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Modeling Metazoan Apoptotic Pathways in Yeast
David T Bloomer1, Tanja Kitevska1, Ingo L Brand1
1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Melbourne, VIC, 3083, Australia.
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.
Insights
Investigate metazoan apoptotic pathways in yeast using effector lethality as a readout. This method identifies apoptotic inhibitors and characterizes caspase activity via reporter gene activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Metazoan apoptosis is crucial for development and disease.
- Characterizing apoptotic pathways requires robust experimental systems.
- Saccharomyces cerevisiae offers a tractable model for studying conserved cellular processes.
Purpose of the Study:
- To describe techniques for characterizing metazoan apoptotic pathways in yeast.
- To establish yeast as a model for identifying apoptotic regulators and inhibitors.
- To develop methods for monitoring caspase activity and substrate specificity.
Main Methods:
- Utilizing the lethality of active caspases, Bax, and Bak in yeast as a readout for effector activity.
- Employing functional yeast-based screens to identify apoptotic inhibitors.
- Monitoring caspase activity through cleavage-dependent reporter gene activation (lacZ).
Main Results:
- Demonstrated that active apoptotic effectors (caspases, Bax, Bak) are lethal to yeast.
- Established a system for investigating proteins and small molecules regulating apoptotic effector activity.
- Developed a reporter system to define caspase cleavage sequences.
Conclusions:
- Saccharomyces cerevisiae serves as an effective model for studying metazoan apoptosis.
- Yeast-based assays enable the discovery of novel apoptotic inhibitors and regulators.
- The described techniques facilitate detailed characterization of caspase activity and substrate specificity.
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