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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

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1437-4315
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Volume 395, Issue 7-8

Issues

Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation

Sebastian Daum / Daniela Krüger / Annette Meister / Jan Auerswald / Simone Prinz
  • Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ John A.G. Briggs
  • Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Kirsten Bacia
Published Online: 2014-07-08 | DOI: https://doi.org/10.1515/hsz-2014-0117

Abstract

As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided.

Keywords: giant unilamellar vesicle; in vitro reconstitution; intracellular transport; membrane fission; protein-lipid interaction; Sar1

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About the article

Corresponding author: Kirsten Bacia, HALOmem, University of Halle, Kurt-Mothes-Strasse 3, 06120 Halle, Germany, e-mail:


Received: 2014-02-05

Accepted: 2014-06-12

Published Online: 2014-07-08

Published in Print: 2014-07-01


Citation Information: Biological Chemistry, Volume 395, Issue 7-8, Pages 801–812, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2014-0117.

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