Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.
Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.
Egaña
Loreto A
LA
Department of Neurobiology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA.
Cuevas
Rolando A
RA
Baust
Tracy B
TB
Parra
Leonardo A
LA
Leak
Rehana K
RK
Hochendoner
Sarah
S
Peña
Karina
K
Quiroz
Marisol
M
Hong
Weimin C
WC
Dorostkar
Mario M
MM
Janz
Roger
R
Sitte
Harald H
HH
Torres
Gonzalo E
GE
D000818:Animals; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats; D013572:Synaptic Vesicles / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D062805:Synaptogyrins; D014162:Transfection
D050483:Dopamine Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C540370:SYNGR3 protein, human; D062805:Synaptogyrins