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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.
Methods in Molecular Biology (Clifton, N.J.)
|April 25, 2016
Summary
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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