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dc.contributor.advisor McNew, James A
dc.creatorBetancourt, Miguel A
dc.date.accessioned 2019-05-16T18:15:30Z
dc.date.available 2019-05-16T18:15:30Z
dc.date.created 2019-05
dc.date.issued 2018-12-18
dc.date.submitted May 2019
dc.identifier.citation Betancourt, Miguel A. "Biochemical Analysis of Atlastin’s Membrane Anchor: Morphology, Dynamics, and Function." (2018) Diss., Rice University. http://hdl.handle.net/1911/105356.
dc.identifier.urihttp://hdl.handle.net/1911/105356
dc.description.abstract This 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.
dc.format.mimetype application/pdf
dc.language.iso eng
dc.subjectEndoplasmic reticulum
biochemistry
cellular biology
atlastin
reticulon
membrane
lipid bilayer
azidophenylalanine
membrane anchor
intramembrane hairpin
lunapark

dc.title Biochemical Analysis of Atlastin’s Membrane Anchor: Morphology, Dynamics, and Function
dc.date.updated 2019-05-16T18:15:30Z
dc.type.genre Thesis
dc.type.material Text
thesis.degree.department Biochemistry and Cell Biology
thesis.degree.discipline Natural Sciences
thesis.degree.grantor Rice University
thesis.degree.level Doctoral
thesis.degree.name Doctor of Philosophy


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