Rat and Drosophila Synaptotagmin 4 Have Opposite Effects during SNARE-catalyzed Membrane Fusion

Zhao Wang,Edwin R Chapman

doi:10.1074/jbc.m110.137745

Abstract

Synaptotagmins (Syt) are a large family of proteins that regulate membrane traffic in neurons and other cell types. One isoform that has received considerable attention is SYT4, with apparently contradictory reports concerning the function of this isoform in fruit flies and mice. SYT4 was reported to function as a negative regulator of neurotrophin secretion in mouse neurons and as a positive regulator of secretion of a yet to be identified growth factor from muscle cells in flies. Here, we have directly compared the biochemical and functional properties of rat and fly SYT4. We report that rat SYT4 inhibited SNARE-catalyzed membrane fusion in both the absence and presence of Ca2+. In marked contrast, fly SYT4 stimulated SNARE-mediated membrane fusion in response to Ca2+. Analysis of chimeric molecules, isolated C2 domains, and point mutants revealed that the C2B domain of the fly protein senses Ca2+ and is sufficient to stimulate fusion. Rat SYT4 was able to stimulate fusion in response to Ca2+ when the conserved Asp-to-Ser Ca2+ ligand substitution in its C2A domain was reversed. In summary, rat SYT4 serves as an inhibitory isoform, whereas fly SYT4 is a bona fide Ca2+ sensor capable of coupling Ca2+ to membrane fusion.

Highlights

Studies indicate that a member of the synaptotagmin family of proteins, SYT4 [3], is a component of synaptic vesicles (SV) [4], whereas other reports rule out the presence of SYT4 on SVs [5, 6]
To begin to discern between these possibilities, we built two rat4A fly4B). We found that this chichimeras between rat SYT1 and rat SYT4: the C2A domain mera was not able to stimulate fusion when tested at concenfrom SYT1 was fused with the C2B domain from rat SYT4
Overexpression of SYT4 reduces the frequency of large dense core vesicles (LDCV) fusion events in PC12 cells, shortens the time from fusion pore opening to dilation [44], increases the frequency and duration of kiss-and-run events [38], modulates LDCV exocytosis in peptidergic nerve terminals of the neurohypophysis [9], and inhibits pre- and postsynaptic brain-derived neurotrophic factor (BDNF) release in neurons [8]

Summary

Introduction

Studies indicate that a member of the synaptotagmin family of proteins, SYT4 [3], is a component of SVs [4], whereas other reports rule out the presence of SYT4 on SVs [5, 6]. A different view arose from similar studies carried out using cultured mouse neurons [8] In this case, SYT4 was shown to co-localize with brain-derived neurotrophic factor (BDNF) in LDCVs that are targeted to both pre- and postsynaptic compartments. SYT4 serves to limit the magnitude of long-term potentiation via its ability to inhibit the release of BDNF [8] It appears that the positive role played by SYT4 at the fly neuromuscular junction and the negative role of SYT4 in the release of BDNF from mouse neurons are contradictory findings. SYT1 binds to target membrane SNARE proteins in a Ca2ϩ promoted manner [23, 24]; it is thought that the Ca2ϩ-independent component of this interaction serves to clamp or inhibit the fusion complex [25].

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Conclusion