Gene Cloning,expression And Interaction Mechanism Of Rab3A And Synaptotagmin ?-C2A Domain

Neurotransmitter release is controlled by a series of proteins.Protein-protein interactions in a synapse represent the key events in such a regulated process.Therefore,investigation of the protein interactions involved in neurotransmitter release is one of the main strategies for revealing the relevant regulatory mechanism.Although many proteins participate in the regulation of neurotransmitter release,a series of independent studies have demonstrated that Rab3A and synaptotagmin play the key regulatory roles.Nevertheless,although much effort has been made on the studies of Rab3 and synaptotagmin,up to now how these two proteins cooperatively regulate the synaptic vesicle exocytosis has not been fully understood.As one of the most abundant subtype in Synaptotagmin family,Synaptotagmin ?(Syt I)is acknowledged as the calcium receptor protein in synaptic vesicle membrane fusing.Syt I functions in the regulation of neurotransmitter release and multiple other cellular processes through its C2 domain binding to other molecules.Rab3A,a subtype member of the small G protein superfamily Rab,is composed of 220 amino acids,and located in brain neurons synaptic vesicles.Rab3A plays important roles in neurotransmitter release and membrane traffic.In our previous study,Rab3A was demonstrated to bind to both C2A and C2B domains of Syt I in a Ca2+-independent manner.Further analysis showed that the key binding site on C2B for Rab3A is KKKK motif and Rab3A may competitively affect C2B binding to the syntaxin in t-SNARE complex,thereby regulating the Ca2+-dependent neurotransmitter release.However,the molecular mechanism for Rab3A interaction with the C2A domain of Syt I has not been clear.In order to localize Rab3-binding site on C2A domain and reveal the relevant regulatory mechanism,in the present study we cloned and heterologously expressed C2A domain and its serial mutant genes,followed by pull-down and western blot analyses.The C2A domain and its mutants,including truncated mutants C2A(143-206)and C2A(207-244),double mutations C2A(R199Q/K200Q),C2A((R233Q/K236Q)and C2A(M173Q/F234Q),were prokaryotically expressed as GST-taged fusion proteins.RabA were prokaryotically expressed as His-taged fusion protein.According to the GST pull down assay and western blot analysis,the truncated mutant C2A(143-206)could bind to Rab3A obviously stronger that C2A(207-244),suggesting that the main binding site of Rab3A on C2A domain may be localized in the N-terminal region of the domain;R199Q/K200Q binding to Rab3A was obviously weakened compared with wild-type C2A domain;R233Q/K236Q and M173Q/F234Q normally bound to Rab3A,indicating that the key Rab3A-binding site is R199/K200 located in the loop 2 of C2A domain.The comprehensive analysis indicated that the Rab3A-binding site on C2A domain does not overlap with most of the known functional sites or residues,such as Ca2+-binding sites formed by the cluster of D residues,conservative YVK motif involved in the Ca2+-dependent binding to syntaxin and phospholipids,hydrophobic residues M173 and F234 mediating C2A domain embedment in the target membrane,etc.Furthermore,it was speculated that the interaction between Rab3A and C2A is not simply based on electrostatic force,and,in view of the reports that R199 is significantly involved in C2A binding to phospholipids,Rab3A regulates C2A-mediated vesicle/presynaptic membrane fusion mainly through competitively affecting the C2A binding to phospholipids in the presynaptic membrane.These results have contributed to the comprehension of action mechanism of Rab3 and synaptotagmin in the regulation of synaptic vesicle exocytosis.