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dc.contributor.advisorRapoport, Tom A.
dc.contributor.authorLiu, Tina Yu
dc.date.accessioned2014-10-21T16:37:31Z
dash.embargo.terms2016-01-01en_US
dc.date.issued2014-10-21
dc.date.submitted2014
dc.identifier.citationLiu, Tina Yu. 2014. Mechanism of endoplasmic reticulum membrane fusion mediated by the Atlastin GTPase. Doctoral dissertation, Harvard University.en_US
dc.identifier.otherhttp://dissertations.umi.com/gsas.harvard.inactive:11745en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:13064987
dc.description.abstractHow organelles acquire their unique shapes is a fundamental question of cell biology. The peripheral endoplasmic reticulum (ER) consists of a vast network of membrane sheets and tubules, the formation of which requires homotypic membrane fusion. Previous studies suggest that the dynamin-like GTPase, atlastin (ATL), mediates ER fusion, but the mechanism by which this occurs is unclear. In this study, I investigate 1) the role of dimerization and conformational changes in the N-terminal domain of ATL, 2) how the C-terminal amphipathic helix and the transmembrane domain of ATL cooperate with the N-terminal domain, and 3) the formation of cis and trans ATL dimers in the fusion mechanism. ATL has a cytosolic N-terminal domain, consisting of a GTPase domain and three-helix bundle (3HB), followed by two transmembrane segments (TMs) and a cytosolic C-terminal tail (CT). Crystal structures of ATL and biochemical experiments suggest that nucleotide-dependent dimerization between ATL molecules sitting in different membranes can tether the membranes together. A subsequent conformational change triggered by GTP hydrolysis could pull the membranes toward one another for fusion. This mechanism is supported by in vitro membrane tethering and fusion assays using vesicles containing full-length Drosophila ATL. The CT and TMs of ATL are also required for efficient membrane fusion. A synthetic peptide corresponding to a conserved amphipathic helix in the CT can act in trans to restore the fusion activity of a tailless ATL mutant. We characterize CT mutants to show that the C-terminal helix promotes fusion by perturbing the lipid bilayer. The TMs of ATL also mediate nucleotide-independent oligomerization, which may allow ATL molecules in the same membrane to synchronously undergo the conformational change leading to fusion. Lastly, we show that continuous GTP hydrolysis is required for membrane tethering, occasionally resulting in fusion. The N-terminal cytosolic domain mediates trans dimer formation between ATL molecules on different membranes. GTP binding induces dimerization through the GTPase domains and 3HBs. We propose that GTP hydrolysis and phosphate release are required not just to drive fusion, but also to dissociate cis dimers that form on the same membrane, thus allowing ATL molecules to form trans dimers.en_US
dc.language.isoen_USen_US
dash.licenseMETA_ONLY
dc.subjectCellular biologyen_US
dc.subjectBiochemistryen_US
dc.subjectBiophysicsen_US
dc.subjectendoplasmic reticulum membrane fusionen_US
dc.subjectendoplasmic reticulum morphologyen_US
dc.subjecthereditary spastic paraplegiaen_US
dc.subjectprotein structureen_US
dc.subjectreconstituted proteoliposomesen_US
dc.subjectSPG3Aen_US
dc.titleMechanism of endoplasmic reticulum membrane fusion mediated by the Atlastin GTPaseen_US
dc.typeThesis or Dissertationen_US
dash.depositing.authorLiu, Tina Yu
dash.embargo.until10000-01-01
thesis.degree.date2014en_US
thesis.degree.disciplineBiology: Medical Sciences, Division ofen_US
thesis.degree.grantorHarvard Universityen_US
thesis.degree.leveldoctoralen_US
thesis.degree.namePh.D.en_US
dc.contributor.committeeMemberGaudet, Rachelleen_US
dc.contributor.committeeMemberWickner, Williamen_US
dc.contributor.committeeMemberFinley, Danielen_US
dc.contributor.committeeMemberMoazed, Daneshen_US
dash.contributor.affiliatedLiu, Tina Yu


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