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Title:The regulation of yeast homotypic membrane fusion by class C ABC transporters
Author(s):Sasser, Terry
Director of Research:Fratti, Rutilio A.
Doctoral Committee Chair(s):Fratti, Rutilio A.
Doctoral Committee Member(s):Gennis, Robert B.; Morrissey, James H.; Tajkhorshid, Emad
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Membrane fusion
Trafficking
ATP-Binding Cassette (ABC) transporter
Abstract:Maintenance of eukaryotic cellular homeostasis requires the fusion of vesicle membranes that is accomplished by a SNARE-mediated mechanism. Membrane fusion is the merger of two lipid bilayers into one continuous membrane. Multiprotein complexes that have been conserved in eukaryotes carry out the basic reactions of fusion. In Saccharomyces cerevisiae, homotypic vacuole fusion occurs in experimentally defined phases. Fusion priming does not involve contact between vacuoles but includes the disassembly of complexes of SNAREs on the same membrane (cis) by Sec18p (NSF) and its cochaperone Sec17p (a-SNAP). Tethering requires Ypt7p (a Rab GTPase) and the HOPS effecter complex. SNARE complexes, including one R SNARE from a donor vacuole and three Q SNAREs from the acceptor vacuole, are formed in trans during docking of vacuoles. The membranes of the docked vacuoles are drawn together to form the “boundary domain” that resembles flat discs. The outer membranes are not in contact and come together at the boundary membrane to form the vertex ring. The vertex microdomain, enriched in fusogenic lipids and proteins, is the origin of fusion where the outer membranes are joined. Fusion culminates with the internalization of the luminal vesicle and mixing of luminal contents. The core fusion machinery has been elucidated in liposomes but many regulatory factors are being discovered in the context of the yeast vacuole. Regulatory proteins and lipids as well as lipid modifiers have been described suggesting a complex system that regulates cellular traffic. The ABC transporter superfamily is present in all organisms and is responsible for actively transporting a wide range of substrates across the lipid bilayer. Several class C ABC transporters interact with factors that are important in the fusion mechanism while others have been implicated in lipid translocation activity. The yeast vacuole contains five ABC transporters of the ABCC1 subfamily: Ycf1p, Bpt1p, Ybt1p, Vmr1 and Nft1. This project sought to identify the role of ABC transporters in membrane fusion as regulators of fusogenic proteins and lipids and their role in the mechanism of remodeling the membrane bilayer as lipid translocators. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities including the translocation of phosphatidylcholine to the vacuole lumen and the regulation of Ca2+ homeostasis. We found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50% relative to wild type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs or recruitment of factors from the cytosol such as Ypt7p and the HOPS tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca2+ transport during fusion. One difference was the prolonged Ca2+ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca2+ efflux. Evidence is presented that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function. Ycf1p, another ABCC family member that was originally characterized as a Cd2+ transporter, has also been found to physically interact with a wide array of proteins including factors that regulate vacuole homeostasis. Here we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild type vacuoles. Plasmid-expressed wild type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation in the Walker A box of the first nucleotide binding domain (Ycf1pK669M) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p regulates fusion through the recruitment Vam7p to vacuolar membranes. Membrane lipids are organized in an asymmetric fashion across the vesicle bilayer and this asymmetry must be maintained through lipid translocation. The translocation or flippase activity can be monitored using fluorescently labeled lipids. In this study, we describe a FRET-based assay to follow the translocation of the rhodamine-labeled phosphatidylethanolamine (RH-PE) in a real-time assay. We found that there is an ATP-dependant Rh-PE flippase on the vacuole and the ABC transporters Ycf1p, Bpt1p and Ypk9p are not responsible for the translocation activity. We also report that pH and osmolyte concentration do not affect the lipid translocation activity. However, modifying the bilayer fluidity with the addition of chlorpromazine (CPZ) abolished lipid translocation activity and propranolol inhibition of phosphatidic acid phosphatase activity increased the rate of lipid translocation.
Issue Date:2013-05-28
URI:http://hdl.handle.net/2142/44801
Rights Information:Copyright 2013 Terry Sasser
Date Available in IDEALS:2013-05-28
2015-05-28
Date Deposited:2013-05


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