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The metabolic growth limitations of petite cells lacking the mitochondrial genome

Nature Metabolism volume 3, pages 1521–1535 (2021)Cite this article

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Abstract

Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. ‘Petite yeasts’, characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.

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Fig. 1: ATP3 variants overcome the slow-growth phenotype of petites.
Fig. 2: Suppressor mutations lead to an increased mitochondrial membrane potential, while morphology and capacity to metabolize glucose remain largely unaffected.
Fig. 3: Amino acid metabolism is perturbed in petites, and their growth defect is rescued by the addition of selected amino acids.
Fig. 4: Aconitase and tricarboxylic acid cycle are inhibited in petites, which is alleviated in evolved petites.
Fig. 5: Iron overload inhibits aconitase, which is required for petite suppression.

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Data availability

Proteomic data are provided in PRIDE under accession number PXD011715. A list of mass spectrometry files is provided in Supplementary Table 5. Processed metabolomics and proteomics data are provided in Supplementary Data 1–3. There is no restriction on data availability.

Code availability

Code used for data analysis and creation of figures is available at https://github.com/JakobV/metabolic-growth-limitations-of-petite-cells/.

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Acknowledgements

We thank our laboratory members and J. Bähler for critical discussion and comments on the manuscript, and C. Kilian for technical support. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001134), the UK Medical Research Council (FC001134) and the Wellcome Trust (FC001134), and received specific funding from the European Research Council (StG 260809 and SYG 951475) and the Wellcome Trust (IA 200829/Z/16/Z), as well as the FWF (Austria) for project P26713 (to M.B.) and a Swiss National Science Foundation Postdoc Mobility fellowship (191052 to J.H.).

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Author notes

  1. These authors contributed equally: Jakob Vowinckel, Johannes Hartl.

Authors and Affiliations

  1. Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK

    Jakob Vowinckel, Johannes Hartl, Kathrin Runggatscher, Markus A. Keller, Michael Mülleder, Nianshu Zhang & Markus Ralser

  2. Biognosys AG, Schlieren, Switzerland

    Jakob Vowinckel

  3. Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Biochemistry, Berlin, Germany

    Johannes Hartl, Michael Mülleder, Andrea Lehmann & Markus Ralser

  4. Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria

    Hans Marx & Diethard Mattanovich

  5. Sequencing Core Facility, Max Planck Institute for Molecular Genetics and Max Planck Unit for the Science of Pathogens, Berlin, Germany

    Martin Kerick & Bernd Timmermann

  6. Institute of Parasitology and Biomedicine ‘López-Neyra’ (IPBLN, CSIC), Granada, Spain

    Martin Kerick

  7. Institute of Human Genetics, Medical University of Innsbruck, Innsbruck, Austria

    Markus A. Keller

  8. The Molecular Biology of Metabolism Laboratory, The Francis Crick Institute, London, UK

    Michael Mülleder, Jason S. L. Yu, Simran Kaur Aulakh & Markus Ralser

  9. Department of Earth Sciences, University of Cambridge, Cambridge, UK

    Jason Day

  10. Department of Biosciences, University of Salzburg, Salzburg, Austria

    Manuela Weber, Mark Rinnerthaler & Michael Breitenbach

  11. Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland

    Cory D. Dunn

  12. Department of Molecular Biology and Genetics, Koç University, İstanbul, Turkey

    Cory D. Dunn

  13. Metabolomics Laboratory, The Francis Crick Institute, London, UK

    James I. MacRae

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  1. Jakob Vowinckel
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Contributions

J.V., J.H. and M. Ralser conceived the project, planned and designed experiments with input from all authors. M. Ralser supervised the project. J.V., J.H. and M. Ralser wrote the manuscript with help from all authors. J.V., J.H., H.M., M.K., K.R., M.A.K., M.M., J.D., M.W., M. Rinnerthaler, J.S.L.Y., S.K.A. and A.L. performed experiments and analysed the data. D.M., B.T., N.Z., C.D.D., J.I.M. and M.B. helped supervise the project and contributed to discussion and interpretation of the results.

Corresponding author

Correspondence to Markus Ralser.

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The authors declare no competing interests.

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Peer review information Nature Metabolism thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Ashley Castellanos-Jankiewicz; Pooja Jha.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Growth of wild-type strain is not affected by adaptive laboratory evolution.

Wild-type yeast evolved in chemostats in parallel to corresponding petites that were sampled during the evolution experiment and spotted on solid F1 medium. Colony sizes remain largely unaffected over the course of the experiment. Shown is one of n = 3 biological replicates.

