Synchronized mitochondrial and cytosolic translation programs

Abstract

Oxidative phosphorylation (OXPHOS) is a vital process for energy generation, and is carried out by complexes within the mitochondria. OXPHOS complexes pose a unique challenge for cells because their subunits are encoded on both the nuclear and the mitochondrial genomes. Genomic approaches designed to study nuclear/cytosolic and bacterial gene expression have not been broadly applied to mitochondria, so the co-regulation of OXPHOS genes remains largely unexplored. Here we monitor mitochondrial and nuclear gene expression in Saccharomyces cerevisiae during mitochondrial biogenesis, when OXPHOS complexes are synthesized. We show that nuclear- and mitochondrial-encoded OXPHOS transcript levels do not increase concordantly. Instead, mitochondrial and cytosolic translation are rapidly, dynamically and synchronously regulated. Furthermore, cytosolic translation processes control mitochondrial translation unidirectionally. Thus, the nuclear genome coordinates mitochondrial and cytosolic translation to orchestrate the timely synthesis of OXPHOS complexes, representing an unappreciated regulatory layer shaping the mitochondrial proteome. Our whole-cell genomic profiling approach establishes a foundation for studies of global gene regulation in mitochondria.

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Figure 1: Expression of dual-origin OXPHOS complexes is induced upon adaptation to respiratory growth.
Figure 2: Mitoribosome profiling provides a genome-wide readout of mitochondrial translation.
Figure 3: Mitochondrial and cytosolic translation of OXPHOS mRNAs is rapidly and synchronously regulated.
Figure 4: Communication between translation systems is unidirectional.

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All data are deposited in Gene Expression Omnibus (accession number GSE74454).

References

  1. 1

    Masters, B. S., Stohl, L. L. & Clayton, D. A. Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. Cell 51, 89–99 (1987)

    CAS  Article  Google Scholar 

  2. 2

    Faye, G. & Sor, F. Analysis of mitochondrial ribosomal proteins of Saccharomyces cerevisiae by two dimensional polyacrylamide gel electrophoresis. Mol. Gen. Genet. 155, 27–34 (1977)

    CAS  Article  Google Scholar 

  3. 3

    Kehrein, K., Bonnefoy, N. & Ott, M. Mitochondrial protein synthesis: efficiency and accuracy. Antioxid. Redox Signal. 19, 1928–1939 (2013)

    CAS  Article  Google Scholar 

  4. 4

    Bonitz, S. G. et al. Codon recognition rules in yeast mitochondria. Proc. Natl Acad. Sci. USA 77, 3167–3170 (1980)

    CAS  ADS  Article  Google Scholar 

  5. 5

    Costanzo, M. C. & Fox, T. D. Control of mitochondrial gene expression in Saccharomyces cerevisiae . Annu. Rev. Genet. 24, 91–113 (1990)

    CAS  Article  Google Scholar 

  6. 6

    Green-Willms, N. S., Butler, C. A., Dunstan, H. M. & Fox, T. D. Pet111p, an inner membrane-bound translational activator that limits expression of the Saccharomyces cerevisiae mitochondrial gene COX2. J. Biol. Chem. 276, 6392–6397 (2001)

    CAS  Article  Google Scholar 

  7. 7

    Herrmann, J. M., Woellhaf, M. W. & Bonnefoy, N. Control of protein synthesis in yeast mitochondria: the concept of translational activators. Biochim. Biophys. Acta 1833, 286–294 (2013)

    CAS  Article  Google Scholar 

  8. 8

    Müller, P. P. et al. A nuclear mutation that post-transcriptionally blocks accumulation of a yeast mitochondrial gene product can be suppressed by a mitochondrial gene rearrangement. J. Mol. Biol. 175, 431–452 (1984)

    Article  Google Scholar 

  9. 9

    Fraenkel, D. G. Yeast Intermediary Metabolism (Cold Spring Harbor Press, 2011)

  10. 10

    Kuhn, K. M., DeRisi, J. L., Brown, P. O. & Sarnow, P. Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source. Mol. Cell. Biol. 21, 916–927 (2001)

