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Hem25p is required for mitochondrial IPP transport in fungi

Nature Cell Biology volume 25, pages 1616–1624 (2023)Cite this article

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Abstract

Coenzyme Q (CoQ, ubiquinone) is an essential cellular cofactor composed of a redox-active quinone head group and a long hydrophobic polyisoprene tail. How mitochondria access cytosolic isoprenoids for CoQ biosynthesis is a longstanding mystery. Here, via a combination of genetic screening, metabolic tracing and targeted uptake assays, we reveal that Hem25p—a mitochondrial glycine transporter required for haem biosynthesis—doubles as an isopentenyl pyrophosphate (IPP) transporter in Saccharomyces cerevisiae. Mitochondria lacking Hem25p failed to efficiently incorporate IPP into early CoQ precursors, leading to loss of CoQ and turnover of CoQ biosynthetic proteins. Expression of Hem25p in Escherichia coli enabled robust IPP uptake and incorporation into the CoQ biosynthetic pathway. HEM25 orthologues from diverse fungi, but not from metazoans, were able to rescue hem25∆ CoQ deficiency. Collectively, our work reveals that Hem25p drives the bulk of mitochondrial isoprenoid transport for CoQ biosynthesis in fungi.

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Fig. 1: A targeted genetic screen identifies Hem25p as a potential transporter of CoQ precursors.
Fig. 2: Chemical genomic screens link HEM25 to the mevalonate pathway.
Fig. 3: Hem25p drives CoQ production independently of its role in haem biosynthesis.
Fig. 4: Hem25p enables IPP import in bacteria.
Fig. 5: Hem25p function is required for bacterial IPP import.
Fig. 6: Hem25p’s role in CoQ biosynthesis is conserved in fungi.

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Data availability

Proteomics raw files have been deposited in the MassIVE repository under accession nos. MSV000089127 and MSV000092359. The lipidomics datasets, including raw files and quantified peak areas, have been deposited in the MassIVE repository and can be accessed with accession no. MSV000092744. Data supporting the findings of this study and materials used in this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank members of the Pagliarini laboratory and K. Henzler-Wildman for helpful discussions and insights throughout this project. This work was supported by NIH R35 GM131795 (D.J.P.), P41 GM108538 (J.J.C. and D.J.P.), F31 AG064891 (J.T.), T32 GM140935 (J.T.), T32 DK007120 (S.W.R.) and the BJC Investigator Program (D.J.P.).

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Authors and Affiliations

  1. Department of Biochemistry, University of Wisconsin–Madison, Madison, WI, USA

    Jonathan Tai & David J. Pagliarini

  2. Morgridge Institute for Research, Madison, WI, USA

    Jonathan Tai, Katherine A. Overmyer, Joshua J. Coon & David J. Pagliarini

  3. Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, USA

    Jonathan Tai, Rachel M. Guerra, Sean W. Rogers, Zixiang Fang & David J. Pagliarini

  4. Department of Chemistry, University of Wisconsin–Madison, Madison, WI, USA

    Laura K. Muehlbauer & Joshua J. Coon

  5. National Center for Quantitative Biology of Complex Systems, Madison, WI, USA

    Evgenia Shishkova, Katherine A. Overmyer & Joshua J. Coon

  6. Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, WI, USA

    Evgenia Shishkova, Katherine A. Overmyer & Joshua J. Coon

  7. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA

    David J. Pagliarini

  8. Department of Genetics, Washington University School of Medicine, St Louis, MO, USA

    David J. Pagliarini

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Contributions

Conceptualization was provided by J.T. and D.J.P, methodology by J.T., R.M.G., S.W.R., Z.F., K.A.O. and D.J.P., formal analysis by J.T., R.M.G., S.W.R., Z.F., L.K.M., E.S. and K.A.O., investigations by J.T., R.M.G., S.W.R., Z.F., L.K.M., E.S. and K.A.O, funding acquisition by D.J.P. and supervision by J.J.C. and D.J.P. The original draft was written by J.T. and D.J.P., and review and editing was carried out by J.T., R.M.G., S.W.R., Z.F., L.K.M., E.S., K.A.O., J.J.C. and D.J.P.

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Correspondence to David J. Pagliarini.

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J.J.C. is a consultant for Thermo Fisher Scientific, 908 Devices and Seer. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Hem25p drives CoQ production independently of its role in heme biosynthesis.