Extended Data Fig. 2 Redox metabolism of wild-type, naive and evolved petites.

a. Wild-type (⍴+) and petite (⍴0) BY4741 or BY4741 Δzwf1 yeast containing empty vector or plasmid-encoded ATP3, ATP3-6 or ATP3-7 were cultured in SC without histidine (SC-H) medium to mid-exponential phase, harvested, and spotted in serial dilutions onto freshly prepared SC-H agar plates ±the oxidant diamide (1.2 mM). Suppressor mutations (ATP3-6, ATP3-7) benefit growth in both BY4741 and BY4741 Δzwf1 background (left panel). Naive petites are slightly more sensitive to diamide compared to evolved petites and wild-type (right panel). In a Δzwf1 background, all strains are hypersensitive to diamide. Shown is one representative experiment n = 3 independent replicates. b.-d. Wild-type (⍴+) and petite (⍴0) YSBN11 strains containing empty vector or plasmid-encoded ATP3, ATP3-6 or ATP3-7 were cultured in SC-H medium to mid-exponential phase and reduced glutathione (GSH) and oxidized glutathione (GSSG) were quantified by LC-MS/MS. In comparison to untreated wild-type controls, treatment with H2O2 (0.5 mM) reduces GSH and increases GSSG levels (positive control). Instead, GSSG and GSH levels are largely unaffected in petites compared to wild-type. Statistics based on two-sided, unpaired t-tests of n = 3 biological replicates comparing wild type to other genotypes. Mean values ± SD. e. Wild-type (⍴+) and petite (⍴0) YSBN11 yeast containing empty vector or plasmid-encoded ATP3-6 were cultured in SM medium, harvested in mid-log phase and re-suspended in medium ± 2 mM tert-BOOH. Superoxide levels were quantified by DHE oxidation after 5 min using fluorescence microscopy. 3 % of wild-type cells, 1 % of naive petite cells and 2 % of evolved petites show DHE fluorescence, while 31 % of wild-type cells challenged with tert-BOOH exhibit fluorescence. Statistics based on two-sided, unpaired t-tests of the following number of biological replicates comparing wild type to other genotypes ⍴+, n = 6 replicates, 13260 cells; ⍴0, n = 11 replicates, 14873 cells; ⍴0 ATP3-6, n = 7 replicates, 17721 cells; ⍴+ test-BOOH, n = 6 replicates, 17721 cells. Mean values ± SEM are shown.

Extended Data Fig. 3 Retrograde response is activated in petites, mitigated in evolved petites, but is not required for suppression of the petite phenotype.

a., b., Wild-type (⍴+) and petite (⍴0) BY4741 strains containing empty vector or plasmid-encoded ATP3-6 and ATP3-7 variants were transformed with a plasmid encoding Pts1-GFP and cultured in SC-HU (histidin, uracil dropout) medium. Cells were harvested in mid-exponential growth phase, fixed with formaldehyde and observed by fluorescence microscopy. Dot-like structures represent peroxisomes (a). Scale bar = 1 μm. Compared to wild-type (10 ± 1) and evolved petites (10 ± 1), naive petites display a significant increase of peroxisomes (17 ± 1) (b). P-values shown based on unpaired, two-sided t-tests, comparing wild type to other genotypes. Mean values ± SEM. Number of cells quantified is indicated by n in the plot. c. Wild-type (⍴+) and petite (⍴0) YSBN11 yeast containing empty vector or plasmid-encoded ATP3-6 were cultured in SM medium. In the mid-exponential growth phase, cells were harvested and subjected to a proteomics workflow. Shown protein fold-changes were normalized to the wild-type. Proteins previously described to be controlled by retrograde response (RTG)1,61,77,78 as well as regulator Rtg2p are significantly upregulated in naive petites (empty plasmid). In comparison, up-regulation in the evolved petite (ATP3-6) is significantly less pronounced. Statistics based on two-sided, unpaired t-tests of n = 3 biological replicates comparing wild type to other genotypes. Mean values ± SD. d. RTG2 or CIT2 are not required for petite suppression. Wild-type (⍴+) BY4741 yeast and strains deleted for RTG2 and CIT2 were transformed with empty plasmid or plasmid encoding for ATP3-6, and mtDNA was depleted. Cells were grown to mid-log growth phase, and equal numbers were spotted in serial dilutions onto SC-H agar. Petites suffer from a growth defect compared to wild-type, which is further amplified in Δrtg2 and Δcit2 strains. Instead, expression of ATP3-6 restores growth irrespective of the genetic background (wild-type, Δrtg2 and Δcit2).

Extended Data Fig. 4 Principal component analysis of proteome changes in response to iron depletion.

Wild-type (⍴+) and petite (⍴0) YSBN11 strains containing empty vector or plasmid-encoded ATP3-6 were cultured in iron-depleted SM medium (- iron) or SM medium containing 200 µg/L iron[III] chloride (+ iron). In the mid-exponential phase, cells were harvested, and proteins were extracted and trypsin-digested. Samples were analyzed by SWATH-MS and protein fold-changes were calculated in comparison to the iron-replete wild-type condition, and data was analyzed using principal component (PC) analysis. In PC1, proteomes were separated according to the petite (slow-growth) phenotype, with evolved petites (expressing ATP3-6) clustering with the wild-type. In PC2, iron-repleted yeast proteomes are separated from iron-depleted proteomes. Combined loadings of all proteins belonging to three GO term groups were plotted (red arrows), showing change of amino acid metabolism along PC1, and up-regulation of iron transport and down-regulation of iron-sulfur proteins (ISPs) along PC2. Data from n = 4 biological replicates per genotype.

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Vowinckel, J., Hartl, J., Marx, H. et al. The metabolic growth limitations of petite cells lacking the mitochondrial genome. Nat Metab 3, 1521–1535 (2021). https://doi.org/10.1038/s42255-021-00477-6

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