Regulation of the anaphase promoting complex (APC/C) in the mitotic and meiotic cell cycle of Saccharomyces cerevisiae

Abstract

Ubiquitin-dependent proteolysis is a crucial process in the eukaryotic cell cycle. A key player of this system is a large complex termed the anaphase promoting complex/cyclosome (APC/C). The APC/C is an ubiquitin ligase, which is essential for the progression through mitosis. The APC/C ligates chains of ubiquitin molecules to lysine residues of its substrate proteins and thereby marks them for degradation by the 26S proteasome. The activity of the APC/C is precisely regulated during the cell cycle. The aim of this work was the characterisation of protein kinases involved in APC/C regulation in the mitotic and meiotic cell cycle of the yeast Saccharomyces cerevisiae. In the first part of this work, control of the APC/C by the protein kinase A (PKA) pathway was analysed. Glucose and activated Ras2 protein both induce PKA activity. Experimental data imply that these two signals are able to affect the APC/C synergistically. PKA of S. cerevisiae is encoded by three different genes, TPK1-3. This work revealed that all three genes have redundant functions in APC/C regulation. In the second part of this work, Ime2, a meiosis-specific protein kinase, was characterised. Ime2 was identified in a screen for negative regulators of the APC/C. Further experiments demonstrate, that Ime2 phosphorylates the APC/C regulator Cdh1 and thereby leads to inhibition of the APC/C. Ectopic expression of IME2 causes stabilisation of cyclins and other APC/C substrates. Furthermore IME2 expression in vegetative cells was shown to arrest cells in mitosis and to inhibit bud formation. These results suggest that Ime2 represents a meiosis-specific regulator of the APC/C. Finally, this work focused on the regulation of Ime2 stability. Ime2 protein levels were shown to fluctuate during meiosis. By the construction and analysis of an IME2 deletion set, the N-terminal region was identified as the part of the protein responsible for kinase activity, whereas the C-terminal 241 amino acids were shown to be essential for Ime2 instability. Smaller deletions did not show a distinct effect on stability of the resulting truncated proteins suggesting that two or more degradation signals are located in the C-terminal region. Deletions resulting in proteins with a truncated C-terminus led to a stabilised and active version of the kinase, which causes cells to form abnormal asci. This result indicates that the instability of Ime2 is important for proper progression through meiosis.