    CAS  Article  Google Scholar 

  11. 11

    Amiott, E. A. & Jaehning, J. A. Mitochondrial transcription is regulated via an ATP “sensing” mechanism that couples RNA abundance to respiration. Mol. Cell 22, 329–338 (2006)

    CAS  Article  Google Scholar 

  12. 12

    Mueller, D. M. & Getz, G. S. Steady state analysis of mitochondrial RNA after growth of yeast Saccharomyces cerevisiae under catabolite repression and derepression. J. Biol. Chem. 261, 11816–11822 (1986)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    DeRisi, J. L., Iyer, V. R. & Brown, P. O. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686 (1997)

    CAS  ADS  Article  Google Scholar 

  14. 14

    Roberts, G. G. & Hudson, A. P. Transcriptome profiling of Saccharomyces cerevisiae during a transition from fermentative to glycerol-based respiratory growth reveals extensive metabolic and structural remodeling. Mol. Genet. Genomics 276, 170–186 (2006)

    CAS  Article  Google Scholar 

  15. 15

    Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 95, 14863–14868 (1998)

    CAS  ADS  Article  Google Scholar 

  16. 16

    Fox, T. D. et al. Analysis and manipulation of yeast mitochondrial genes. Methods Enzymol. 194, 149–165 (1991)

    CAS  Article  Google Scholar 

  17. 17

    Ashe, M. P., De Long, S. K. & Sachs, A. B. Glucose depletion rapidly inhibits translation initiation in yeast. Mol. Biol. Cell 11, 833–848 (2000)

    CAS  Article  Google Scholar 

  18. 18

    Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009)

    CAS  ADS  Article  Google Scholar 

  19. 19

    Rooijers, K., Loayza-Puch, F., Nijtmans, L. G. & Agami, R. Ribosome profiling reveals features of normal and disease-associated mitochondrial translation. Nature Commun. 4, 2886 (2013)

    ADS  Article  Google Scholar 

  20. 20

    Vignais, P. V., Stevens, B. J., Huet, J. & Andre, J. Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits. J. Cell Biol. 54, 468–492 (1972)

    CAS  Article  Google Scholar 

  21. 21

    Wolin, S. L. & Walter, P. Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J. 7, 3559–3569 (1988)

    CAS  Article  Google Scholar 

  22. 22

    Poutre, C. G. & Fox, T. D. PET111, a Saccharomyces cerevisiae nuclear gene required for translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit II. Genetics 115, 637–647 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Simpson, C. E. & Ashe, M. P. Adaptation to stress in yeast: to translate or not? Biochem. Soc. Trans. 40, 794–799 (2012)

    CAS  Article  Google Scholar 

  24. 24

    Poyton, R. O., Bellus, G., McKee, E. E., Sevarino, K. A. & Goehring, B. In organello mitochondrial protein and RNA synthesis systems from Saccharomyces cerevisiae . Methods Enzymol. 264, 36–42 (1996)

    CAS  Article  Google Scholar 

  25. 25

    Lamb, A. J., Clark-Walker, G. D. & Linnane, A. W. The biogenesis of mitochondria. 4. The differentiation of mitochondrial and cytoplasmic protein synthesizing systems in vitro by antibiotics. Biochim. Biophys. Acta 161, 415–427 (1968)

    CAS  Article  Google Scholar 

  26. 26

    Rak, M. et al. Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p. Mol. Biol. Cell (2016)

  27. 27

    Sun, T. & Zhang, Y. Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation. Nucleic Acids Res. 36, 1654–1664 (2008)

    CAS  Article  Google Scholar 

  28. 28

    Zhang, Y., Bell, A., Perlman, P. S. & Leibowitz, M. J. Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae . RNA 6, 937–951 (2000)

    CAS  Article  Google Scholar 

  29. 29

    Liu, B. & Qian, S. B. Translational reprogramming in cellular stress response. Wiley Interdiscip. Rev. RNA 5, 301–315 (2014)

    CAS  Article  Google Scholar 

  30. 30

    Pavlov, M. Y. & Ehrenberg, M. Optimal control of gene expression for fast proteome adaptation to environmental change. Proc. Natl Acad. Sci. USA 110, 20527–20532 (2013)

