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The ARG8m Reporter for the Study of Yeast Mitochondrial Translation

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The Mitoribosome

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2661))

Abstract

Mitochondrial translation is an intricate process involving both general and mRNA-specific factors. In addition, in the yeast Saccharomyces cerevisiae, translation of mitochondrial mRNAs is coupled to assembly of nascent polypeptides into the membrane. ARG8m is a reporter gene widely used to study the mechanisms of yeast mitochondrial translation. This reporter is a recodified gene that uses the mitochondrial genetic code and is inserted at the desired locus in the mitochondrial genome. After deletion of the endogenous nuclear gene, this reporter produces Arg8, an enzyme necessary for arginine biosynthesis. Since Arg8 is a soluble protein with no relation to oxidative phosphorylation, it is a reliable reporter to study mitochondrial mRNAs translation and dissect translation form assembly processes. In this chapter, we explain how to insert the ARG8m reporter in the desired spot in the mitochondrial DNA, how to analyze Arg8 synthesis inside mitochondria, and how to follow steady-state levels of the protein. We also explain how to use it to find spontaneous suppressors of translation defects.

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Acknowledgements

This work was supported by research grants from Consejo Nacional de Ciencia y Tecnología (CONACyT) [47514 to XP-M] and fellowship [777444 to D. F-M]; and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, (PAPIIT), UNAM [IN202720 and IN223623 to XP-M].

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Authors and Affiliations

  1. Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

    Daniel Flores-Mireles, Yolanda Camacho-Villasana & Xochitl Pérez-Martínez

Authors
  1. Daniel Flores-Mireles
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  2. Yolanda Camacho-Villasana
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  3. Xochitl Pérez-Martínez
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Corresponding author

Correspondence to Xochitl Pérez-Martínez .

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Editors and Affiliations

  1. Miller School of Medicine, University of Miami, Miami, FL, USA

    Antoni Barrientos

  2. Miller School of Medicine, University of Miami, Miami, FL, USA

    Flavia Fontanesi

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Flores-Mireles, D., Camacho-Villasana, Y., Pérez-Martínez, X. (2023). The ARG8m Reporter for the Study of Yeast Mitochondrial Translation. 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_16

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  • DOI: https://doi.org/10.1007/978-1-0716-3171-3_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3170-6

  • Online ISBN: 978-1-0716-3171-3

  • eBook Packages: Springer Protocols

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