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Title:Lipid composition and membrane physical parameters regulate priming and SNARE function in yeast vacuole membrane fusion
Author(s):Karunakaran, Surya Priya
Director of Research:Fratti, Rutilio A.
Doctoral Committee Chair(s):Fratti, Rutilio A.
Doctoral Committee Member(s):Gennis, Robert B.; Morrissey, James H.; Nair, Satish K.
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Membrane fusion
regulatory lipids
curvature
chlorpromazine (CPZ)
phosphatidic acid (PA)
diacylglycerol (DAG)
Sec18
Abstract:Fusion at highly specialized membrane microdomains is a prerequisite for processes like hormone and neurotransmitter release, antigen presentation and viral infection. Apart from a family of highly conserved proteins called SNAREs, membrane fusion also requires a set of regulatory lipids that includes ergosterol, diacylglycerol, phosphatidic acid and phosphoinositides. Although a lot is known about the SNARE protein machinery, the role of lipids and the lipid environment of the membrane are not clear. For long, lipids had been thought to play a passive role by binding protein factors through their lipid binding domains. However, recent studies have shown that a subset of lipids play critical regulatory role by modulating the SNARE fusion machinery. The regulatory role of lipids on the fusion machinery can be divided into at least two mechanisms. The first mechanism depends on the direct chemical interaction of lipids with the protein fusion catalysts. Here, we report that phosphatidic acid serves to bind a key AAA+ family ATPase, Sec18p that effects priming in yeast vacuolar membrane fusion. The yeast PA phosphatase Pah1p alters the ratio of PA to DAG on the membrane during priming and serves to recruit Sec18p to the cis-SNARE complexes at the site of priming. The second mechanism is indirect and physical and is dictated by the physical parameters of the membrane such as curvature, fluidity and lateral tension. We report that inducing negative curvature on the vacuole membrane lowers the force threshold required for fusion, thus allowing non-canonical SNARE complexes to support fusion. On the other hand, increasing the positive curvature of the membrane increases the force threshold such that even canonical SNARE complexes do not support fusion. We also report that the formation of the SNARE bundle and the presence of a transmembrane domain serve to transmit the pulling force generated from the SNARE bundle to the membrane, which serves to distort the lipid bilayer leading to fusion. Either a defect in the SNARE complex or the absence of a transmembrane leads to a break in force transmission and thus results in fusion stalled at the hemifusion stage. The knowledge gained from this study sheds more light on the mechanism by which membrane lipids regulate fusion. We have demonstrated that regulatory lipids are important during distinct stages of fusion and serve to recruit key protein catalysts at the site of fusion. The lipid composition of the membrane also dictates membrane parameters such as curvature and fluidity. We demonstrate that membrane curvature, fluidity and lateral tension are essential factors that can affect the core fusion machinery. The contribution of the membrane in membrane fusion is only beginning to be understood and we hope that this study will contribute to a growing sea of knowledge in the field of membrane fusion.
Issue Date:2013-05-24
URI:http://hdl.handle.net/2142/44246
Rights Information:Copyright 2013 Surya Priya Karunakaran
Date Available in IDEALS:2013-05-24
Date Deposited:2013-05


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