    CAS  ADS  Article  Google Scholar 

  31. 31

    Sonenberg, N. & Hinnebusch, A. G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136, 731–745 (2009)

    CAS  Article  Google Scholar 

  32. 32

    Gruschke, S. et al. The Cbp3-Cbp6 complex coordinates cytochrome b synthesis with bc 1 complex assembly in yeast mitochondria. J. Cell Biol. 199, 137–150 (2012)

    CAS  Article  Google Scholar 

  33. 33

    Perez-Martinez, X., Butler, C. A., Shingu-Vazquez, M. & Fox, T. D. Dual functions of Mss51 couple synthesis of Cox1 to assembly of cytochrome c oxidase in Saccharomyces cerevisiae mitochondria. Mol. Biol. Cell 20, 4371–4380 (2009)

    CAS  Article  Google Scholar 

  34. 34

    Ehrenreich, I. M. et al. Dissection of genetically complex traits with extremely large pools of yeast segregants. Nature 464, 1039–1042 (2010)

    CAS  ADS  Article  Google Scholar 

  35. 35

    Gaisne, M., Becam, A. M., Verdiere, J. & Herbert, C. J. A ‘natural’ mutation in Saccharomyces cerevisiae strains derived from S288c affects the complex regulatory gene HAP1 (CYP1). Curr. Genet. 36, 195–200 (1999)

    CAS  Article  Google Scholar 

  36. 36

    Baruffini, E., Lodi, T., Dallabona, C. & Foury, F. A single nucleotide polymorphism in the DNA polymerase gamma gene of Saccharomyces cerevisiae laboratory strains is responsible for increased mitochondrial DNA mutability. Genetics 177, 1227–1231 (2007)

    CAS  Article  Google Scholar 

  37. 37

    Young, M. J. & Court, D. A. Effects of the S288c genetic background and common auxotrophic markers on mitochondrial DNA function in Saccharomyces cerevisiae . Yeast 25, 903–912 (2008)

    CAS  Article  Google Scholar 

  38. 38

    Alani, E., Cao, L. & Kleckner, N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116, 541–545 (1987)

    CAS  Article  Google Scholar 

  39. 39

    Moqtaderi, Z. & Struhl, K. Expanding the repertoire of plasmids for PCR-mediated epitope tagging in yeast. Yeast 25, 287–292 (2008)

    CAS  Article  Google Scholar 

  40. 40

    Storici, F. & Resnick, M. A. The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods Enzymol. 409, 329–345 (2006)

    CAS  Article  Google Scholar 

  41. 41

    Dimitrov, L. N., Brem, R. B., Kruglyak, L. & Gottschling, D. E. Polymorphisms in multiple genes contribute to the spontaneous mitochondrial genome instability of Saccharomyces cerevisiae S288C strains. Genetics 183, 365–383 (2009)

    CAS  Article  Google Scholar 

  42. 42

    Gouget, K., Verde, F. & Barrientos, A. In vivo labeling and analysis of mitochondrial translation products in budding and in fission yeasts. Methods Mol. Biol. 457, 113–124 (2008)

    CAS  Article  Google Scholar 

  43. 43

    Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nature Protocols 7, 1534–1550 (2012)

    CAS  Article  Google Scholar 

  44. 44

    Brar, G. A. et al. High-resolution view of the yeast meiotic program revealed by ribosome profiling. Science 335, 552–557 (2012)

    CAS  ADS  Article  Google Scholar 

  45. 45

    Ingolia, N. T. Genome-wide translational profiling by ribosome footprinting. Methods Enzymol. 470, 119–142 (2010)

    CAS  Article  Google Scholar 

  46. 46

    Martin, M. Cutadapt removes adaptor sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011)

    Article  Google Scholar 

  47. 47

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

    Article  Google Scholar 

  48. 48

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

    Article  Google Scholar 

  49. 49

    Li, G. W., Burkhardt, D., Gross, C. & Weissman, J. S. Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell 157, 624–635 (2014)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank F. Winston, T. Fox, G. Brar and members of the Churchman lab for advice and discussions; members of the O’Shea, Novina, and Springer labs for use of equipment and advice; and M. Hickman and D. Botstein for sharing the HAP1+ strain. Research supported by a Damon Runyon Cancer Research Foundation Frey Award (to L.S.C.), a Burroughs Wellcome Fund Career Award at the Scientific Interface (to L.S.C.), an Ellison Medical Foundation New Scholar in Aging Award (to L.S.C.), the National Institutes of Health F32 (to M.T.C.), and a Boehringer Ingelheim Fonds PhD Fellowship (to G.S.).

