Biochemical Analysis of Atlastin’s Membrane Anchor: Morphology, Dynamics, and Function

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dc.contributor.advisorMcNew, James Aen_US
dc.creatorBetancourt, Miguel Aen_US
dc.date.accessioned2019-05-16T18:15:30Zen_US
dc.date.available2019-05-16T18:15:30Zen_US
dc.date.created2019-05en_US
dc.date.issued2018-12-18en_US
dc.date.submittedMay 2019en_US
dc.date.updated2019-05-16T18:15:30Zen_US
dc.description.abstractThis project sheds new light into the endoplasmic reticulum (ER) fusion protein, atlastin. We studied its membrane anchor and its interaction with the lipid bilayer. The endoplasmic reticulum is composed flattened sheets and interconnected tubules that extend throughout the cytosol and contact other organelles. These discrete ER morphologies require specialized proteins that drive membrane curvature, dynamics, and mediate their maintenance. The GTPase atlastin is required for homotypic fusion of ER tubules. All atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal tail. We analyzed atlastin’s hydrophobic anchor with recombinant atlastin and different mutants reconstituted into preformed liposomes, as model membranes. While traditionally atlastin’s membrane anchor was assumed to be two transmembrane segments that fully span the lipid bilayer; we have found it consists of two intramembranous hairpin loops. The topology of these hairpins remains static during membrane fusion and do not appear to play an active role in lipid mixing. We also analyzed the membrane domain topology of the mitochondrial fusion protein mitofusin-1 and ER resident protein Sac1 and found that they also have a dual intramembranous hairpin membrane anchor. This points to a conserved topology that may be expanded to ER resident protein and atlastin homologs. We were also able to recapitulate an ER-like network with atlastin proteoliposomes in polylysine coated coverslips; thus, showing that atlastin’s can form and maintain tubular structures, this result is consistent with the dual hairpin intramembrane loop topology. We also determined that co-reconstitution of atlastin with reticulon, an ER tube-forming protein, did not influence GTPase activity or membrane fusion, however both have the propensity to inhabit high curvature membranes. We also analyzed atlastin’s GTP binding pocket and found that inter- and intra-molecular salt bridging is important in GTP hydrolysis.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.citationBetancourt, Miguel A. "Biochemical Analysis of Atlastin’s Membrane Anchor: Morphology, Dynamics, and Function." (2018) Diss., Rice University. <a href="https://hdl.handle.net/1911/105356">https://hdl.handle.net/1911/105356</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/105356en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectEndoplasmic reticulumen_US
dc.subjectbiochemistryen_US
dc.subjectcellular biologyen_US
dc.subjectatlastinen_US
dc.subjectreticulonen_US
dc.subjectmembraneen_US
dc.subjectlipid bilayeren_US
dc.subjectazidophenylalanineen_US
dc.subjectmembrane anchoren_US
dc.subjectintramembrane hairpinen_US
dc.subjectlunaparken_US
dc.subjecten_US
dc.titleBiochemical Analysis of Atlastin’s Membrane Anchor: Morphology, Dynamics, and Functionen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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