Abstract: Objective Synaptic vesicles complete of synaptic vesicles cycle by exocytosis and endocytosis. The retrieval of synaptic vesicle membrane after exocytosis is essential for the maintenance of synaptic transmission in central nervous system synapses. Dynamin-1 is a 96kD multidomain GTPase enzyme that is crucial for the fission stage of synaptic vesicle recycling and vesicle traffick. Several models range from viewing Dynamin-1 strictly as a mechanochemical enzyme to considering it as a regulatory protein for the recruitment of the downstream binding partners responsible for scission. To address the role of Dynamin-1 and its interaction proteins in synaptic vesicle endocytosis, we screened and identified interaction proteins of Dynamin-1 PRD functional domain in rat brain synaptosomes. Methods pGEX-4T-2-PRD, a prokaryotic expression plasmid of PRD functional domain, was constructed into Dynamin-1. GST-PRD fusion proteins were obtained by Escherichia coli (E.coli) expression system combined glutathione agarose purified column. Rat brain synaptosomal fractions were isolated by ultracentrifugation. Glutathione S-transferase(GST) pull-down assay was employed to screen interaction proteins between rat brain synaptosome and GST-PRD fusion proteins. Subsequently, these proteins were identified using liquid chromatography spectroscopy (LC-MS). Result We successfully purified the GST-PRD fusion proteins and extracted rat brain synaptosome. Thirty-five interaction proteins of Dynamin-1 PRD functional domain in rat synaptosome were isolated and identified, consisting of synaptic vesicle-associated proteins, cytoskeletal proteins, metabolic enzymes and other proteins. Conclusion Here we reported a comprehensive set of candidate proteins that are closely related to synaptic vesicle recycling. The study has laid the foundation for clarifying the function, regulatory mechanism of Dynamin-1 and cracking the pathway/protein network of synaptic vesicle recycling.

Key words: Brain synaptosome, Dynamin-1, PRD functional domain, Synaptic vesicle recycling, Liquid chromatography, Rat