Abstract
Exocytotic release of glutamate depends upon loading of the neurotransmitter into synaptic vesicles by vesicular glutamate transporters, VGLUTs. The major isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in synapses of the adult rodent brain that correlates with the probability of release and potential for plasticity. Indeed, expression of different VGLUT protein isoforms confers different properties of release probability. Expression of VGLUT1 or 2 protein also determines the kinetics of synaptic vesicle recycling. To identify molecular determinants that may be related to reported differences in VGLUT trafficking and glutamate release properties, we investigated some of the intrinsic differences between the two isoforms. VGLUT1 and 2 exhibit a high degree of sequence homology, but differ in their N- and C-termini. While the C-termini of VGLUT1 and 2 share a dileucine-like trafficking motif and a proline-, glutamate-, serine-, and threonine-rich PEST domain, only VGLUT1 contains two polyproline domains and a phosphorylation consensus sequence in a region of acidic amino acids. The interaction of a VGLUT1 polyproline domain with the endocytic protein endophilin recruits VGLUT1 to a fast recycling pathway. To identify trans-acting cellular proteins that interact with the distinct motifs found in the C-terminus of VGLUT1, we performed a series of in vitro biochemical screening assays using the region encompassing the polyproline motifs, phosphorylation consensus sites, and PEST domain. We identify interactors that belong to several classes of proteins that modulate cellular function, including actin cytoskeletal adaptors, ubiquitin ligases, and tyrosine kinases. The nature of these interactions suggests novel avenues to investigate the modulation of synaptic vesicle protein recycling.
Highlights
The high frequency of neurotransmitter release observed at many synapses requires mechanisms to recycle synaptic vesicle membrane, proteins, and transmitter locally at the nerve terminal
It requires the recruitment of a clathrin coat by adaptor proteins (APs), the acquisition of curvature mediated by endophilin, epsin and other cytosolic proteins, scission of the nascent vesicle from the plasma membrane orchestrated by dynamin, followed by uncoating triggered by the phosphatidylinositol phosphatase synaptojanin [1,5,6]
We have previously shown that interaction of PP2 with endophilins accelerates VGLUT1 recycling, in a manner dependent on the dileucine-like trafficking motif present in the C-terminus [18]
Summary
The high frequency of neurotransmitter release observed at many synapses requires mechanisms to recycle synaptic vesicle membrane, proteins, and transmitter locally at the nerve terminal. Reformation of synaptic vesicles from the plasma membrane by classical clathrin-mediated endocytosis is very similar to endocytosis occurring in non-neural cells. It requires the recruitment of a clathrin coat by adaptor proteins (APs), the acquisition of curvature mediated by endophilin, epsin and other cytosolic proteins, scission of the nascent vesicle from the plasma membrane orchestrated by dynamin, followed by uncoating triggered by the phosphatidylinositol phosphatase synaptojanin [1,5,6]. Synaptic vesicle recycling is driven by a sequence of protein interactions and enzymatic activities [10,11]
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