Studying the ArfGAP-Dependent Conformational Changes in SNAREs

Vesicle SNAREs (v-SNAREs) are included with high fidelity into each transport vesicle generated in the cell. These SNAREs determine the fate of vesicles, as they are the key factors deciding with which compartment a particular vesicle will fuse. The mechanism of high fidelity inclusion of SNAREs into transport vesicles is very difficult to study in vivo. Therefore, we use in vitro assays aiming to recapitulate SNARE uptake into vesicles. One of the key assays is a pull-down with SNARE tails fused to GST in the presence or absence of ArfGAPs and coat components such as the small GTPase Arf1 and coatomer. This in vitro assay allowed us to show that the ArfGAPs Glo3 and Gcs1 can induce a conformational change in SNAREs. Protease protection assays were used to confirm the conformational change and can also be used to address the question about the nature of the different conformations in SNARE proteins.