Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

10-POS Board 4 THE TWO SNARE MOTIFS OF THE SNARE PROTEIN SNAP25 ARE STRUCTURALLY AND FUNCTIONALLY DISTINCT

Katelyn N Kraichely 1 ; Volker Kiessling 1 ; Binyong Liang 1 ; Lukas K Tamm 1 ; 1 University of Virginia Health System, Molecular Physiology and Biological Physics, Charlottesville, VA, USA

All eukaryotic cells maintain membrane and protein homeostasis through secretory pathways that transport vesicular cargo and lipids between compartments. The membrane fusion events in these pathways are catalyzed by a large and conserved family of membrane-anchored or associated SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins. SNARE proteins each contribute one or two regions of 60-70 residues termed “SNARE motifs” that assemble into a four helix, coiled-coil bundle to drive membrane fusion. Fusion is particularly tightly regulated during neurotransmitter exocytosis at neuronal synapses, which utilize a SNARE core complex composed of syntaxin-1a and SNAP25 (target membrane or “tSNAREs”) on the plasma membrane and synaptobrevin-2 on the vesicle membrane. The tSNARE SNAP25 contains two SNARE motifs connected by a flexible linker and anchored to the plasma membrane through four palmitoylation sites. Only a subset of cellular cognate SNAREs utilize this tandem SNARE motif arrangement, raising the question of the functional and evolutionary significance of SNAP25’s two-domain topology. Our data using magnetic resonance spectroscopy shows that the two SNARE motifs contained in the SNAP25 sequence are structurally distinct and independent from one another. Additionally, we utilize a reconstituted single-particle fusion assay to demonstrate that SNAP25 fragments containing only one of the two SNARE motifs are able to catalyze fusion with different fusion efficiencies and kinetics from one another and the full-length protein. Lastly, FRET and fluorescence self quenching experiments investigate how the different SNARE motifs of SNAP25 influence the lateral organization of Syntaxin-1a in the membrane. Combining these experimental perspectives provides fundamental new insight into the mechanism of assembly and function of the SNARE complex and implications for how the neuronal SNARE complex is optimized for fine control of synaptic exocytosis.

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