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M.T.C. and L.S.C. designed the research and wrote the manuscript. M.T.C. conducted the experiments with help from I.C.S. who performed mitoribosome profiling with drug treatments, and G.S., who created and performed mitoribosome profiling on the pet111Δ strain. M.T.C. analysed the data. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to L. Stirling Churchman.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks P. Van Damme and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 OXPHOS proteins are induced during mitochondrial biogenesis.

Western blot analysis of mitochondrial (Cob, Cox1, Cox2) and nuclear (Cox4) OXPHOS proteins compared to Flag-tagged mitoribosome small subunit protein MrpS17. Gapdh was used as a loading control. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 Dynamics of non-OXPHOS RNAs during mitochondrial biogenesis.

ac, RNA levels (reads per kb) normalized to spike-in controls and plotted as fold changes compared to levels in log phase glucose growth for all nuclear-encoded structural components of the complexes shown (a); intron-encoded maturases (b); and nuclear and mitochondrial-encoded mitoribosome subunits (c). To calculate values for maturase transcripts, only reads not overlapping the main ORF (COX1 or COB) were considered. Group II intron splicing intermediates stably accumulate and may not represent translation-competent transcripts.

Extended Data Figure 3 Optimization of affinity purification for intact mitoribosomes.

a, Serial dilution spot tests verifying tagged mitoribosome subunits are functional as they support respiratory growth on glycerol (YPG). ρ0 is a strain without mitochondrial DNA. b, Frequency of petite colonies in our corrected S288c strain (see Methods) after growth for 5 days on 0.1% glucose and 3% glycerol. BY4742 is S288c background with designer auxotrophies. Σ1278b is a strain with wild-type HAP1, and a high-fidelity allele of MIP1, MIP1[Σ], along with other differences compared to S288c. Error bars show variation due to counting, with 175–750 colonies counted for each sample. c, Lysis and immunoprecipitation buffer conditions affect mitoribosome subunit association and thus footprint retention. Left: silver staining after immunoprecipitation of the large subunit (LSU) with Mrp20–Flag and of the small subunit (SSU) with Mrps17–Flag in condition 1 (20 mM Tris pH 8.0, 200 mM KCl, 5 mM MgCl2, 0.5% lauryl maltoside), and in condition 2 (10 mM Tris pH 8.0, 50 mM NH4Cl, 10 mM MgCl2, 0.5% lauryl maltoside). Arrowheads indicate bands that appear in both immunoprecipitations in condition 2 that can be assigned to the LSU or SSU by comparison to condition 1. Asterisks mark the expected mobility of the tagged proteins. Right: northern blotting of the co-purifying RNA in each condition. For gel source data, see Supplementary Fig. 1. d, Western blot showing fractions from immunoprecipitation using optimized buffer conditions. Flag immunoprecipitation targeting the mitoribosome SSU co-purifies a haemagglutinin (HA)-tagged LSU protein. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 Mitoribosome profiling is robust, reproducible, and does not require translation inhibitors.

a, Mapping statistics for representative mitoribosome and cytoribosome profiling libraries from log phase glycerol-grown cells. b, Fraction of reads mapping to each frame of mitochondrial ORFs (left) and nuclear ORFs (right) in mitoribosome profiling and cytoribosome profiling data, respectively. RNA-seq reads in the left panel were treated identically to footprint reads, including size selection for library generation. c, Reproducibility between biological replicates. Each dot corresponds to the number of reads mapped to a particular position on mRNA (RPM, left), or summed number of reads mapped across each mRNA then normalized to length (RPKM, right). d, Reproducibility with and without translation inhibitors thiamphenicol (50 μg ml−1) and GTP analogue GMPPNP (1 mM).

