טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMittelman Karin
SubjectMapping of Ynd1 Domains Required for Adenovirus E4orf4-
Induced Toxicity in Yeast
DepartmentDepartment of Medicine
Supervisor Professor Tamar Kleinberger
Full Thesis textFull thesis text - English Version


Abstract

The Adenovirus protein E4orf4 is involved in various cellular processes, including induction of non-classical, p53-independent apoptosis.  E4orf4 induces apoptosis in a more efficient manner in mammalian cells that have undergone oncogenic transformation in comparison with normal cells.

PP2A is a major serine/threonine phosphatase in the cell, composed of three subunits: a scaffolding A subunit, a catalytic C subunit, and a regulatory B subunit.  The B subunit has many isoforms encoded by four unrelated gene families: B/B55, B’/B56, B’’ and B’’’.  The interaction of E4orf4 with PP2A complexes containing the B55 subunit is required for induction of apoptosis.

E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae, and this growth arrest is PP2A-dependent.  As in mammalian cells, CDC55, the yeast homolog of the B55 subunit, is essential for mediating E4orf4-induced growth arrest.  Therefore, S. cerevisiae may serve as a fine model system for the study of the E4orf4-induced signal transduction pathway.

A genomic library screen in S. cerevisiae revealed that deletion of the gene YND1 confers partial resistance to E4orf4-induced toxicity.  Ynd1 belongs to the apyrase family and regulates protein and lipid glycosylation.  Ynd1 is localized to the Golgi apparatus.  It has a long N-terminus that is located in the Golgi lumen, one transmembrane domain and a short cytosolic tail.

Experiments in S. cerevisiae have revealed a genetic interaction between YND1 and CDC55.  These two genes have an additive effect on the growth arrest induced by E4orf4.  Ynd1 and Cdc55 physically interact, and the association between them is disrupted by E4orf4, which binds both of them.

In this work, we attempted to further characterize the role of Ynd1 in the signal transduction pathway induced by E4orf4 in yeast.  We mapped the domains of Ynd1 required for mediation of E4orf4-induced toxicity using several mutagenesis methods.  We generated a series of mutants, all of which were examined by several assays to determine their ability to mediate E4orf4-induced toxicity, their ability to bind E4orf4 and Cdc55, and their subcellular localization.  Our results suggest that the localization of Ynd1 to the Golgi membrane may be essential for mediating E4orf4-induced toxicity.  Furthermore, we show that 14 amino acids located in the cytosolic tail of Ynd1 may comprise the binding site of both E4orf4 and Cdc55 on Ynd1.  These results could explain the disruption of the association of Ynd1 and Cdc55 by E4orf4.