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MitoPho8Δ60 Assay as a Tool to Quantitatively Measure Mitophagy Activity

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Mitophagy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1759))

Abstract

Mitophagy, a selective type of macroautophagy (hereafter referred to as autophagy), specifically mediates the vacuole/lysosome-dependent degradation of damaged or surplus mitochondria. Because this process regulates the number and quality of mitochondria, it is vital for proper cellular homeostasis. Mitophagy also plays critical roles in the clearance of paternal mitochondria in C. elegans embryos, in erythroid cell maturation, and in the prevention of neurodegenerative disease and cancer. In order to study the molecular mechanism and regulation of mitophagy, sensitive assays are necessary to quantitatively measure mitophagy activity. In the budding yeast, Saccharomyces cerevisiae, a “mitoPho8Δ60” assay was developed to study mitophagy. In this assay, Pho8, a vacuolar phosphatase protein, is genetically engineered to be targeted to mitochondria. When mitophagy is induced, the phosphatase protein, along with mitochondria, is conveyed to the vacuole, where its C-terminal propeptide is removed and the phosphatase activity becomes activated; under growing conditions only a background level of delivery occurs. For this reason, the enzymatic activity of mitoPho8Δ60 is correlated with the amount of mitochondria delivered to the vacuole. Thus, this assay serves as a very convenient tool to quantitatively monitor mitophagy activity in yeast.

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Acknowledgments

This work was supported by NIH grant GM053396 to D.J.K. and a Rackham Predoctoral Fellowship to X.L.

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Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Life Sciences Institute, University of Michigan, Room 6036, 210 Washtenaw Avenue, Ann Arbor, MI, 48109-2216, USA

    Zhiyuan Yao, Xu Liu & Daniel J. Klionsky

Authors
  1. Zhiyuan Yao
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  2. Xu Liu
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  3. Daniel J. Klionsky
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Corresponding author

Correspondence to Daniel J. Klionsky .

Editor information

Editors and Affiliations

  1. Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan

    Nobutaka Hattori

  2. Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan

    Shinji Saiki

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© 2017 Springer Science+Business Media New York

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Yao, Z., Liu, X., Klionsky, D.J. (2017). MitoPho8Δ60 Assay as a Tool to Quantitatively Measure Mitophagy Activity. In: Hattori, N., Saiki, S. (eds) Mitophagy. Methods in Molecular Biology, vol 1759. Humana Press, New York, NY. https://doi.org/10.1007/7651_2017_12

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  • DOI: https://doi.org/10.1007/7651_2017_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7749-9

  • Online ISBN: 978-1-4939-7750-5

  • eBook Packages: Springer Protocols

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