Kramer, Lawrence B.. The role of phosphorylation and ubiquitination in the regulation of glutamate receptor trafficking in caenorhabditis elegans. Retrieved from https://doi.org/doi:10.7282/T3833RPT
DescriptionThe regulation of AMPA-type glutamate receptor (AMPAR) 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 in vivo significance of factors
involved in 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, and in a related project find a possible link between AMPAR phosphorylation and ubiquitination-driven internalization. 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 elongated accretions of GLR-1 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. We provide evidence that GLR-1 accumulates at RAB-10-containing endosomes in uev-1 mutants. 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, we find that mutations in ubc-13 result in accumulations of GLR-1 in a similar pattern to that found in uev-1 mutants. Our results suggest that UEV-1 may regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking. In another project, we used a reverse genetics approach to
study the regulation of phosphorylation state on the trafficking of GLR-1. We found that mutation of a single GLR-1 serine (S891) to alanine is sufficient to
strongly reduce GLR-1 puncta in the ventral nerve cord. We also determined that this effect is precluded if the C-terminal portion of GLR-1 is mutated such that it can no longer be ubiquitinated and endocytosed. This suggests that S891 phosphorylation may regulate endocytosis and/or ubiquitination and subsequent
degradation of GLR-1. This may provide a link between ubiquitination and phosphorylation in the trafficking of GLR-1, with possible implications for similar
links in mammalian AMPAR trafficking.