Charged Residues that Control the Electrostatic Association of Synaptotagmin 1 C2AB with Membranes

Abstract

Signaling in the central nervous system depends on the fusion of synaptic vesicles to the presynaptic membrane of the neuron. This fusion event is mediated by membrane-anchored SNAREs (Soluble NSF Attachment Protein Receptors), but requires a number of additional protein factors including munc18, complexin and Synaptotagmin 1 (Syt1). Synaptotagmin 1 has been shown to act as the Ca2+ sensor in neuronal fusion, and it could act by bridging bilayers and assisting SNARE-mediated fusion, or it might directly interact with the SNAREs to assist SNARE assembly. Recent work has indicated that two arginine residues (R398 and R399) on the face opposite the calcium binding region in C2B domain are critical for the function of Syt1 in neurotransmitter release (Xue et al. (2008), NSMB 15, 1160) and it has been proposed that these residues function in associating this region to the membrane interface. Using site-directed spin labeling and progressive power saturation, we have shown that in a soluble fragment of Syt1 containing the two C2 domains (C2AB), that the region opposite the Ca2+-binding sites in the C2B domain associates with the membrane interface and allows Syt1 to bridge two bilayers. When the single mutation R398Q and the double mutation R398Q R399Q are made to C2AB, power saturation measurements indicate that this region no longer associates with the membrane interface. Work is currently underway to test the role of these arginine residues in SNARE association. This work was supported by a grant from NIGMS, GM 072694.