טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRainis Liat
SubjectE4orf4 Induced Growth Arrest in Yeast: A Tool for the
Study of Protein Phosphatase 2A (PP2A)
DepartmentDepartment of Medicine
Supervisor Professor Tamar Kleinberger


Abstract

E4orf4 (adenovirus type 5 E4 open reading frame 4) is a 14 kDa protein encoded by adenovirus. This protein induces transformed cell-specific and p53-independent apoptosis. The viral protein interacts with PP2A, one of the major serine/threonine phosphatases in the cell. The interaction with a specific subpopulation of PP2A molecules that contain the B subunit is required for induction of apoptosis.

It was recently demonstrated that E4orf4 induces a PP2A-dependent growth arrest in S. cerevisiae by targeting PP2A to the APC/cyclosome and inhibiting the activity of the APC/cyclosome in mitosis. Similarly to mammalian cells, the E4orf4-induced growth arrest in yeast requires an interaction of the viral protein with CDC55 (the B subunit homolog) and not Rts1 (the B’ subunit homolog). It has also been shown that the yeast system can be utilized to select for non-apoptotic mutants of E4orf4 that do not bind PP2A. These results indicate that a biological selection assay based on E4orf4 toxicity in yeast can yield mutations in the E4orf4-PP2A complex, which affect interactions within the complex.

Our goal is to better understand the structure and function of PP2A and the functional consequences of its interaction with E4orf4. A genetic selection in yeast was employed to identify B subunit domains involved in its interactions with the core enzyme and with E4orf4.

CDC55 was subjected to PCR mutagenesis and transformed into cdc55D yeast expressing E4orf4. Mutants were selected based on their inability to mediate E4orf4-induced toxicity. The screening process, as well as the characterization of the 14 mutants obtained, will be presented and discussed.