Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes

  1. Xinyu Zhu
  2. Aren Boulet
  3. Katherine M Buckley
  4. Casey B Phillips
  5. Micah G Gammon
  6. Laura E Oldfather
  7. Stanley A Moore
  8. Scot C Leary
  9. Paul A Cobine  Is a corresponding author
  1. Auburn University, United States
  2. University of Saskatchewan, Canada
  3. University Saskatchewan, Canada

Abstract

The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modelling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated a L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.

Data availability

All data generated or analyzed during this study are included in the manuscript, supplemental file, and available on GenBank.

The following previously published data sets were used
    1. Alasdair C Ivens 1
    2. Christopher S Peacock
    3. Elizabeth A Worthey
    4. Lee Murphy
    5. Gautam Aggarwal
    6. Matthew Berriman
    7. Ellen Sisk
    8. Marie-Adele Rajandream
    9. Ellen Adlem
    10. Rita Aert
    11. Atashi Anupama
    12. Zina Apostolou
    13. Philip Attipoe
    14. Nathalie Bason
    15. Christopher Bauser
    16. Alfred Beck
    17. Stephen M Beverley
    18. Gabriella Bianchettin
    19. Katja Borzym
    20. Gordana Bothe
    21. Carlo V Bruschi
    22. Matt Collins
    23. Eithon Cadag
    24. Laura Ciarloni
    25. Christine Clayton
    26. Richard M R Coulson
    27. Ann Cronin
    28. Angela K Cruz
    29. Robert M Davies
    30. Javier De Gaudenzi
    31. Deborah E Dobson
    32. Andreas Duesterhoeft
    33. Gholam Fazelina
    34. Nigel Fosker
    35. Alberto Carlos Frasch
    36. Audrey Fraser
    37. Monika Fuchs
    38. Claudia Gabel
    39. Arlette Goble
    40. André Goffeau
    41. David Harris
    42. Christiane Hertz-Fowler
    43. Helmut Hilbert
    44. David Horn
    45. Yiting Huang
    46. Sven Klages
    47. Andrew Knights
    48. Michael Kube
    49. Natasha Larke
    50. Lyudmila Litvin
    51. Angela Lord
    52. Tin Louie
    53. Marco Marra
    54. David Masuy
    55. Keith Matthews
    56. Shulamit Michaeli
    57. Jeremy C Mottram
    58. Silke Müller-Auer
    59. Heather Munden
    60. Siri Nelson
    61. Halina Norbertczak
    62. Karen Oliver
    63. Susan O'neil
    64. Martin Pentony
    65. Thomas M Pohl
    66. Claire Price
    67. Bénédicte Purnelle
    68. Michael A Quail
    69. Ester Rabbinowitsch
    70. Richard Reinhardt
    71. Michael Rieger
    72. Joel Rinta
    73. Johan Robben
    74. Laura Robertson
    75. Jeronimo C Ruiz
    76. Simon Rutter
    77. David Saunders
    78. Melanie Schäfer
    79. Jacquie Schein
    80. David C Schwartz
    81. Kathy Seeger
    82. Amber Seyler
    83. Sarah Sharp
    84. Heesun Shin
    85. Dhileep Sivam
    86. Rob Squares
    87. Steve Squares
    88. Valentina Tosato
    89. Christy Vogt
    90. Guido Volckaert
    91. Rolf Wambutt
    92. Tim Warren
    93. Holger Wedler
    94. John Woodward
    95. Shiguo Zhou
    96. Wolfgang Zimmermann
    97. Deborah F Smith
    98. Jenefer M Blackwell
    99. Kenneth D Stuart
    100. Bart Barrell
    101. Peter J Myler
    (2011) ASM272v2
    GCA_000002725.2.

Article and author information

Author details

  1. Xinyu Zhu

    Biological Sciences, Auburn University, Auburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Aren Boulet

    Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
    Competing interests
    The authors declare that no competing interests exist.
  3. Katherine M Buckley

    Biological Sciences, Auburn University, Auburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Casey B Phillips

    Biological Sciences, Auburn University, Auburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Micah G Gammon

    Biological Sciences, Auburn University, Auburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Laura E Oldfather

    Biological Sciences, Auburn University, Auburn, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Stanley A Moore

    Biochemistry, Microbiology and Immunology, University Saskatchewan, Saskatoon, Canada
    Competing interests
    The authors declare that no competing interests exist.
  8. Scot C Leary

    Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
    Competing interests
    The authors declare that no competing interests exist.
  9. Paul A Cobine

    Biological Sciences, Auburn University, Auburn, United States
    For correspondence
    paul.cobine@auburn.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6012-0985

Funding

National Institutes of Health (R01GM120211)

  • Scot C Leary
  • Paul A Cobine

National Science Foundation (EF 2021886)

  • Katherine M Buckley

Alabama Agricultural Experiment Station

  • Paul A Cobine

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Zhu et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,520
    views
  • 368
    downloads
  • 27
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Xinyu Zhu
  2. Aren Boulet
  3. Katherine M Buckley
  4. Casey B Phillips
  5. Micah G Gammon
  6. Laura E Oldfather
  7. Stanley A Moore
  8. Scot C Leary
  9. Paul A Cobine
(2021)
Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes
eLife 10:e64690.
https://doi.org/10.7554/eLife.64690

Share this article

https://doi.org/10.7554/eLife.64690

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Eva Herdering, Tristan Reif-Trauttmansdorff ... Ruth Anne Schmitz
    Research Article

    Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Joar Esteban Pinto Torres, Mathieu Claes ... Yann G-J Sterckx
    Research Article

    African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.