Extended Data Figure 5 Mitochondrial and cytosolic protein synthesis on OXPHOS mRNAs is rapidly regulated.

a, b, Fold changes in relative protein synthesis (footprint RPKM values) compared to log phase glucose growth for the OXPHOS subunits synthesized in mitochondria (a; values are means of two experiments) and cytosol (b). Asterisks on heat maps indicate the subunits shown in the line plots.

Extended Data Figure 6 Mitoribosome translation efficiency fold changes are reproducible.

Fold change data identical to those shown in Fig. 3a, but including range bars for two experiments performed from independent cultures on different days (left), and fold change translation efficiency data plotted as a scatter with the Pearson correlation coefficient (right). Dotted lines mark twofold difference. RNA-seq data used in calculating translation efficiency are from a single experiment.

Extended Data Figure 7 Global translation is transiently inhibited upon shift to glycerol.

Polysome profiles from samples used for cytoribosome profiling, but without RNase I treatment. Gradients were loaded with lysate from equal cell numbers, allowing overall ribosome abundances to be compared between samples. Doubling time during log phase is ~1.2 h in glucose and ~3.7 h in glycerol.

Extended Data Figure 8 Cytosolic translation controls mitochondrial translation response.

a, FACS analysis of yeast cultures treated with CCCP. Wild-type cultures were grown in glucose to mid-log phase and treated with 40 μM CCCP for the indicated times. Mitochondrial membrane potential (ΔΨm) was assessed using 1 μM tetramethylrhodamine (TMRM), which is taken up by only a fraction of the cell population (17.9% in this experiment). TMRM accumulates inside negatively charged mitochondria, producing increased fluorescence intensity (102). Loss of membrane potential dissipates probe, measured as loss of high-intensity fluorescence. b, Representative northern blots for data in Fig. 4b, c and panel e. For quantification, northern signals were normalized by relative mitoribosome recovery measured by Mrps17–Flag signal in western blots. For gel source data, see Supplementary Fig. 1. c, Northern blotting of total RNA for the indicated transcripts. For gel source data, see Supplementary Fig. 1. d, Quantification of viability assay. Cells were grown in YPD (Glu) or YPG (Gly) with or without drug for the time indicated. Cells were washed out of drug and plated on YPD for calculation of colony-forming units. CCCP (40 μM); CHX (100 μg ml−1); pentamidine (Pent; 10 μM). e, Mitochondrial translation response to inhibition of mitochondrial import with CCCP, measured by northern blotting for footprints (see b). f, Fold change in synthesis measured by cytoribosome profiling of nuclear-encoded mitochondrial mRNA-specific translational activators (colour-coded by mRNA target). For each mitochondrial mRNA, the names of the known translational activators are listed. Source data

Extended Data Figure 9 Cytosolic OXPHOS translation response is independent of mitochondrial gene expression.

a, Metabolic labelling to measure mitochondrial translation (Mito Tln), detectable only in the presence of CHX, and cytosolic translation (Cyto Tln). Samples generated in the absence of CHX were diluted 15-fold before loading the gel compared to samples generated with CHX. Mitochondrial translation products are labelled. b, c, Full data set for experiment presented in Fig. 4d, e, showing fold changes in translation efficiency of all nuclear-encoded complex III, complex IV, and ATP synthase subunits measured by cytoribosome profiling without (−Pent) or with (+Pent) inhibition of mitochondrial translation (b) and in ρ0 cells (c), which have neither mitochondrial translation nor functional OXPHOS complexes.

Extended Data Figure 10 Verification of mtDNA loss in ρ0 strain.

a, Spot tests verifying that the ρ0 strain generated by overnight growth in ethidium bromide (see Methods) cannot respire (no growth on YPG). b, PCR (left) and qPCR (right) verifying loss of mitochondrial-encoded genes COX1, COX3, and the 21S mitochondrial rRNA gene. MRPS17 is nuclear-encoded. Bars show s.e.m. for technical triplicates.

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Couvillion, M., Soto, I., Shipkovenska, G. et al. Synchronized mitochondrial and cytosolic translation programs. Nature 533, 499–503 (2016). https://doi.org/10.1038/nature18015

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