In vitro systems to study the relationship between apoptosis in multicellular organisms and yeast : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Turitea, Palmerston North
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Date
2000
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Massey University
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Apoptosis is a distinct form of cell death that is characterised by specific morphological and biochemical markers, such as chromatin condensation and internucleosomal DNA cleavage. This type of cell death is evolutionarily conserved in higher eukaryotes. Homologues of the main apoptosis regulators, such as the Bcl-2 family of proteins and caspases, have been found in multicellular organisms. However, homologues of these proteins have not been found in the unicellular organism Saccharomyces cerevisiae, although in certain circumstances S. cerevisiae will exhibit features of apoptosis. In this project, we developed in vitro systems to explore the relationship between mammalian apoptosis and any similar mechanism that may be present in yeast. Components derived from yeast and mammalian cells were incubated together in vitro and assessed for the activation of apoptosis. Rat cytochrome c activates apoptosis in mammalian cell-free extracts (human neuroblastoma SY5Y cells). Internucleosomal DNA cleavage was observed in S. cerevisiae spheroplasts when they were incubated in mammalian cell-free extracts activated by rat cytochrome c. Although yeast cytochrome c is similar to rat cytochrome c, it failed to induce apoptosis in mammalian cell-free extracts. Yeast cytosol caused internucleosomal DNA cleavage in PCl2 nuclei. This cleavage was enhanced by rat cytochrome c and was mostly inhibited by the caspase inhibitor DEVD-CHO, but only in the presence of rat cytochrome c. Yeast cytosol did not cause chromatin condensation in PCl2 nuclei or cleavage of Parp (a downstream caspase substrate). Yeast cytosol was therefore unable to induce apoptosis in PCl2 nuclei. Mitochondria play a central role in most forms of mammalian apoptosis. We developed a cell-free system in which we could examine the role of mitochondria in apoptosis. We attempted to activate apoptosis in SY5Y cytosol by the addition of mitochondria subjected to rupture-inducing treatment, with limited success. However, we found that mitochondria purified from healthy SY5Y cells protected PCl2 nuclei from undergoing apoptosis in vitro.
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Apoptosis