Abstract
The regulation of AMPA-type glutamate receptor (AMPAR) membrane trafficking is a key mechanism by which neurons regulate synaptic strength and plasticity. AMPAR trafficking is modulated through a combination of receptor phosphorylation, ubiquitination, endocytosis, and recycling, yet the factors that mediate these processes are just beginning to be uncovered. Here we identify the ubiquitin-conjugating enzyme variant UEV-1 as a regulator of AMPAR trafficking in vivo. We identified mutations in uev-1 in a genetic screen for mutants with altered trafficking of the AMPAR subunit GLR-1 in C. elegans interneurons. Loss of uev-1 activity results in the accumulation of GLR-1 in elongated accretions in neuron cell bodies and along the ventral cord neurites. Mutants also have a corresponding behavioral defect—a decrease in spontaneous reversals in locomotion—consistent with diminished GLR-1 function. The localization of other synaptic proteins in uev-1-mutant interneurons appears normal, indicating that the GLR-1 trafficking defects are not due to gross deficiencies in synapse formation or overall protein trafficking. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants, and that receptors arrive at these endosomes independent of clathrin-mediated endocytosis. UEV-1 homologs in other species bind to the ubiquitin-conjugating enzyme Ubc13 to create K63-linked polyubiquitin chains on substrate proteins. We find that whereas UEV-1 can interact with C. elegans UBC-13, global levels of K63-linked ubiquitination throughout nematodes appear to be unaffected in uev-1 mutants, even though UEV-1 is broadly expressed in most tissues. Nevertheless, ubc-13 mutants are similar in phenotype to uev-1 mutants, suggesting that the two proteins do work together to regulate GLR-1 trafficking. Our results suggest that UEV-1 could regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking.
Highlights
Excitatory synaptic communication in the central nervous system is mediated by the neurotransmitter glutamate and the glutamate receptor ion channels that receive and propagate glutamatergic signaling at the postsynaptic membrane [1]
We have identified an in vivo role for UEV-1 in a metazoan, C. elegans, in regulating the trafficking of an AMPA-type glutamate receptor subunit, GLR-1
We find that while UEV-1 is broadly expressed in C. elegans cells and tissues, it causes a specific defect in the localization of GLR-1, which can be rescued cell autonomously in GLR-1-expressing interneurons by expression of a uev-1 cDNA
Summary
Excitatory synaptic communication in the central nervous system is mediated by the neurotransmitter glutamate and the glutamate receptor ion channels that receive and propagate glutamatergic signaling at the postsynaptic membrane [1]. The trafficking of glutamate receptors, AMPA-type receptors (AMPARs), in and out of the postsynaptic membrane is emerging as a key mechanism underlying synaptic and behavioral plasticity [5,7]. AMPARs are tetrameric [10], and each subunit has cytoplasmic tail sequences that determine its subcellular trafficking [11,12,13,14,15,16]. These tail sequences direct both AMPAR endocytosis and exocytosis in response to neural activity by interacting with kinases and phosphatases, PDZ scaffolding molecules, and the endocytosis machinery [2,17,18]
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