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.
Acknowledgments
We thank Gerd Hause for his support with electron microscopy at the University of Halle, Karin Breunig and Markus Pietzsch for advice concerning yeast cell culture, Randy Schekman and Bob Lesch for protocols, expression strains and discussions and Claudia Müller for technical assistance. Financial support from the Deutsche Forschungsgemeinschaft within Graduiertenkolleg 1026, from the Bundesministerium für Bildung und Forschung (FKZ 03Z2HN22) and from the state of Saxony-Anhalt (Landesgraduiertenfoerderung and European Regional Development Grants 124112001 and 1241090001) is gratefully acknowledged.
Conflict of interest statement
Funding: Bundesministerium für Bildung und Forschung, (Grant/Award Number: ‘03Z2HN22’) Deutsche Forschungsgemeinschaft, (Grant/Award Number: ‘GRK 1026’). State of Saxony-Anhalt (Landesgraduiertenförderung and <softenter;European Regional Developments (Grant/Award Numbers 124112001 and 1241090001).
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