Intracellular cell trafficking is mediated by transport vesicle fusion, which is driven by the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNAREs are classified as R-SNAREs (Syntaxins and SNAPs (soluble N-ethylmaleimide-sensitive factor attachment protein)) and Q-SNAREs (VAMPs). Syntaxin molecules from the transport vesicles, bind to SNAPs which inturn bind to VAMPs on target vesicles, triggering the membrane fusion. Different SNAREs are specific to their cell type and the fusion of syntaxins and synaptobrevins determines the specificity of the vesicle fusion and also catalyses the fusion process. Fusion complexes, viz Stx1A-SNAP25-VAMP2, Stx4-SNAP23-VAMP8, Stx4-SNAP23-Vamp2 and Stx13-SNAP25/29-Vamp2/3 have previously been studied. There are total of 15 syntaxins, four SNAPs and seven VAMPs known to us, accounting for total of 613 possible interactions. However a full description of the selectivity of SNARE assembly is yet to be determined.

In our studies, we use Amplified Luminescent Proximity Homogeneous Assay screen (ALPHA screen) coupled with Single molecular Fluorescence to determine pair-wise SNARE interactions. SNAREs are expressed in cell-free expression system and assays are performed to determine their binding partners. The lipid content of the cell-free lysate and the presence of vesicles allows us to mimic the biological condition in a cell-free system. We have focused not only on one-to-one Stx-VAMP and SNAP-VAMP interactions but also on the Stx-SNAP-VAMP tri-complex.

Our current approach allows us to identify the SNARE assembly partners using cell-free expression system, but also study their binding affinities, providing an insight in the biological processes. We will discuss the specificity of SNARE interactions and the factors that can modify the composition of the tri-complexes such as Munc18 and α-synuclein.