In the budding yeast Saccharomyces cerevisiae, the G1-specific cyclin-dependent kinases (Cdks) Cln1-, Cln2-Cdc28 and Pcl1-, Pcl2-Pho85 are essential for ensuring that DNA replication and cell division are properly linked to cell polarity and bud morphogenesis. However, like most genes in S. cerevisiae, individual cyclin genes are not required for viability, and the phenotypes associated with deletion of any single cyclin gene tend to be subtle. My goal was to dissect the cellular roles of the G1 cyclins by systematically identifying their genetic interactions. To do this, I conducted Synthetic Genetic Array (SGA) screens using strains deleted for different combinations of cyclin genes. The results of screens with strains deleted for the G1 cyclin pairs, CLN1, CLN2, or PCL1, PCL2, confirmed a role for these cyclins in cell polarity regulation and identified novel G1 Cdk substrates, which I examined in more detail.
One cell polarity regulator that showed an interesting pattern of genetic interactions with G1 cyclins was BNI1, which encodes a yeast formin protein. Overexpression of BNI1 caused an Synthetic Dosage Lethal interaction in the absence of both G1 cyclin pairs while its deletion caused synthetic lethality specifically in the absence of PCL1, PCL2. Consistent with these genetic interactions, phosphorylation of Bni1 was partially dependent on CLN1, CLN2. It has been proposed that Bni1 is regulated by intramolecular interactions. In an effort to discover how phosphorylation might affect Bni1 function, I developed assays to test for intramolecular interactions. In my experiments I found no evidence that Bni1 is regulated by intramolecular binding, as was proposed from parallels with its mouse homolog mDia1.
I also found that deletion of BNI4, which encodes an adaptor protein that targets several proteins to the bud neck, results in severe growth defects in the absence of the Cdc28 cyclins Cln1 and Cln2, and overexpression of BNI4 was toxic in yeast cells lacking the Pho85 cyclins Pcl1 and Pcl2. I discovered that Bni4 was phosphorylated by Pcl1- and Pcl2-Pho85 in vitro and that phosphorylation of Bni4 was dependent on PCL1 and PCL2 in vivo. Further analysis showed that phosphorylation of Bni4 by Pcl-Pho85 is necessary for its localization to bud neck, and the bud neck structure can be disrupted by overexpressing BNI4 in pcl1pcl2 mutant cells. I propose that if Bni4 cannot be regulated by phosphorylation, it may titrate away an essential component that resides at the bud neck, thus causing catastrophic morphogenesis defects. The relationship between G1 Cdk activity and the polarity regulator Bni4 serves as a bridge to link the cell cycle machine to the regulation of cell.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/19263 |
| Date | 03 March 2010 |
| Creators | Zou, Jian |
| Contributors | Andrews, Brenda |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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