|M.Sc Student||Ziv Keren|
|Subject||Study of the Mechanisms Underlying Induction of Apoptosis|
by the Adenovirus E4orf4 Protein in a
S.Cerevisiae Model System
|Department||Department of Medicine||Supervisor||Professor Tamar Kleinberger|
|Full Thesis text - in Hebrew|
E4orf4 is an adenoviral protein involved in various cellular processes, including induction of cell death. Cell death induced by E4orf4 is a non-classical p53-independent type of apoptosis since it is caspase-independent in many cell lines. E4orf4 induces cell death in a more efficient manner in mammalian cells that have undergone oncogenic transformation in comparison to normal cells, suggesting it has a therapeutic potential. To find a system that would facilitate a genetic analysis of the E4orf4 apoptotic pathway, we performed experiments in the budding yeast S. cerevisiae. E4orf4 induces an irreversible growth arrest in WT budding yeast, which shares an extensive homology with E4orf4-induced apoptosis in mammalian cells. In a genomic library screen in S. cerevisiae, it was discovered that deletion of the gene YND1 confers partial resistance to E4orf4-induced growth arrest. Ynd1 is localized in the Golgi membrane and plays a role in regulation of protein and lipid glycosylation.
In the first part of this work, we mapped the domains of Ynd1 required for mediation of E4orf4-induced toxicity. A series of mutants was generated, lacking several domains of the Ynd1 protein. Quantitative assays of the ability of WT Ynd1 and Ynd1 mutants to mediate the E4orf4 toxic signal suggested that the cytosolic tail of Ynd1 transduced the E4orf4 toxic signal at least as well as WT Ynd1, if not better. Immunoprecipitation assays and a Proteinase K digestion protection assay performed on E4orf4 indicated that the majority of E4orf4 protein localized to the cytosolic face of the membrane, and that the interaction between E4orf4 and Ynd1 occurs in the cytosol. In the second part of this work, we attempted to examine how the lack of proteins physically associated with Ynd1 affects the E4orf4 toxic signal. These proteins include polypeptides involved in ER to Golgi vesicle-mediated transport (Erv29 and Yip3), vesicle organization (Sys1) or ER retention of cargo proteins (Erd1). Pmp2 regulates the activity of plasma membrane H()-ATPase. We found that some of these proteins were required for E4orf4-induced toxicity, whereas others were not. Future work will determine the nature of the contribution of these proteins to E4orf4-induced toxicity.