Abstract
This chapter describes techniques for characterizing metazoan apoptotic pathways using Saccharomyces cerevisiae. Active forms of the major apoptotic effectors—caspases, Bax and Bak—are all lethal to yeast. Using this lethality as a readout of caspase/Bax/Bak activity, proteins and small molecules that directly or indirectly regulate the activity of these effectors can be investigated in yeast, and apoptotic inhibitors can be identified using functional yeast-based screens. Caspase activity can also be monitored in yeast by cleavage-dependent liberation of a transcription factor from the plasma membrane, enabling it to activate the lacZ reporter gene. This system can be used to define the sequences that can be efficiently cleaved by particular caspases.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Madeo F, Herker E, Wissing S, Jungwirth H, Eisenberg T, Frohlich KU (2004) Apoptosis in yeast. Curr Opin Microbiol 7(6):655–660
Puryer MA, Hawkins CJ (2006) Human, insect and nematode caspases kill Saccharomyces cerevisiae independently of YCA1 and Aif1p. Apoptosis 11(4):509–517
Hawkins CJ, Wang SL, Hay BA (1999) A cloning method to identify caspases and their regulators in yeast: identification of Drosophila IAP1 as an inhibitor of the Drosophila caspase DCP-1. Proc Natl Acad Sci 96(6):2885–2890
Hawkins CJ, Silke J, Verhagen AM, Foster R, Ekert PG, Ashley DM (2001) Analysis of candidate antagonists of IAP-mediated caspase inhibition using yeast reconstituted with the mammalian Apaf-1-activated apoptosis mechanism. Apoptosis 6(5):331–338
Sikorski R, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27
Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110(1):119–122
Karim AS, Curran KA, Alper HS (2013) Characterization of plasmid burden and copy number in Saccharomyces cerevisiae for optimization of metabolic engineering applications. FEMS Yeast Res 13(1):107–116, 110/1111/1567-1364.12016. Epub 12012 Nov 12020
Srinivasula S, Ahmad M, MacFarlane M, Luo Z, Huang Z, Fernandes-Alnemri T, Alnemri E (1998) Generation of constitutively active recombinant caspases-3 and -6 by rearrangement of their subunits. J Biol Chem 273(17):10107–10111
Ho PK, Jabbour AM, Ekert PG, Hawkins CJ (2005) Caspase-2 is resistant to inhibition by inhibitor of apoptosis proteins (IAPs) and can activate caspase-7. FEBS J 272(6):1401–1414
Wang SL, Hawkins CJ, Yoo SJ, Muller HA, Hay BA (1999) The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by REAPER, HID and GRIM, which disrupt DIAP1-caspase interactions. Cell 98(4):453–463
Thornberry N, Rano T, Peterson E, Rasper D, Timkey T, Garcia-Calvo M, Houtzager V, Nordstrom P, Roy S, Vaillancourt J, Chapman K, Nicholson D (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem 272(29):17907–17911
Srinivasula S, Ahmad M, Fernandes-Alnemri T, Alnemri E (1998) Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell 1(7):949–957
Kluck RM, Martin SJ, Hoffman BM, Zhou JS, Green DR, Newmeyer DD (1997) Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. EMBO J 16(15):4639–4649
Brand IL, Civciristov S, Taylor NL, Talbo GH, Pantaki-Eimany D, Levina V, Clem RJ, Perugini MA, Kvansakul M, Hawkins CJ (2012) Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria. PLoS One 7(6):39248. doi:10.1371/journal.pone.0039248
Brand IL, Green MM, Civciristov S, Pantaki-Eimany D, George C, Gort TR, Huang N, Clem RJ, Hawkins CJ (2011) Functional and biochemical characterization of the baculovirus caspase inhibitor MaviP35. Cell Death Dis 2, e242
Jabbour AM, Ekert PG, Coulson EJ, Knight MJ, Ashley DM, Hawkins CJ (2002) The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis. Cell Death Differ 9(12):1311–1320
Beaumont TE, Shekhar TM, Kaur L, Pantaki-Eimany D, Kvansakul M, Hawkins CJ (2013) Yeast techniques for modeling drugs targeting Bcl-2 and caspase family members. Cell Death Dis 4:e619, 10.1038/cddis.2013.143
Khoury CM, Greenwood MT (2008) The pleiotropic effects of heterologous Bax expression in yeast. Biochim Biophys Acta 1783(7):1449–1465
Walensky LD, Gavathiotis E (2011) BAX unleashed: the biochemical transformation of an inactive cytosolic monomer into a toxic mitochondrial pore. Trends Biochem Sci 36(12):642–652. doi:10.1016/j.tibs.2011.08.009
Kitevska T, Roberts SJ, Pantaki-Eimany D, Boyd SE, Scott FL, Hawkins CJ (2014) Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate. Biosci Rep 17:17
Westein SJ, Scott FL, Hawkins CJ (2008) Analysis of the minimal specificity of CED-3 using a yeast transcriptional reporter system. Biochim Biophys Acta 1783(3):448–454
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153(1):163–168
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Bloomer, D.T., Kitevska, T., Brand, I.L., Jabbour, A.M., Nguyen, H., Hawkins, C.J. (2016). Modeling Metazoan Apoptotic Pathways in Yeast. In: Puthalakath, H., Hawkins, C. (eds) Programmed Cell Death. Methods in Molecular Biology, vol 1419. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3581-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3581-9_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3579-6
Online ISBN: 978-1-4939-3581-9
eBook Packages: Springer Protocols