Wells, Glenn Paul; (2002) Swi6 protein interactions and the regulation of the yeast cell cycle. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In budding yeast, the cell cycle is initiated at a point in G1 called Start, which marks the irrevocable commitment to a round of mitotic cell division. At Start there is an increase in the expression of over 200 cell cycle-regulated genes depending in part, on the Start-specific transcription factor, Swi6p. The aim of this work was to identify factors that interact with Swi6p and thereby regulate its activity. The search for novel Swi6p protein-protein interactions was performed using the new and relatively untested Sos Recruitment System (SRS). This allowed not only the investigation of Swi6p interactions, but also the assessment of the effectiveness of the SRS system in yeast. The results of the screening ultimately proved inconclusive, as putative interactions could not be verified further by alternative m vivo or in vitro systems. The type of proteins identified from the screening, suggested a bias in the SRS system towards detecting conditional interactions. Nevertheless, during the investigation of the results from the SRS screen, a novel Swi6p interaction was discovered serendipitously by other means. In vitro assays with affinity-purified proteins demonstrated that Swi6p was a substrate of the major yeast cyclin dependent kinase, Cdc28p resulting in the specific phosphorylation of Swi6p at serine 160. Earlier work had identified phosphorylation at serine 160 as the signal for nuclear export of Swi6p but concluded also that this was Cdc28p independent. However, phosphorylation of serine 160 is at a canonical Cdc28p-phosphorylation site and is mainly catalysed by Clb5p/6p- Cdc28p complexes in vitro. In vivo, peak activity of Clb5p and Clb6p corresponds to the time of Swi6 phosphorylation and nuclear export. Furthermore, there is a delay in Swi6p nuclear export in clb5 clb6 mutants as judged by cellular observations of Swi6p-GFP fusion protein throughout the cell cycle. The fact the expression of CLB5 and CLB6 is regulated by Swi6p at Start suggests a negative feedback loop promoting cell cycle progression by removing Swi6p from the nucleus after Gl. Interestingly, phosphorylated serine 160 is a specific in vitro substrate of the essential mitotic phosphatase, Cdcl4p that is active at the M to G1 transition. The ability of Cdcl4p to dephosphorylate Swi6p suggests a feed forward mechanism whereby completion of anaphase directly promotes passage through Start by stimulating import of Swi6p back into the nucleus.

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