A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding.

B A Davletov,T C Südhof

doi:10.1016/s0021-9258(19)74326-9

Abstract

Synaptotagmin I is a Ca(2+)- and phospholipid-binding protein of synaptic vesicles with an essential function in neurotransmission. Ca2+/phospholipid binding by synaptotagmin I may be mediated by its C2 domains, sequence motifs that have been implicated in the Ca2+ regulation of a variety of proteins. However, it is currently unknown if C2 domains are sufficient for Ca2+/phospholipid binding or if they even directly participate in Ca2+/phospholipid binding. In order to address this question, we have studied the Ca2+/phospholipid-binding properties of the first C2 domain of synaptotagmin I. Our results show that this C2 domain by itself binds Ca2+ and phospholipids with high affinity (half-maximal binding at 4-6 microM free Ca2+) and exhibits strong positive cooperativity. The C2 domain is specific for negatively charged phospholipids and for those divalent cations that are known to stimulate synaptic vesicle exocytosis (Ca2+ > Sr2+, Ba2+ >>> Mg2+). These studies establish that C2 domains can serve as independently folding Ca2+/phospholipid-binding domains. Furthermore, the cation specificity and the cooperativity of Ca2+ binding by the C2 domain from synaptotagmin I support a role for this protein in mediating the Ca2+ signal in neurotransmitter release.

Highlights

Synaptotagmin I is a Ca2+- and phospholipid-bindingunidentified, hction for synaptotagmin in neurotransmitter protein of synaptivcesicles with an essential functionrelienase
Our results indicate thaCtz domains aresufficient for Ca2+lphospholipid binding.the highaffinity of the first C2 domain from synaptotagmin I for Ca2+ (4-6 p ~ E,CS0) and its cooperativity in Ca2+ binding agreewell with a Ca2+-dependentfunction in synaptic gesting a role for synaptotagmin I in Ca2+-dependent synaptic vesicle exocytosis (Littleton et al, 1993)
The presence of sequences homologous to this domain in synaptotagmins and a variety of other proteins suggests that this domain may represent a widely shared functional motif (Vogel et al, 1988; Perin et al, 1990; Clark et al, 1991;Maruyama and Brenner, 1991).Transfection experiments demonstratedthat theCa2+-dependentregulation of PKC depends on the presence of the C2domain (Kaibuchi et al, 1989;Akita et al, 1990),and purified PKC and synaptotagmin I were shown to bind Ca2+and phospholipids (Bazzi and Nelsestuen, 1990; Brose et al, 1992)

Summary

Introduction

Synaptotagmin I is a Ca2+- and phospholipid-bindingunidentified, hction for synaptotagmin in neurotransmitter protein of synaptivcesicles with an essential functionrelienase. The binding of 3H-labeled P W C liposomes to the first Cz domain of synaptotagmin I fused to GST (GST-SytA; toppanel ) or to GST alone (bottompanel ) were studied as a function of different cations.

Results

Conclusion