UEV-1 Is an Ubiquitin-Conjugating Enzyme Variant That Regulates Glutamate Receptor Trafficking in C. elegans Neurons Lawrence B. Kramer1,4, Jaegal Shim2, Michelle L. Previtera3,4, Nora R. Isack1, Ming-Chih Lee1, Bonnie L. Firestein3, Christopher Rongo 1 * 1 The Waksman Institute, Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America, 2 Cancer Experimental Resources Branch, National Cancer Center, Goyang, Korea, 3 Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America, 4 Graduate Program in Molecular Biosciences, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America 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. Citation: Kramer LB, Shim J, Previtera ML, Isack NR, Lee M-C, et al. (2010) UEV-1 Is an Ubiquitin-Conjugating Enzyme Variant That Regulates Glutamate Receptor Trafficking in C. elegans Neurons. PLoS ONE 5(12): e14291. doi:10.1371/journal.pone.0014291 Editor: Brian D. McCabe, Columbia University, United States of America Received June 2, 2010; Accepted November 18, 2010; Published December 13, 2010 Copyright: 2010 Kramer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The manuscript was funded by a grant from the National Institutes of Health (R01 NS42023) to CGR and a grant from the National Science Foundation (IBN-0548543) to BLF. MLP was supported by the IGERT Program on Biointerfaces, by National Science Foundation grant DGE-0333196, and by a Louis Bevier Dissertation Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rongo@waksman.rutgers.edu Introduction 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]. Glutamatergic synapses show substantial plasticity, becoming dynamically weakened or strengthened in an activity dependent manner [2–6]. The trafficking of glutamate receptors, particularly AMPA-type recep- tors (AMPARs), in and out of the postsynaptic membrane is emerging as a key mechanism underlying synaptic and behavioral plasticity [5,7]. Thus, robust molecular and cellular models for learning and memory will require a full understanding of the various mechanisms and molecules that regulate AMPAR trafficking. AMPAR trafficking involves several steps, including initial delivery to the synaptic membrane, anchoring, endocytosis, and finally sorting either to the lysosomal pathway for degradation or to the synaptic membrane via various recycling pathways [8,9]. AMPARs are tetrameric [10], and each subunit has cytoplasmic tail sequences that determine its subcellular trafficking [11–16]. 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]. The Ubiquitin Proteasome System (UPS) also regulates AMPAR trafficking [18–20]. AMPARs, like many membrane proteins, can be post-translationally modified by the addition of single ubiquitin molecules (monoubiquitination) [21]. Monoubi- quitination often serves as a signal for their internalization [19,22,23]. In addition, polyubiquitination can occur through the addition of new ubiquitin molecules to the final ubiquitin molecule in a growing polyubiquitin chain that is initially attached PLoS ONE | www.plosone.org 1 December 2010 | Volume 5 | Issue 12 | e14291