Rapid Fractionation of Mitochondria from Mouse Liver and Heart Reveals In vivo Metabolite Compartmentation

Overview

Journal FEBS Lett

Specialty Biochemistry

Date 2022 Oct 11

PMID 36217875

Authors

Fay M Allen

Ana S H Costa

Anja V Gruszczyk

Georgina R Bates

Hiran A Prag

Efterpi Nikitopoulou

Carlo Viscomi

Christian Frezza

Andrew M James

Michael P Murphy

Affiliations

Soon will be listed here.

Abstract

The compartmentation and distribution of metabolites between mitochondria and the rest of the cell is a key parameter of cell signalling and pathology. Here, we have developed a rapid fractionation procedure that enables us to take mouse heart and liver from in vivo and within ~ 30 s stabilise the distribution of metabolites between mitochondria and the cytosol by rapid cooling, homogenisation and dilution. This is followed by centrifugation of mitochondria through an oil layer to separate mitochondrial and cytosolic fractions for subsequent metabolic analysis. Using this procedure revealed the in vivo compartmentation of mitochondrial metabolites and will enable the assessment of the distribution of metabolites between the cytosol and mitochondria during a range of situations in vivo.

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References

Martin J, Costa A, Gruszczyk A, Beach T, Allen F, Prag H . Succinate accumulation drives ischaemia-reperfusion injury during organ transplantation. Nat Metab. 2020; 1:966-974. PMC: 7212038. DOI: 10.1038/s42255-019-0115-y. View

Murphy M, Chouchani E . Why succinate? Physiological regulation by a mitochondrial coenzyme Q sentinel. Nat Chem Biol. 2022; 18(5):461-469. PMC: 9150600. DOI: 10.1038/s41589-022-01004-8. View

Murphy M, ONeill L . Krebs Cycle Reimagined: The Emerging Roles of Succinate and Itaconate as Signal Transducers. Cell. 2018; 174(4):780-784. DOI: 10.1016/j.cell.2018.07.030. View

Hansen S, Andersen M, Birkedal H, Cornett C, Wibrand F . The important role of taurine in oxidative metabolism. Adv Exp Med Biol. 2006; 583:129-35. DOI: 10.1007/978-0-387-33504-9_13. View

Murphy M, ONeill L . How should we talk about metabolism?. Nat Immunol. 2020; 21(7):713-715. DOI: 10.1038/s41590-020-0691-8. View

Klement P, Nijtmans L, Van den Bogert C, Houstek J . Analysis of oxidative phosphorylation complexes in cultured human fibroblasts and amniocytes by blue-native-electrophoresis using mitoplasts isolated with the help of digitonin. Anal Biochem. 1995; 231(1):218-24. DOI: 10.1006/abio.1995.1523. View

Rogers G, Brand M, Petrosyan S, Ashok D, Elorza A, Ferrick D . High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS One. 2011; 6(7):e21746. PMC: 3143121. DOI: 10.1371/journal.pone.0021746. View

Gremse D, Dean B, Kaplan R . Effect of pyridoxal 5'-phosphate on the function of the purified mitochondrial tricarboxylate transport protein. Arch Biochem Biophys. 1995; 316(1):215-9. DOI: 10.1006/abbi.1995.1030. View

Ruprecht J, Kunji E . Structural Mechanism of Transport of Mitochondrial Carriers. Annu Rev Biochem. 2021; 90():535-558. DOI: 10.1146/annurev-biochem-072820-020508. View

10.

Murphy M . Mitochondrial thiols in antioxidant protection and redox signaling: distinct roles for glutathionylation and other thiol modifications. Antioxid Redox Signal. 2011; 16(6):476-95. DOI: 10.1089/ars.2011.4289. View

11.

Scarlett J, Packer M, Porteous C, Murphy M . Alterations to glutathione and nicotinamide nucleotides during the mitochondrial permeability transition induced by peroxynitrite. Biochem Pharmacol. 1996; 52(7):1047-55. DOI: 10.1016/0006-2952(96)99426-5. View

12.

Ryan D, Murphy M, Frezza C, Prag H, Chouchani E, ONeill L . Coupling Krebs cycle metabolites to signalling in immunity and cancer. Nat Metab. 2019; 1:16-33. PMC: 6485344. DOI: 10.1038/s42255-018-0014-7. View

13.

Ruprecht J, Kunji E . The SLC25 Mitochondrial Carrier Family: Structure and Mechanism. Trends Biochem Sci. 2019; 45(3):244-258. PMC: 7611774. DOI: 10.1016/j.tibs.2019.11.001. View

14.

Coty W, Pedersen P . Phosphate transport in rat liver mitochondria. Membrane components labeled by N-ethylmaleimide during inhibition of transport. J Biol Chem. 1975; 250(9):3515-21. View

15.

Williamson J, Anderson J, Browning E . Inhibition of gluconeogenesis by butylmalonate in perfused rat liver. J Biol Chem. 1970; 245(7):1717-26. View

16.

Williams N, ONeill L . A Role for the Krebs Cycle Intermediate Citrate in Metabolic Reprogramming in Innate Immunity and Inflammation. Front Immunol. 2018; 9:141. PMC: 5807345. DOI: 10.3389/fimmu.2018.00141. View