Study On The Molecular Mechanism Of Interaction Between Rab3A And Synaptotagmin 1

Rab3, a small molecular weight GTP-binding protein, is highly enriched in the nervous system and can be specifically localized on the membrane of synapticvesicles. Like other Rabs, Rab3 cycles on and off its target membrances according to its GTP- or GDP- bound state and plays critical roles in the later stages of vesicles transportation. Synaptotagmin isa Ca2+-bingding protein anchored to the membrane of secretory vesicles via a single transmembrane domainnear its N terminus. It senses calcium influx and triggers synaptic vesicle fusion and neurotransmitter release. Given their unique features, Rab3 and synaptotagmin have been considered as Yin and Yang of membrane fusion, respectively. Rab3 A is one of the most abundant Rab3 isoforms in brain. There have been experimental results showing that the Rab3 A limits the number of Ca2+-mediated vesicle fusion and regulates the synaptic vesicle exocytosis. Synaptotagmin 1 is the major synaptotagmin in brain. It binds Ca2+through its C2 domains, thus triggering membrane fusion. Up to now, the molecular mechanism of the interaction between Rab3 A and synaptotagmin 1 as well as its effects on the release of neurotransmitters have not been completely clear.In order to study the molecular mechanism of the interaction between Rab3 A and synaptotagmin 1 and probe the effects of such an interaction on neurotransmitter release, in our present research we extracted the total RNA from the hippocampal tissues of Rats and obtained their c DNA by reverse transcription. The specific primers for molecular cloning of Rab3 A, synaptotagmin 1 and their mutants were chemically synthesize and the respective recombinant genes were preapred.After the recombinant genes were cloned into respective express vectors, three domain mutants C2 AB, C2 A and C2 B of Synaptotagmin 1 were prokaryotically expressed as GST fusion proteins; the whole-length Rab3 A andits mutants Rab3A-Q81 L, Rab3A-T36 N, Rab3A-N(1-80), Rab3A-C(81-220) were prokaryotically expressed as His-tag fusion proteins. Then GST Pull-Down experiments were employed to probethe interactions of synaptotagmin 1 domains with Rab3 A and its mutants in the presence and absence of GTP as well as Ca2+, respectively. The results showed that the C2 A and C2 B could interact with Rab3 A,which was not significantly affected by Ca2+; C2 A domain of synaptotagmin 1 interaction with Rab3 A is GTP-dependent, and C2 B domain of synaptotagmin 1 interactiopn with Rab3 A is independent of GTP. In addition, Rab3 A was demonstrated to bind to the C2 domains of synaptotagmin 1 via its N-terminal sequence(1-80 amino acids).In conclusion, the present studydemonstrated that Rab3 A interacts with synaptotagmin 1 directly via its N-terminal sequence in a Ca2+-independent manner; the interaction between Rab3 A and C2 A domain of synaptotagmin 1 depends on GTP, and, however, the interaction between Rab3 A and C2 B domain of synaptotagmin 1 does not depend on GTP, suggesting a differenmt interaction mechnism. The present study has preliminarily revealed the molecular mechanism of the interaction between Rab3 A and synaptotagmin 1, and provided new data for probing into the effects of the interaction between Rab3 A and synaptotagmin 1 on neurotransmitter release.