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Title: A study of the regulation and function of Fab1p, a phosphatidylinositol 3-phosphate 5 kinase in Saccharomyces cerevisiae
Author: Phelan, John P.
ISNI:       0000 0001 3486 9917
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2005
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The Saccharomyces cerevisiae protein Fab1p is the archetypal type III phosphatidyl inositol phosphate kinase. This family of enzymes is universal to all eukaryotes and is responsible for the synthesis of phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3- phosphate. In S. cerevisiae , Fab1p regulates a number of cellular processes via the production of phosphatidylinositol 3,5-bisphosphate including: vacuole acidification, protein trafficking to the vacuole lumen, vacuole membrane recycling and apical bud formation. It is now clear that these processes are regulated independently however the molecular details of Fab1p regulation have yet to be identified. Using yeast two-hybrid analysis in a systematic screen with Fab1p and its domains, we have identified over 300 potential interactors. Phenotypic analyses on 17 corresponding deletion mutants of these proteins have identified 8 that display phenotypes consistent with loss of Fab1p. We have focused on Apl2p, which appears to be a bona fide activator of Fab1p. Apl2p, and Apl4p, which interacts with the Fab1p regulator Vacl4p, are part of the heterotetrameric complex AP-1 involved in clathrin mediated trafficking from the trans-Golgi network. We show that AP-1 regulates phosphatidylinositol 3,5-bisphosphate production in vivo and is required for the trafficking of the ubiquitinated cargoes CPS and Phm5p to the vacuole lumen, a process that requires Fab1p. Over-expression of Fab1p in AP-1 mutants reverts these trafficking defects. Using point mutants of AP-1 we show that these trafficking events are clathrin-dependant. AP-1 is not required for Fab1p-dependant vacuole morphology and vacuole acidity. Thus, we speculate that Fab1p is regulated by AP-1 for the maintenance of a pool of phosphatidylinositol 3,5-bisphosphate required for trafficking of cargoes to the vacuole. AP-1 is responsible for the retention of proteins at the trans-Golgi network therefore we speculate that retention to this compartment might also be a Fab1p-dependent mechanism.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available