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Title: Further characterisation of the Rim101 pathway in Saccharomyces cerevisiae
Author: Goodman, Daniel
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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The Rim101 pathway is a signalling pathway that has been primarily, but not exclusively, associated with the response to alkaline stress in yeast. The mechanistic basis for activation of this signalling pathway is yet to be determined and the function of the pathway has only been partially characterised. This study has screened a range of novel stress conditions for Rim101 pathway activation using a Western-based assay. The application of high extracellular concentrations of KCl was found to induce Rim101p processing. This activation was found to be potassium-specific since Rim101p processing was not promoted by administration of NaCl or sorbitol. The potassium-induced Rim101p cleavage was established to occur via the canonical Rim101 pathway and with similar dynamics to that induced by alkaline stress. Since both alkaline stress and high extracellular potassium concentrations dramatically alter the resting membrane potential, intracellular pH and intracellular potassium levels of the yeast cell, the potential involvement of the Rim101 pathway in membrane potential, intracellular pH and potassium homeostases was characterised. The role of the pathway in membrane potential homeostasis was investigated using a potentiometric dye and depolarising agents. Proton extrusion activity, which is the predominant generator of membrane potential, was assessed in rim mutants using an external pH electrode that measured acidification of the extracellular medium. Toxic cation sensitivity was assessed in rim mutants in a series of phenotypic screens. A potential function for the Rim101 pathway in cytosolic pH homeostasis was investigated by expressing a pH-sensitive modified form of GFP in wild-type and rim mutant cells. A role in vacuolar pH homeostasis was analysed using a pH-sensitive dye that sequesters to the vacuole. The ‘steady-state’ pH values of the cytosol and vacuole were assessed over a broad range of extracellular pH values and the dynamic changes upon application of glucose and KCl were recorded. A functional association between the Rim101 pathway and intracellular potassium homeostasis was analysed through assessment of the growth of rim mutants over a wide range of extracellular potassium concentrations. The internal potassium levels of wild-type and rim mutant cells were determined under a broad range of conditions through analysis of yeast cell extracts using ion chromatography. The rim101 and rim8 mutants were found to exhibit hyperpolarised membrane potentials. The rim101 mutant displayed membrane potential changes in response to the application of depolarising agents that were quantitatively different from those exhibited by the rim8 mutant, implicating a novel role for the full-length form of Rim101p, which had previously been thought to be inactive. The level of proton extrusion was assessed in the mutants and was surprisingly found to be only modestly higher than that of the wild-type strain. The rim101 mutant displayed an alkalinised cytosol and an acidified vacuole compared to the wild-type strain over a broad range of extracellular pH values. The rim8 mutant exhibited an alkalinised cytosol but presented a vacuolar pH that was very similar to that of the wild-type strain. The rim101 and rim8 mutants were seen to grow significantly better than the wild-type at very low concentrations of potassium. Preliminary data suggests that the rim mutants may unexpectedly accumulate less potassium than the wild-type strain, implicating a reduced requirement for this ion in these mutants. This study has found novel functional roles for the Rim101 pathway in membrane potential, intracellular pH and potassium homeostasis.
Supervisor: Haynes, Ken ; Tang, Christoph ; Arst, Herb ; Bignell, Elaine Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available