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Free keywords:
giant unilamellar vesicle
in vitro reconstitution
intracellular
transport
membrane fission
protein-lipid interaction
Sar1
purified cytosolic proteins
guanine-nucleotide-binding
endoplasmic-reticulum
transport vesicles
gtp hydrolysis
golgi
transport
membrane coat
er
liposomes
complex
Biochemistry & Molecular Biology
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.
