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Title: Studies on the regulation and role of the cell integrity pathway of Saccharomyces cerevisiae
Author: Sabetnia, Sahar Z. S.
ISNI:       0000 0001 3544 2042
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2002
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The Pkc1p-MAPK pathway of S. cerevisiae is required for proper cell surface construction and is hence referred to as the cell integrity pathway. The cell integrity pathway is known to be activated in response to cell surface damage, such as heat, hypo- osmotic shock, mating pheromone treatment, calcofluor white, zymolyase, secretion blocks/tunicamycin treatment and addition of membrane deforming compound, chlorpromazine. HCS77 and MID2 encode type-I transmembrane proteins that are putative receptors of cell surface damage. Hcs77p is known to be required for activation of the Pkc1p-MAPK pathway during heat shock. In this study we identify Mid2p as an upstream regulator of Pkc1p-MAPK pathway during cell surface stress. Furthermore, we find that Mid2p and Hcs77p are redundant activators of the cell integrity pathway. First, it we find that MID2 genetically interacts with the Pkc1p-MAPK pathway, functioning upstream of PKCl and the MAPK cascade. Second, M1D2 is a suppressor of the cell lysis defect of hcs77Delta cells at elevated temperatures. Third, mid2Deltahcs77Delta cells are inviable in the S288C strain background and viable only on osmotically stabilised medium in the EG 123 strain background. Fourth, Mpklp activation assays show that Mid2p and Hcs77p function redundantly to activate the cell integrity pathway in response to heat shock and mating pheromone. Mid2p and indeed the whole of the Pkc1p-MAPK pathway are required to maintain viability of cells during the mating response. First, mid2Delta cells display a pheromone induced death. Second, death of mid2Delta cells in presence of mating pheromone is suppressed by over-expression of Pkc1p-MAPK pathway components. Third, HCS77 and MPK1 are also involved in the mating response to maintain cell integrity, as null mutants of these components show loss of cell integrity during pheromone treatment. Fourth, the pheromone-induced death of hcs77Delta cells can be suppressed by over-expression of the Pkc1p-MAPK pathway components. Fifth, Mid2p and Hcs77p function as redundant activators of the Pkc1p-MAPK pathway in response to mating pheromone treatment. Sixth, death of Pkc1p-MAPK pathway mutants is as a result of cell lysis, probably due to defects in the cell wall. Seventh, the death of Pkc1p-MAPK pathway mutants in a-factor is not osmotically remedial. Eighth, the defect in the cell wall is not one associated with improper chitin deposition during pheromone treatment. Ninth, Pkc1p-MAPK pathway mutants are not defective in actin localisation. Tenth, the pheromone-induced death of these mutants is not a consequence of cell polarisation. All the data presented indicate that the Pkc1p-MAPK pathway has an important role to play in maintaining cellular integrity during the mating response, independent of actin localisation and chitin deposition. However, the role that Pkc1p pathway plays during the mating response appears to be not associated with regulation of cell-cell fusion. In this study, we also find that the Pkc1p-MAPK pathway and the calcineurin pathway are co-regulated in response to cell surface stresses, by independent sensing mechanisms. Calcineurin is a Ca2+/calmodulin regulated protein phosphatase that is conserved from yeast to mammalian cells. Calcineurin activity is stimulated during pheromone treatment, heat shock comparable to the Pkc1p-MAPK pathway. First, Using a calcineurin reporter construct, the calcineurin pathway is found to be activated in response to cell surface stress associated with tunicamycin, chlorpromazine, calcofluor white and zymolyase. Second, activation of the calcineruin pathway in response to cell surface stress is mediated by an increase in calcium influx, partly via the Mid Ip putative mechanosensitive calcium channel. Third, regulation of calcineurin activity in response to cell surface stress is not dependent upon the Pkc1p-MAPK pathway. Fourth, the whole of the Pkc1p-MAPK pathway is involved in modulation of basal calcineurin activity, as mpk1Delta and mid2Deltahcs77Delta cells display a reduced basal calcineurin activity. Fifth, the Pkc1p-MAPK pathway modulates calcineurin activity by regulating calcium efflux and hence maintaining cellular calcium homeostasis. Sixth, the Pkc1p-MAPK pathway regulates calcium efflux by a mechanism, which is independent of the transcription factor Rlmlp. Our data suggest that the Pkc1p-MAPK pathway functions upstream of the calcium-signaling pathway. To support this proposal, our Mpk1p activation assays, show that calcium is not required for activation of Mpk1p in response to cell surface stress.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Cell surface stress