Probing SNARE assembly and disassembly in vitro and in live cells

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PROJECT ABSTRACT/SUMMARY Intracellular vesicle fusion is primarily mediated by SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), which consist of vesicle membrane protein synaptobrevin II (syb2, VAMP2 or v- SNARE), and target membrane proteins syntaxin (syx) and SNAP-25 (collectively called t-SNAREs). As the vesicle approaches the target membrane, the v-SNARE and t-SNAREs assemble in a zipper-like manner to form a vesicle fusion machine, enabling release of the molecular cargo. After fusion, the assembled SNARE complex is disassembled by the recycling machinery, composed of NSF and α-SNAP, into individual proteins for the next round of fusion. Since vesicle fusion is a ubiquitous process and is critical for cell-cell communication, mutations in SNAREs lead to numerous neuronal and metabolic diseases. Over the past few decades, extensive studies have revealed great details of individual steps in the SNARE cycle. However, the molecular mechanism that orders the sequence of each step remains unclear. A pressing need exists to probe this relationship. Here, we seek to understand the assembly and disassembly of SNAREs in vitro and in live cells. In Aim 1, we will develop a hydrogen deuterium exchange-mass spectrometry (HDX-MS) approach to analyze SNARE complex formation and deformation in vitro under physiologically relevant conditions. We have shown that the sequence coverage collected after MS of the individual SNARE proteins includes their SNARE motifs, supporting that subsequent HDX-MS will reveal details into their mechanism. We will use this approach to study SNARE binary and ternary complex to examine assembly, then introduce the recycling proteins to assess complex disassembly. In Aim 2, our objective is to understand the spatiotemporal regulation of the SNARE cycle during vesicle exocytosis by developing a genetically encoded intensity-based conformational sensor for SNAREs assembly and disassembly (icsenSNARE). Using reconstituted assays, we have identified the first design, icsenSNARE000 that showed a 1.5-fold fluorescence increase upon assembly of the SNARE complex and could be reversed by the disassembly machinery. Finally, we plan to employ icsenSNARE in live cells to reveal how the spatiotemporal regulation of the SNARE cycle is coupled to vesicle fusion. Together, the proposed study will advance mechanistic understandings of vesicle exocytosis, develop a new approach for studying SNARE assembly and disassembly, and generate a new probe for the SNARE proteins. Achievement of this work has the potential to help study SNARE-related pathologies to improve human health.