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

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.

Insights into the Mechanisms of Membrane Curvature and Vesicle Scission by the Small GTPase Sar1 in the Early Secretory Pathway

“…The mechanism for COPII vesicle scission, particularly the role and relevance of GTP hydrolysis, remains elusive [12]. In previous studies, in vitro reconstitution reactions using a minimal set of yeast COPII proteins and liposomes have yielded COPII-coated vesicles [5,13].…”

“…Straight and rigid membranous extensions were only observed under non-hydrolyzing conditions [16]. Thus, the ability of Sar1 to form tubular extensions of different morphologies is regulated by the nucleotide-bound state [12]. …”

Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy

“…Liposomes were prepared from a complex mixture of lipids (termed the 'major-minor-mix') that was previously established in COPII in vitro reconstitution experiments (23)(24)(25). It consists of 34.4 mol% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 14.8 mol% of 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE), 3.4 mol% of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 5.4 mol% of L-α-phosphatidylinositol from soy (soy-PI), 1.5 mol% of L-α-phosphatidylinositol-4-phosphate from porcine brain (PI(4)P), 0.5 mol% of L-α-phosphatidylinositol-4,5-bisphosphate from porcine brain (PI(4,5)P 2 ), 1.3 mol% of 1,2-dioleoyl-sn-glycero-3-(cytidine diphosphate) (CDP-DAG) and 33.3 mol% of ergosterol.…”

“…The protein Sar1p from Saccharomyces cerevisiae was expressed and purified as previously described (24). The protein variant Sar1pS147C/C171S was prepared in the same way, but additionally labeled with Alexa Fluor 488 maleimide (Invitrogen/Thermo-Fisher, Calsbad, CA).…”

“…The protein variant Sar1pS147C/C171S was prepared in the same way, but additionally labeled with Alexa Fluor 488 maleimide (Invitrogen/Thermo-Fisher, Calsbad, CA). Both labeled and unlabeled Sar1p proteins were functional in a COPII recruitment and membrane deformation assay (24) and with respect to GTPase enzyme activity. Protein concentrations were determined by UV absorption, using linear unmixing of the protein spectrum and the nucleotide spectrum.…”