synaptotagminMutants Reveal Essential Functions for the C2B Domain in Ca²⁺-Triggered Fusion and Recycling of Synaptic VesiclesIn Vivo

Abstract

Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

Conserved Prefusion Protein Assembly in Regulated Exocytosis

“…The severity of the AD3 mutation was initially explained through the inability of SYT to oligomerize (Littleton et al, 2001), but this has been subsequently challenged (Borden et al, 2005). In addition, calcium-induced oligomerization of a vesicular protein cannot readily account for the change in the distribution of the vesicles at the plasma membrane in resting nerve terminals (Reist et al, 1998).…”

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

“…Initial studies suggested that multimerization of synaptic SNARE complexes could be achieved via domain swapping, whereby one of the two SNAP-25 helices could be substituted by the equivalent helix from a neighboring complex (Kweon et al, 2002). Alternative models proposed the involvement of accessory proteins, such as synaptotagmin (Littleton et al, 2001) or complexin (Tokumaru et al, 2001), or the transmembrane domains of syb2 and syntaxin 1A (Laage et al, 2000), in synaptic SNARE complex multimerization. However, SNARE complexes assembled from recombinant coils and lacking transmembrane domains are able to associate with each other (Fasshauer et al, 1997;Fasshauer et al, 1998;Margittai et al, 2001;Ernst and Brü nger, 2003), arguing that at least some of the interactions that support multimerization may require neither accessory proteins nor transmembrane domains.…”

Effects of synaptotagmin reveal two distinct mechanisms of agonist‐stimulated internalization of the M4 muscarinic acetylcholine receptor

“…The best characterized isoform is the neuronal-specific synaptotagmin I (Augustine, 2001). Synaptotagmin I is present in synaptic vesicle membranes, where it is likely to function as the Ca 2 þ sensor for exocytosis at the nerve terminal (Augustine, 2001;Ferna´ndez-Chaco´n et al, 2001;Littleton et al, 2001). Synaptotagmin I spans the vesicle membrane once, and its cytoplasmic domain contains two C2 domains, both of which bind Ca 2 þ (Davletov & Su¨dhof, 1993;Sutton et al, 1995;Desai et al, 2000;Ubach et al, 2001).…”

“…It has also been shown that tyrosinebased endocytotic motifs of membrane proteins can enhance the binding of AP-2 to the C2B domain of synaptotagmin (Haucke & De Camilli, 1999), suggesting a way in which coated pit formation may be coupled to the selection of 'cargo' proteins. The significance of the C2B domain with respect to endocytosis is highlighted by the finding that its disruption appears to inhibit synaptic vesicle recycling at the nerve terminal in both Caenorhabditis elegans (Jorgensen et al, 1995) and Drosophila (Littleton et al, 2001).…”