Synapsin is a novel Rab3 effector protein on small synaptic vesicles. I. Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals.
Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.
Giovedì
Silvia
S
Department of Experimental Medicine, Section of Human Physiology, University of Genova, Via Benedetto XV, 16132, Italy.
Vaccaro
Paola
P
Valtorta
Flavia
F
Darchen
François
F
Greengard
Paul
P
Cesareni
Gianni
G
Benfenati
Fabio
F
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli; D015723:Gene Library; D009411:Nerve Endings / Q000502:physiology; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016704:Synapsins / Q000302:isolation & purification / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000502:physiology*; D020692:rab3 GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D016704:Synapsins; D020692:rab3 GTP-Binding Proteins