Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

26-POS Board 26 BIOPHYSICAL FEATURES UNDERLYING THE EXTREME CALCIUM SENSITIVITY OF SYNAPTOTAGMIN-7 Jefferson Knight 1 ; Hai Tran 1 ; Lauren Anderson 1 ; 1 University of Colorado Denver, Dept. of Chemistry, Denver, Colorado, USA Synaptotagmin (Syt) family proteins are crucial regulators of membrane fusion during exocytosis. Eight of the seventeen Syt isoforms bind membranes in a Ca 2+ -dependent manner via their tandem C2 domains, C2A and C2B. Among these, Syt-7 is the most Ca 2+ sensitive, binding anionic lipids and triggering SNARE-mediated membrane fusion even at sub- micromolar Ca 2+ concentrations. Our goal was to understand how the C2A and C2B domains of Syt-7 work together to achieve this extreme Ca 2+ sensitivity. We compared membrane binding properties of individual and tandem C2 domains between Syt-7 and Syt-1 using fluorescence- based liposome binding assays, stopped flow dissociation kinetics, insertion depth measurements, and aggregation assays. Results indicate that the greatest difference between the two proteins is the Ca 2+ -dependent membrane binding strength of the C2A domains: Syt-7 C2A bound physiological liposomes with ~25-fold greater Ca 2+ sensitivity and dissociated ~100-fold more slowly than Syt-1 C2A. The C2B domain of Syt-7 also bound membranes somewhat more strongly than Syt-1 C2B, both in the presence and absence of the polyanionic lipid phosphatidylinositol-(4,5)-bisphosphate (PIP 2 ), although differences between the C2B domains were not as stark as between the C2A domains. Perhaps surprisingly, Syt-7 exhibited less interdomain cooperativity than Syt-1, as assessed via direct comparisons of chelator-induced dissociation kinetics from model liposomes lacking PIP 2 . We suggest this is due to the relative energetic dominance of the C2A domain in Syt-7 but the C2B domain in Syt-1. Inclusion of 1% PIP 2 in the lipid composition potentiated the ability of the Syt-7 C2AB tandem to bind and aggregate liposomes even in the absence of Ca 2+ , an effect which is presumably prevented in vivo by other competing interactions. Overall, our results suggest conserved structural features that support divergent membrane binding energetics and Ca 2+ sensitivities between these two important synaptotagmins.

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