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Title: Studies on the Cell Integrity MAP kinase of yeast
Author: King, Victoria Ann
ISNI:       0000 0001 3599 9406
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2006
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The yeast cell integrity MAP kinase SIt2p binds the Hsp90 molecular chaperone (Millson el aI., 2005), making it an ideal model client protein of Hsp?O in the easily manipulatable yeast Saccharomyces cerevisiae. This study was aimed at investigating several aspects of the control ofSlt2p. A two-stage purification method, using IMAC and TAP, was employed to isolate the Hsp90:S1t2p complex to gain insight into the Hsp90 facilitated activation of SIt2p (MiIlson el al., 2005). This met limiting success, isolating only Hsp82, and potentially Hsc82, in the final purification stages. TAP alone using SIt2p as the bait detected the presence of both Hsp90 and the Hsp90 co-chaperone Sti Ip. Limitations with this work can be attributed to constraints amounts of starting protein extract. Studies were conducted into the nuclear localisation of Slt2p. The S. cerevisiae ~importin pse1-1 mutant was shown to display a cell i.ntegrity defect phenotype, that was rescued by osmotic stabilisation. Using two hybrid and protein binding techniques Pse Ip and SIt2p were shown to interact proposing Pse Ip as the nuclear importin for Slt2p. Further investigation showed that the pse1-1 mutation compromised localisation of SIt2p to the nucleus and nuclear target, RIm Ip. activation confirming this. The final part of this work identified the largely uncharacterised protein, Ecm 15p as a multicopy suppressor of the caffeine sensitivity of the pse1-1 mutant and as having a link to the cell integrity pathway. Multicopy ECM15 also rescued the caffeine sensitivity of the Rim Ip null mutant, BY4743 rim/b.. Expression levels of an RIm Ip-responsive LacZ-reporter indicated rescue of growth on caffeine being attributable to restoration of cell integrity transcription. Rescue in the RIm Ip null mutant suggests that this is due to an alternative transcription factor. Both Pse Ip and Slt2p were shown to bind Ecm 15p in a non-stress inducible manner supporting a role for Ecm 15p in the cell integrity pathway.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available