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Title:The regulation of yeast homotypic vacuole fusion by phosphatidic acid and diacylglycerol
Author(s):Starr, Matthew
Director of Research:Fratti, Rutilio A
Doctoral Committee Chair(s):Fratti, Rutilio A
Doctoral Committee Member(s):Gennis, Robert B; Jin, Hong; Zhang, Kai
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Membrane Fusion
ATPase
Lipids
Bilayer
Chaperone
Sec18
Sec17
Ypt7
Protein-lipid
Phospholipids
Lipid Regulation
Phosphatidic Acid
Diacylglycerol
Abstract:Eukaryotic membrane fusion is a highly conserved process that is necessary to maintain cellular homeostasis. Ultimately, two membrane compartments are brought to each other, direct contact is established, and the bilayers and lumenal contents mix. In Saccharomyces cerevisiae, homotypic vacuole fusion occurs through a series of well-defined phases. SNAREs are activated by their co-chaperones Sec18 (mammalian NSF) and Sec17 (mammalian α-SNAP) in an ATP dependent priming stage. Tethering first brings two fusing compartments into contact with each other via interactions between the Rab GTPase Ypt7 and its effector HOPS complex. Once tethered, compartments are pulled into tight contact with each other through the formation of trans-SNARE complexes during a docking stage. The final fusion stage proceeds through a hemifusion intermediate to pore formation and lumenal content mixing. The roles of proteins that carry out membrane fusion stages are well established, however the roles regulatory lipids at the membrane play are still unclear. Many specific regulatory lipids localize to the site of fusing vacuoles termed the “vertex ring” implying an important role for them throughout the fusion process. It has been shown that the minimal membrane lipid content necessary for fusion to occur includes ergosterol, phosphatidic acid (PA), diacylglycerol (DAG), and phosphoinositides. However, the interactions and effects each of these lipids has throughout the stages of membrane fusion has not been elucidated. This project aimed to identify specific regulatory roles for the lipids PA and DAG. Previous work identified the interconversion of the two lipids by the DAG kinase Dgk1 and PA phosphatase Pah1 may be important for vacuole fusion to effectively occur. Pah1 conversion of PA to DAG was shown to be necessary for SNARE priming activity and localization of key protein factors. We found that Sec18, the SNARE chaperone that carries out priming, is a PA binding protein. Addition of a PA-specific binding domain blocked this binding and displaced Sec18 from vacuolar membranes. Furthermore, exogenous PA addition blocked Sec18 priming activity similarly to chemical inhibition of Pah1. Vacuoles lacking Pah1 showed decreased recruitment of Sec18 to inactive SNAREs. This recruitment defect was reversible upon the introduction of Pah1 by complementation or exogenous addition. Taken together, our data suggest a direct role for the regulation of SNARE priming at the vacuole by membrane PA. We further investigated the effects PA has on Sec18 protein conformation by looking at effects on the protein’s structure during binding. We found that PA induced a conformation change in Sec18 protomers, but active hexameric Sec18 does not readily bind PA. Sec18 binding to PA resulted in different protein interactions with 1,8 ANS and different limited proteolysis profiles suggesting changes in protein conformation. No significant changes in secondary structure were determined by circular dichroism and no shifts in intrinsic tryptophan fluorescence of Sec18 were observed in the presence of PA. Molecular dynamic simulations predicted that significant protomer conformation changes occur between the monomeric and hexameric forms of Sec18. Furthermore, simulations predicted that PA binds to Sec18 at the protein’s hexamer interface. Together, our data suggest that PA may regulate Sec18 function by stabilizing its inactive monomeric form preventing formation of an active hexamer. At the endoplasmic reticulum, Dgk1 and Pah1 have been shown to direct oppose each other in the interconversion of DAG and PA. We deleted DGK1 and observed no reversal of vacuolar morphology from pah1∆ cells indicating the two enzymes do not directly offset each other at the vacuole. However, vacuoles from dgk1∆ cells did show enhanced fusion. This elevated fusion was attributed to an increase in the fusogenic lipid DAG at the vacuole. These vacuoles also had an increased resistance to inhibitors of the Rab GTPase Ypt7. Taken together, our results suggest a role for Dgk1 in regulating vacuole fusion by reducing membrane DAG levels and altering Ypt7 activity.
Issue Date:2018-09-07
Type:Thesis
URI:http://hdl.handle.net/2142/102890
Rights Information:Copyright 2018 Matthew Starr
Date Available in IDEALS:2019-02-08
Date Deposited:2018-12


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