Abstract
Redox cycles have been reported in ultradian, circadian and cell cycle-synchronized systems. Redox cycles persist in the absence of transcription and cyclin-CDK activity, indicating that cells harbor multiple coupled oscillators. Nonetheless, the causal relationships and molecular mechanisms by which redox cycles are embedded within ultradian, circadian or cell division cycles remain largely elusive. Yeast harbor an ultradian oscillator, the yeast metabolic cycle (YMC), which comprises metabolic/redox cycles, transcriptional cycles and synchronized cell division. Here, we reveal the existence of robust cycling of H2O2 and peroxiredoxin oxidation during the YMC and show that peroxiredoxin inactivation disrupts metabolic cycling and abolishes coupling with cell division. We find that thiol-disulfide oxidants and reductants predictably modulate the switching between different YMC metabolic states, which in turn predictably perturbs cell cycle entry and exit. We propose that oscillatory H2O2-dependent protein thiol oxidation is a key regulator of metabolic cycling and its coordination with cell division.
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All datasets generated or analyzed during this study are included in this Article and its Supplementary Information. Source data are provided with this paper.
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Acknowledgements
B.M. acknowledges generous financial support from the Deutsche Forschungsgemeinschaft in the framework of the SPP1710 (MO 2774/2-1) and IRTG1830 programmes, as well as funding from the Technische Universität Kaiserslautern Nachwuchsring and the Forschungsinitiative Rhineland-Pfalz BioComp. G.Y.M. is funded by the Georg Forster Research Fellowship, awarded from the Alexander von Humboldt Foundation. We thank W. Zachariae (Max Planck Institute of Biochemistry) for providing the Clb2 antibody and B. Luke (Institute of Molecular Biology) for providing the Sic1 antibody. We thank T. Dick, J. Herrmann, J. Riemer, L. Prates Roma and F. Hannemann for invaluable discussions and for helpful and insightful comments on the manuscript. We thank V. Nehr for valuable technical assistance.
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B.M., P.S.A. and Z.S. designed all experiments and wrote the manuscript. P.S.A., J.Z., M.M. and S.M. performed metabolic cycle and online roGFP2-Tsa2ΔCR-based measurements, as well as experiments to assess the impact of redox compounds on oxygen and roGFP2-Tsa2ΔCR cycling. They also performed the experiments to assess the impact of genetic and chemical perturbation of the YMC on cell division using flow cytometry-based analysis of DNA content. P.S.A. performed tetrad dissection and the experiments associated with auxin degron-based regulation of peroxiredoxin level. G.Y. performed all budding index experiments and western blot analyses of cell cycle markers. P.S.A., T.M. and B.M. performed the correlation analyses and statistical analyses of all datasets. All authors contributed to data interpretation.
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Extended data
Extended Data Fig. 1 Oxygen consumption and cytosolic H2O2 levels oscillate in phase.
a–c, The media oxygen saturation and roGFP2-Tsa2ΔCR oxidation was monitored for three complete cycles in three independent YMC-synchronized cultures of wild-type cells (datasets presented in Fig. 1c and Supplementary Fig. 2b). Autocorrelation analysis of the oxygen and roGFP2-Tsa2ΔCR oxidation revealed robust and in-phase periodicity of the two signals. d, Plot showing time points of the oxygen saturation and roGFP2-Tsa2ΔCR oxidation minima and maxima. Data were fitted by linear regression and significance, as tested by a two-sided ANOVA, revealed p ≈ 0 (4.9 × 10−324).
Extended Data Fig. 2 LOC to HOC switching correlates with probe reduction after diamide treatment.
a–c, RoGFP2-Tsa2ΔCR probe oxidation following the addition of 2 mM diamide towards the end of LOC phase in three independent YMC-synchronized cultures.
Extended Data Fig. 3 Peroxiredoxin deletion perturbs the YMC.
a–d, Oxygen traces to show YMC cycling in either wild-type cells or cells deleted for the genes encoding the indicated peroxiredoxins. Each trace is derived from a completely independent YMC-synchronized culture. e, Cartoon illustrating the subcellular localization of the peroxiredoxins presented in a–d. f, Graph showing the average YMC periods determined from the datasets presented in a–d. n = 3 independent YMC-synchronized cultures. Error bars, mean ± s.d. P values are derived from an unpaired two-tailed Student’s t-test.
Extended Data Fig. 4 Combined deletion of AHP1 and TSA1 is lethal in CEN.PK yeast.
a, Scheme illustrating the mating, sporulation and tetrad dissection procedure. b, Images of tetrad dissection plates for all 33 tetrads dissected. c, Images showing growth of cells from all recovered viable spores on media containing the indicated antibiotics to assess for the presence of the antibiotic resistance cassettes used for gene deletion. d, Table showing the eight possible genotypes and the number of spores recovered with each genotype.
Extended Data Fig. 5 A prolonged switch to HOC phase leads to accumulation of cells with two buds.
a, Representative microscopy images of DAPI stained cells with 2 buds, isolated from YMC-synchronized cultures ~10 hours after addition of 5 mM DTT, the scale bar represents 2 µM. n = 3 independent experimental repeats.
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Amponsah, P.S., Yahya, G., Zimmermann, J. et al. Peroxiredoxins couple metabolism and cell division in an ultradian cycle. Nat Chem Biol 17, 477–484 (2021). https://doi.org/10.1038/s41589-020-00728-9
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DOI: https://doi.org/10.1038/s41589-020-00728-9