Synapsin Is a Novel Rab3 Effector Protein on Small Synaptic Vesicles: II. FUNCTIONAL EFFECTS OF THE Rab3A-SYNAPSIN I INTERACTION

Silvia Giovedì,François Darchen,Flavia Valtorta,Paul Greengard,Fabio Benfenati

doi:10.1074/jbc.m404168200

Abstract

Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedi, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J. Biol. Chem. 279, 43760-43768). We have analyzed the functional consequences of this interaction on the biological activities of both proteins and on their subcellular distribution within nerve terminals. The presence of synapsin I stimulated GTP binding and GTPase activity of both purified and endogenous synaptic vesicle-associated Rab3A. Conversely, Rab3A inhibited synapsin I binding to F-actin, as well as synapsin-induced actin bundling and vesicle clustering. Moreover, the amount of Rab3A associated with synaptic vesicles was decreased in synapsin knockout mice, and the presence of synapsin I prevented RabGDI-induced Rab3A dissociation from synaptic vesicles. The results indicate that an interaction between synapsin I and Rab3A exists on synaptic vesicles that modulates the functional properties of both proteins. Given the well recognized importance of both synapsins and Rab3A in synaptic vesicles exocytosis, this interaction is likely to play a major role in the modulation of neurotransmitter release.

Highlights

Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedı, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J
It has been demonstrated recently that GTP/GDP exchange is not a prerequisite for Rab3A binding to the SV membrane and that GDP-bound Rab3A binds to a protein component of SV that competes with ferase; LRh-PE, N-[lissamine rhodamine B sulfonyl] L-␣-phosphatidylethanolamine; MAPK, mitogen-associated protein kinase Erk 1/2; NBD-PE, N-[4-nitrobenzo-2-oxa-1,3-diazole] L-␣-phosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; protein kinase A (PKA), cAMP-dependent protein kinase; RSV, synapsin-rebound synaptic vesicles; synapsin-depleted SV (SSV), synapsin-depleted synaptic vesicles; untreated SV (USV), untreated synaptic vesicles; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate
Effects of Synapsin I on the Biochemical Properties of Rab3A—The rate of the Rab3 cycle within the nerve terminal is regulated by the key events of GDP/GTP exchange, which follows the association of Rab3 with SV, and GTP hydrolysis that promotes Rab3 dissociation from SV during priming and fusion

Summary

FUNCTIONAL EFFECTS OF THE Rab3A-SYNAPSIN I INTERACTION*

A family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals Under conditions of high frequency stimulation, Rab3A knockout mice exhibit a decrease in the recruitment of SV at the presynaptic membrane and a delayed recovery of release after stimulation [15], whereas train and paired-pulse facilitations were increased after injection of constitutively active Rab3A into Aplysia neurons [10] These effects, together with the described interaction between the Rab effector Rabphilin-3 and the actin bundling protein ␣-actinin [16], implicate Rab in the activity-dependent trafficking of SV upstream of SV fusion, possibly by affecting the dynamics of SV binding to the actin cytoskeleton. Synapsin stimulated the Rab3A cycle by increasing GTP binding, GTPase activity, and Rab3A recruitment to the SV membrane, and Rab3A inhibited the actin binding and SV clustering activity of synapsin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION