Abstract
Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.
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References
Kehrein K, Schilling R, Möller-Hergt BV et al (2015) Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies. Cell Rep 10:843–853. https://doi.org/10.1016/J.CELREP.2015.01.012
Singh AP, Salvatori R, Aftab W et al (2020) Molecular connectivity of mitochondrial gene expression and OXPHOS biogenesis. Mol Cell 79:1051–1065.e10. https://doi.org/10.1016/j.molcel.2020.07.024
Desai N, Brown A, Amunts A, Ramakrishnan V (2017) The structure of the yeast mitochondrial ribosome. Science (80-) 355:528–531. https://doi.org/10.1126/science.aal2415
Gruschke S, Gröne K, Heublein M et al (2010) Proteins at the polypeptide tunnel exit of the yeast mitochondrial ribosome. J Biol Chem 285:19022–19028. https://doi.org/10.1074/jbc.M110.113837
Salvatori R, Kehrein K, Singh AP et al (2020) Molecular wiring of a mitochondrial translational feedback loop. Mol Cell 77:887–900.e5. https://doi.org/10.1016/J.MOLCEL.2019.11.019
Zeng R, Smith E, Barrientos A (2018) Yeast mitoribosome large subunit assembly proceeds by hierarchical incorporation of protein clusters and modules on the inner membrane. Cell Metab 27:645–656.e7. https://doi.org/10.1016/j.cmet.2018.01.012
Couvillion MT, Soto IC, Shipkovenska G, Churchman LS (2016) Synchronized mitochondrial and cytosolic translation programs. Nature 533:499–503. https://doi.org/10.1038/nature18015
Price CA (1982) Particle abstraction in biology—an introduction. In: Centrifugation in density gradients. Academic Press, pp 1–11
Meisinger C, Pfanner N, Truscott KN (2006) Isolation of yeast mitochondria. Methods Mol Biol 313:33–39. https://doi.org/10.1385/1-59259-958-3:033
Gruschke S, Römpler K, Hildenbeutel M et al (2012) The Cbp3–Cbp6 complex coordinates cytochrome b synthesis with bc1 complex assembly in yeast mitochondria. J Cell Biol 199:137. https://doi.org/10.1083/JCB.201206040
Gruschke S, Kehrein K, Römpler K et al (2011) Cbp3-Cbp6 interacts with the yeast mitochondrial ribosomal tunnel exit and promotes cytochrome b synthesis and assembly. J Cell Biol 193:1101–1114. https://doi.org/10.1083/jcb.201103132
Acknowledgment
This work was supported by the Swedish Research Council and the Knut and Alice Wallenberg foundation (to Martin Ott) and the Austrian Science Fund FWF (J4398-B to Andreas Aufschnaiter).
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Aufschnaiter, A., Carlström, A., Ott, M. (2023). Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation. In: Barrientos, A., Fontanesi, F. (eds) The Mitoribosome. Methods in Molecular Biology, vol 2661. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3171-3_8
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DOI: https://doi.org/10.1007/978-1-0716-3171-3_8
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