Dong, Kangzhen (2010)
Ph.D. thesis, University of Birmingham.
The endo-membrane system of eukaryotic cells is a dynamic network of membrane-bound organelles that exists in a constant state of flux as they exchange membrane and protein cargoes. The yeast vacuole is a highly dynamic organelle that changes shape in response to both hypo and hyper-osmotic stresses and so has been used to study both membrane fusion and fission. Fusion is relatively well characterised but fission is not well understood. This work focuses on the molecular events that co-ordinate vacuole fission during hyper-osmotic stress. One polyphosphoinositide pathway was known to regulate vacuole fission before this work began: the Fab1p pathway. The Fab1p pathway is activated during hyper-osmotic stress and causes the vacuole to fragment into many tiny sub-compartments by a process of membrane fission. I characterised a number of the components of this pathway to learn how they work together, including Vac7p, Vac14p, Fig4p and Ymr1p. The specificity and localisation of several of these proteins were characterised and a greater understanding of this protein network emerges from this work. I then focused on understanding how fission of the vacuole is maintained and fusion inhibited in hyper-osmotic media. It was previously reported that Yck3p, a vacuolar protein casein kinase, was activated by hyper-osmotic stress to phosphorylate Vps41p, a component of the vacuole fusion machinery. The effect of this phosphorylation and the upstream signalling pathway controlling Yck3p were unknown. I started by looking if any known signalling systems might control Yck3p and ruled out both the Fab1p and Hog1p osmotic stress response pathways as upstream activators. I then found that another polyphosphoinositide signalling system controls Yck3p: namely the Plc1p phospholipase pathway that produces a series of water soluble inositol polyphosphate second messengers. I further found that deletion of any of the inositol polyphosphate kinases in the Plc1p pathway prevented Yck3p signalling, suggesting that the inositol pyrophosphates control Yck3p activity in some fashion. I also identified the Yck3p phosphorylation sites on Vps41p by FT-ICR mass spectrometry and showed that phosphorylation of these sites was required to block vacuole fusion during hyper-osmotic stress. vps41 mutants lacking these sites showed aberrant behavior: their vacuoles underwent normal fission in response to hyper-osmotic stress but then immediately re-fused in an apparent “futile cycle”. Thus Plc1p controls a novel signalling pathway mediated by Yck3p that prevents vacuole fusion by phosphorylating thus inhibiting a vital component of the fusion machinery: Vps41p.
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