing antisera, B. Westermann, University of Bayreuth, for providing the plasmid pVT100U-mt GFP and S. W. Hell for continuous support. We are grateful to R. Schmitz-Salue and G. Heim for excellent technical assistance and J. Jethwa for proofreading the manuscript. This work was supported by the Swedish Research Council, the Knut and Alice Wallenberg Foundation (both to M.O.), the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain and the DFG funded SFB1190 (project P01) (both to S.J.).
The authors declare no competing interests.
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Integrated supplementary information
Images of representative mitochondrial segments of ten different yeast cells expressing Cbp3-GFP (a), Coa1-GFP (b), Atp25-GFP (c), mitochondria targeted GFP (mtGFP) (d), Atp4-GFP (e), and Mic60-GFP (f) fusion proteins from the respective endogenous genomic locus (or from a plasmid in case of mtGFP). Cells were immunolabelled with an antiserum against GFP and imaged with STED nanoscopy and diffraction limited confocal microscopy, as indicated. All nanoscopy experiments were done in triplicate, persistently giving similar results. Scale bars: 200 nm
Supplementary Figure 2 The majority of all CM fragments seen on 2D slices are part of larger cristae.
For the analysis, a FIB-SEM data stack recorded on wild type S. cerevisiae cells grown in galactose medium was scrutinized. We recorded one FIB-SEM stack containing several dozen yeast cells and we reconstructed mitochondrial membranes out of three cells, all displaying similar ultrastructures. (a) We define the size of a given crista as the largest contour length of this crista in a 2D section; by this, we will underestimate the size of very large cristae, but will reliably detect all small cristae. To determine the largest contour length of an individual crista, we analyzed 2D sections in a reconstructed 3D FIB-SEM data stack. Only the largest contour length of a crista was used for further analysis. The analysis was performed on mitochondria of two randomly chosen cells, giving similar results. In the example given in (a), the arrowheads point to the same crista in different slices of the FIB-SEM data stack. The contour length of the crista shown in slice 216 (marked in magenta) is used to characterize this crista. (The mitochondrial outer membrane is depicted in orange, the CMs in green). (b) Distribution of measured largest contour lengths of cristae. 112 cristae in two different cells were analyzed. Note that only ~6 % (7 in 112 cristae) were smaller than 50 % of the median. We define these cristae as small cristae. (c) The contour lengths of the cristae were arranged into classes and the frequency of these classes is shown. (d) A part of the mitochondrial network (21 slices) that incorporates the largest of the small cristae (see a-c) was reconstructed in 3D. The small crista is highlighted in magenta, all other cristae in cyan, the outer mitochondrial membrane in light gray. (e) The CM lengths in the 21 2D slices that lead to the 3D reconstruction shown in (d) were measured. The pie-chart displays the amount of CMs belonging to large cristae (blue) and small cristae (red). Scale bars: 200 nm (a), 100 nm (d)
Shown are Western Blots of whole cell extracts decorated with an antiserum against GFP, as indicated. As a loading control of each western blot, a porin specific serum was used. Outlines indicate different exposure times that were used to ensure optimal signal to noise ratios for proteins with different expression levels on the same blot. All experiments were repeated at least two times with similar results. The full blots are shown in Supplementary Figure 6
Supplementary Figure 4 Genomic tagging of the proteins of interest with GFP did not influence respiration.
Ten-fold dilutions of logarithmically growing cells with the indicated genotypes were spotted onto agar plates containing glucose (YPD; fermentable), glycerol (YPG; non-fermentable) or glycerol/ethanol (SGE; non-fermentable) as the carbon sources. All experiments were repeated at least two times with similar results. The plates were incubated for 4 d at 30 °C before photography
Supplementary Figure 5 Quantitative immuno-gold EM of mitochondrial proteins on cryo-sectioned cells.
Shown are representative EM images (right) and the quantifications taken from a larger number of images (left). The distributions of Cor1 and Cox2 were determined with antiserum against these proteins; all other sections were decorated with antiserum against GFP. Cells were grown at 30 °C in galactose containing medium. The arrowheads point to gold particles at the IBM (black) or at the CM (grey). For each analyzed protein localization, at least three individual slices were decorated and analyzed; n indicates the number of analyzed gold particles. Scale bars: 100 nm
(a-i) corresponds to the labelling in Supplementary Figure 3. Reflected light images of the blot membranes containing the size marker are combined with the camera images of the recorded enhanced chemiluminescence (ECL) signals. Membranes were not cut
Supplementary Figures 1–6, Supplementary Table and Supplementary Video legends, and Supplementary References.
Proteins investigated in this study and their role in mitochondrial translation and OXPHOS assembly.
Primary antibodies used in this study.
Primers used for epitope tagging and gene knockouts.
Mitochondrial inner architecture.
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Stoldt, S., Wenzel, D., Kehrein, K. et al. Spatial orchestration of mitochondrial translation and OXPHOS complex assembly. Nat Cell Biol 20, 528–534 (2018). https://doi.org/10.1038/s41556-018-0090-7
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