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.
Similar content being viewed by others
References
Deretic V, Levine B (2009) Autophagy, immunity, and microbial adaptations. Cell Host Microbe 5:527–549
Klionsky DJ, Codogno P (2013) The mechanism and physiological function of macroautophagy. J Innate Immun 5:427–433
Xie Z, Klionsky DJ (2007) Autophagosome formation: core machinery and adaptations. Nat Cell Biol 9:1102–1109
Johansen T, Lamark T (2011) Selective autophagy mediated by autophagic adapter proteins. Autophagy 7:279–296
Jin M, Liu X, Klionsky DJ (2013) SnapShot: selective autophagy. Cell 152:368–368.e2
Kanki T, Wang K, Cao Y, Baba M, Klionsky DJ (2009) Atg32 is a mitochondrial protein that confers selectivity during mitophagy. Dev Cell 17:98–109
Okamoto K, Kondo-Okamoto N, Ohsumi Y (2009) Mitochondria-anchored receptor Atg32 mediates degradation of mitochondria via selective autophagy. Dev Cell 17:87–97
Motley AM, Nuttall JM, Hettema EH (2012) Pex3-anchored Atg36 tags peroxisomes for degradation in Saccharomyces cerevisiae. EMBO J 31:2852–2868
Scott SV, Guan J, Hutchins MU, Kim J, Klionsky DJ (2001) Cvt19 is a receptor for the cytoplasm-to-vacuole targeting pathway. Mol Cell 7:1131–1141
Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39:359–407
Sato M, Sato K (2011) Degradation of paternal mitochondria by fertilization-triggered autophagy in C. elegans embryos. Science 334:1141–1144
Sandoval H, Thiagarajan P, Dasgupta SK, Schumacher A, Prchal JT, Chen M, Wang J (2008) Essential role for Nix in autophagic maturation of erythroid cells. Nature 454:232–235
Youle RJ, Narendra DP (2011) Mechanisms of mitophagy. Nat Rev Mol Cell Biol 12:9–14
Ashrafi G, Schwarz TL (2013) The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ 20:31–42
Klionsky DJ, Emr SD (1989) Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO J 8:2241–2250
Kanki T, Wang K, Klionsky DJ (2010) A genomic screen for yeast mutants defective in mitophagy. Autophagy 6:278–280
Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH (2002) A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res 30:e23
Kanki T, Wang K, Baba M, Bartholomew CR, Lynch-Day MA, Du Z, Geng J, Mao K, Yang Z, Yen WL, Klionsky DJ (2009) A genomic screen for yeast mutants defective in selective mitochondria autophagy. Mol Biol Cell 20:4730–4738
Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96
Cheong H, Klionsky DJ (2008) Biochemical methods to monitor autophagy-related processes in yeast. Methods Enzymol 451:1–26
Mao K, Wang K, Liu X, Klionsky DJ (2013) The scaffold protein Atg11 recruits fission machinery to drive selective mitochondria degradation by autophagy. Dev Cell 26:9–18
Acknowledgments
This work was supported by NIH grant GM053396 to D.J.K. and a Rackham Predoctoral Fellowship to X.L.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/7651_2017_12
Published:
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