a, Relative protein abundances of hem25∆ cells compared to WT cells versus statistical significance. CoQ biosynthetic proteins and Complex Q proteins are highlighted. Raw data from the Y3K dataset33 (respiration-RDR condition) are displayed as the mean from three biologically independent samples with two-sided Student’s t-test used. b, Relative transcript abundances of CoQ biosynthetic genes from hem25∆ with ALA supplementation compared to WT cells without ALA supplementation (mean ± SD, n = 3 biologically independent samples). c, Relative CoQ6 abundances in WT and hem25∆ yeast. Cells were treated with either vehicle (ethanol) or 10 µM CoQ6. CoQ6 supplementation raised the cellular CoQ levels of WT and hem25∆ yeast to similar levels (p = 0.3642 WT+CoQ6 vs hem25∆+CoQ6, mean ± SD, n = 3 biologically independent samples). d, Relative protein abundances of WT cells with CoQ6, hem25∆ cells without CoQ6, and hem25∆ cells with CoQ6 compared to WT cells without CoQ6 (mean, n = 3 biologically independent samples, two-sided Student’s t-test). e, Relative abundance of unlabeled PPHB and CoQ in WT, hem25∆, coq6∆, and coq6∆hem25∆ yeast from the experiment in Fig. 2d (mean ± SD, n = 3 biologically independent samples); ND, not detected. f, Normalized abundance of unlabeled PPHB levels in isolated coq6∆ and coq6∆hem25∆ mitochondria (mean ± SD, n = 3 biologically independent samples). g, Coq1p levels in isolated mitochondria from each replicate, assessed by immunoblotting. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 4-HB import by pcaK and unadjusted IPP uptake kinetics.

a, Immunoblot for MBP-Hem25p, MBP-SLC25A38, and MBP-Yhm2p in E. coli membranes preparations. b, Steady-state kinetics of [1-14C]-IPP uptake in cells expressing MBP-Hem25p or the empty vector. Initial rates (nmol/min) values represent the mean ± SD of three biologically independent samples. c, Time course of 50 µM [phenyl-14C6]-4-HB uptake by E. coli cells expressing the Pseudomonas putida PcaK or the empty vector (*p = 0.0002, **p = 0.0042, ***p = 0.0006, ****p = 0.0011 empty vector vs pcaK, mean ± SD, n = 3 biologically independent samples, two-sided Student’s t-test). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 Hem25p function is required for CoQ biosynthesis.

a, Relative PPHB abundances in hem25∆ yeast carrying WT and mutant HEM25FLAG constructs under the control of the endogenous HEM25 promoter. Levels are relative to WT yeast carrying the empty expression vector (*p = 0.0011, **p = 0.0006, ***p = 0.0116, ****p = 0.0009 HEM25FLAG vs mutants or empty vector, mean ± SD, n = 3 biologically independent samples, two-sided Student’s t-test). b, c, Immunoblots of whole cell lysates (b) or isolated mitochondria (c) from WT, coq6∆, or hem25∆ cells expressing various EV and Hem25p-FLAG constructs under the control of the endogenous HEM25 promoter. d, e, Relative CoQ (d) and PPHB (e) abundances in hem25∆ yeast carrying WT and mutant HEM25FLAG constructs under the control of the constitutive GPD promoter. Levels are relative to WT yeast carrying the empty expression vector (*p = 0.0006, **p = 0.0005, ***p = 0.0009, ****p = 0.0019, #p = 0.0012, ##p = 0.0014, ###p = 0.0012, ####p = 0.0012 HEM25FLAG vs mutants or empty vector, mean ± SD, n = 3 biologically independent samples, two-sided Student’s t-test). f, g, Immunoblots of whole cell lysates (f) or isolated mitochondria (g) from WT, coq6∆, or hem25∆ cells expressing various EV and Hem25p-FLAG constructs under the control of the constitutive GPD promoter. h, Time course of 50 µM [1-14C]-IPP uptake by E. coli cells expressing WT MBP-Hem25p, mutant MBP-Hem25p, or the empty vector (*p = 0.0494, **p = 0.0023, ***p = 0.0136, ****p = 0.0063 empty vector vs WT, mean ± SD, n = 3 biologically independent samples, two-sided Student’s t-test). i, Immunoblot for WT and mutant MBP-Hem25p in E. coli membranes preparations. j, k Normalized abundance of unlabeled OPP (j) and CoQ8 (k) in E. coli cells expressing the EV, WT MBP-Hem25p, or the R181P MBP-Hem25p mutant (mean ± SD, n = 3 biologically independent samples). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 SLC25A38 does not contribute to CoQ biosynthesis.

a, Normalized PPHB abundance in hem25∆ cells expressing Hem25p-FLAG or SLC25A38. (mean ± SD, n = 3 biologically independent samples) b, Relative lipid abundances in hem25∆ yeast compared to WT versus statistical significance with CoQ10 and the CoQ10 biosynthetic intermediate demethoxy-coenzyme Q (DMQ10,) highlighted. c, Relative protein abundance SLC25A38KO cells compared to WT cells versus statistical significance with CoQ-related (COQ3-COQ9) and OxPhos-related proteins highlighted. For panels (b) and (c), raw lipidomic and proteomic data, respectively, from the MITOMICS resource46 are displayed as the mean from three independent samples with two-sided Welch’s t-test used. Source numerical data are available in source data.

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Supplementary information

Supplementary Information

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Reporting Summary

Peer Review File

Supplementary Table 1

Combined file containing all Supplementary Tables. Table 1. Primers for qPCR of COQ genes. Table 2. Plasmid and fusion protein sequences for bacterial uptake assays. Table 3. Primers used in this Study for site-directed mutagenesis. Table 4. Primers used in this Study for amplification of HEM25 or the promoter region.

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Tai, J., Guerra, R.M., Rogers, S.W. et al. Hem25p is required for mitochondrial IPP transport in fungi. Nat Cell Biol 25, 1616–1624 (2023). https://doi.org/10.1038/s41556-023-01250-5

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