Abstract
Due to its unique biology the mitochondrion of Trypanosoma brucei has attracted a lot of interest since many decades, making it arguably the best studied mitochondrion outside yeast and mammals. Here we describe a method allowing purification of mitochondria from procyclic trypanosomes that yields highly enriched and functional organelles. The method is based on isotonic lysis of cells by nitrogen cavitation, DNase I digestion, differential centrifugation and Nycodenz gradient centrifugation. The method is scalable and can be adapted to culture volumes a small as 100 mL or as large as 24 L.
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References
Friedman JR, Nunnari J (2014) Mitochondrial form and function. Nature 505:335–543
Burki F (2014) The eukaryotic tree of life from a global phylogenomic perspective. Cold Spring Harb Perspect Biol 6(5):a016147
Murcha MW, Wang Y, Narsai R, Whelan J (2013) The plant mitochondrial protein import apparatus – the differences make it interesting. Biochim Biophys Acta 1840:1233–1245
Schneider A (2001) Unique aspects of mitochondrial biogenesis in trypanosomatids. Int J Parasitol 31:1403–1415
Verner Z, Basu S, Benz C, Dixit S, Dobakova E, Faktorova D et al (2015) Malleable mitochondrion of Trypanosoma brucei. Int Rev Cell Mol Biol 315:73–151
Schneider A, Ochsenreiter T (2018) Failure is not an option – mitochondrial genome segregation in trypanosomes. J Cell Sci 131(18):pii: jcs221820
Siegel TN, Gunasekera K, Cross GA, Ochsenreiter T (2011) Gene expression in Trypanosoma brucei: lessons from high-throughput RNA sequencing. Trends Parasitol 27:434–441
Read LK, Lukes J, Hashimi H (2016) Trypanosome RNA editing: the complexity of getting U in and taking U out. Wiley Interdiscip Rev RNA 7:33–51
Schneider A (2011) Mitochondrial tRNA import and its consequences for mitochondrial translation. Annu Rev Biochem 80:1033–1053
Alfonzo JD, Söll D (2009) Mitochondrial tRNA import – the challenge to understand has just begun. Biol Chem 390:717–722
Lukes J, Basu S (2015) Fe/S protein biogenesis in trypanosomes – a review. Biochim Biophys Acta 1853:1481–1492
Schnaufer A, Domingo GJ, Stuart K (2002) Natural and induced dyskinetoplastic trypanosomatids: how to live without mitochondrial DNA. Int J Parasitol 32:1071–1084
Dean S, Gould MK, Dewar CE, Schnaufer AC (2013) Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes. Proc Natl Acad Sci U S A 110:14741–14746
Harsman A, Schneider A (2017) Mitochondrial protein import in trypanosomes - Expect the unexpected. Traffic 18:96–109
Kaurov I, Vancova M, Schimanski B, Cadena LR, Heller J, Bily T et al (2018) The diverged trypanosome MICOS complex as a hub for mitochondrial cristae shaping and protein import. Curr Biol 28:3393–3407. e5
Ramrath DJF, Niemann M, Leibundgut M, Bieri P, Prange C, Horn EK et al (2018) Evolutionary shift toward protein-based architecture in trypanosomal mitochondrial ribosomes. Science 362(6413):eaau7735
Schneider A, Charrière F, Pusnik M, Horn EK (2007) Isolation of mitochondria from procyclic Trypanosoma brucei. Methods Mol Biol 372:67–80
Brun R, Schönenberger M (1979) Cultivation an in vitro cloning of procyclic culture forms of Trypansoma brucei in a semi-defined medium. Acta Trop 36:289–292
Peikert CD, Mani J, Morgenstern M, Käser S, Knapp B, Wenger C et al (2017) Charting organellar importomes by quantitative mass spectrometry. Nat Commun 8:15272
Bochud-Allemann N, Schneider A (2002) Mitochondrial substrate level phosphorylation is essential for growth of procyclic Trypanosoma brucei. J Biol Chem 277:32849–32854
Allemann N, Schneider A (2000) ATP production in isolated mitochondria of procyclic Trypanosoma brucei. Mol Biochem Parasitol 111:87–94
Schneider A, Bouzaidi-Tiali N, Chanez A-L, Bulliard L (2007) ATP production in isolated mitochondria of procyclic Trypanosoma brucei. Methods Mol Biol 372:379–387
Hauser R, Pypaert M, Häusler T, Horn EK, Schneider A (1996) In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae. J Cell Sci 109:517–523
Mani J, Desy S, Niemann M, Chanfon A, Oeljeklaus S, Pusnik M et al (2015) Mitochondrial protein import receptors in kinetoplastids reveal convergent evolution over large phylogenetic distances. Nat Commun 6:6646
Pusnik M, Schmidt O, Perry AJ, Oeljeklaus S, Niemann M, Warscheid B et al (2015) Mitochondrial preprotein translocase of trypanosomatids has a bacterial origin. Curr Biol 21:1738–1743
Niemann M, Wiese S, Mani J, Chanfon A, Jackson C, Meisinger C et al (2013) Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology. Mol Cell Proteomics 12:515–528
Farine L, Niemann M, Schneider A, Butikofer P (2015) Phosphatidylethanolamine and phosphatidylcholine biosynthesis by the Kennedy pathway occurs at different sites in Trypanosoma brucei. Sci Rep 5:16787
Chomczyinski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
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Niemann, M., Schneider, A. (2020). A Scalable Purification Method for Mitochondria from Trypanosoma brucei. In: Michels, P., Ginger, M., Zilberstein, D. (eds) Trypanosomatids. Methods in Molecular Biology, vol 2116. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0294-2_36
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DOI: https://doi.org/10.1007/978-1-0716-0294-2_36
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