to a substrate protein [24]. Typically this occurs through the formation of a covalent link between the carboxy-terminus of the incoming ubiquitin and the lysine 48 (K48) residue of the final ubiquitin in the chain. Once four or more ubiquitins are added, the polyubiquitinated protein becomes a degradation substrate for the 26S proteasome. Polyubiquitin chains can also be formed through linkages other than K48. For example, polyubiquitin chains can be assembled through K63 linkages. K63-linked polyubiquitination is generally not thought to target substrates to the proteasome; rather, it appears to have a more regulatory role [25]. For example, K63- ubiquitination has emerged as an additional mechanism by which endocytosed proteins become shunted to Multivesicular Bodies (MVBs) for degradation [26–30]. One family of proteins implicated in K63-linked ubiquitination is the UEV protein family. UEV proteins share sequence similarity with ubiquitin E2 conjugating enzymes, but lack the catalytic cysteine present in functional E2 enzymes [31–35]. One well-studied UEV protein is the ESCRT complex subunit Vps23, which routes ubiquitinated membrane receptors from the endosome to MVBs and eventually to lysosomes for degradation [36,37]. Other known UEV proteins form heterodimers with the E2 enzyme Ubc13 to catalyze the formation of K63-linked polyubiquitin chains [38]. UEV/Ubc13 heterodimers are implicated in several biological processes, including the immune response [39], lysosomal targeting [27,30,40,41], the formation of protein inclusions [42,43], DNA repair [44], protein transport [45], and oncogenic transformation [46]. While UEV proteins have been examined extensively in yeast and in mammalian cell culture, little is known about their function in vivo in metazoans. Moreover, it is unknown whether K63-linked ubiquitination plays a role in AMPAR trafficking. The role of ubiquitination in AMPAR trafficking can be studied in vivo in the model organism C. elegans. The C. elegans genome contains two AMPA-type subunits: GLR-1 and GLR-2 [47–49]. Fluorescently tagged versions of GLR-1 are functional and localized to postsynaptic elements when expressed in nematodes [50]. GLR-1 is required in the interneuron circuit that regulates backward movement and direction reversal, a key element in the foraging behavior of animals. Mutants with depressed GLR-1 synaptic levels rarely reverse direction, whereas mutants with elevated synaptic levels reverse direction with increased frequency [47–49,51,52]. The synaptic abundance of GLR-1 is regulated by a combination of clathrin-dependent endocytosis [21,53,54] and clathrin-independent endocytosis [55]. Ubiquitination plays sev- eral critical roles in regulating GLR-1 trafficking, including the direct ubiquitination of GLR-1 itself on four critical lysines, as well as the ubiquitination of several of the key kinases and signaling molecules that regulate GLR-1 trafficking [21,53,56,57]. Whereas K63-linked ubiquitination specifically has not been implicated in GLR-1 regulation, the C. elegans genome does encode several UEV proteins [58], including UEV-1, which can interact in vitro with UBC-13 [59] and has been implicated in regulating polyglutamine aggregates in C. elegans [60]. Here we identify UEV-1 as a regulator of AMPAR trafficking. We show that uev-1 mutants have defects in GLR-1 localization and GLR-1-mediated behavior that are consistent with an intracellular endosomal accumulation of GLR-1. UEV-1 is expressed broadly in most tissues, yet regulates GLR-1 trafficking cell autonomously in the command interneurons. The localization of other synaptic proteins to the central synapses of these neurons appears unaffected in uev-1 mutants. We provide evidence that GLR-1 receptors accumulate in endosomes in uev-1 mutants via clathrin-independent endocytosis, but likely cannot escape such endosomes to either recycle back to the synaptic membrane or be UEV-1 and Glutamate Receptors transported for degradation to MVBs and lysosomes. We show that UEV-1 can bind to C. elegans UBC-13, and that ubc-13 mutants have a similar phenotype to uev-1 mutants. Our results suggest that UEV-1 and UBC-13 function together in C. elegans to regulate GLR-1 trafficking, but that UEV-1 is unlikely to be the sole mediator of global K63-linked polyubiquitination in C. elegans. Results GLR-1 accumulates in uev-1 mutants We performed a genetic screen for mutants with defects in the localization of GLR-1 receptors tagged with green fluorescent protein (GLR-1::GFP). GLR-1::GFP receptors are normally localized to postsynaptic clusters at neuron–neuron synapses within C. elegans ventral cord neurites [21,50,61]. In wild-type animals, these receptor clusters are generally seen as small (,0.5– 0.7 micron) puncta (Figure 1A). We identified an allele, od10, of a gene that we determined to be uev-1 (please see Materials and Methods for details on mapping and cloning). Mutants for uev- 1(od10) accumulate large, elongated accretions (,4–6 microns) of GLR-1::GFP, especially in the retrovesicular ganglion region of the ventral nerve cord immediately posterior to the nerve ring (Figure 1B). The average size of puncta and accretions taken together in uev-1 mutants is twice that of wild-type animals (Figure 1I), and there is a slight decrease in the number of GLR-1 puncta (normalized for neurite length; Figure 1J). We also analyzed GLR-1 accretions alone by identifying them based on their size and elongated morphology. Whereas wild-type animals nearly lack these accretions, uev-1 mutants have significant numbers of them (Figure 2G). These results indicate that UEV-1 is involved in the regulation of GLR-1-containing glutamate receptors in ventral cord neurites. The change in the pattern of GLR-1 localization could be a consequence of uev-1 mutants having general defects in protein trafficking or in synapse formation. To test this possibility, we examined the localization of other synaptic proteins: SNB-1 (synaptobrevin), UNC-43 (CaMKII), and LIN-10 (Mint2). We used transgenes that express GFP-tagged versions of these proteins using the glr-1 promoter [50,61-63]. We introduced these transgenes into wild type and uev-1 mutants to observe in their ventral cord neurites the subcellular localization of the proteins they encode. SNB-1::GFP is localized to presynaptic terminals [50,64], and we found no significant change in the size or number of SNB-1::GFP-labeled terminals in uev-1 mutants compared with wild type (Figure 1, C, D, I, and J). UNC-43::GFP and LIN- 10::GFP colocalize with GLR-1 at postsynaptic elements in the ventral cord [61-63], and we found no significant difference in the size or number of either LIN-10::GFP (Figure 1, E, F, I, and J) or UNC-43::GFP (Figure 1, G–J) puncta in uev-1 mutants compared to wild type. These results lead us to conclude that the aberrant accumulation of GLR-1 in uev-1 mutants is not because of gross defects in synaptic protein trafficking or in the formation of synapses to GLR-1-expressing neurons. In addition, because the glr-1 promoter was used to express all of these synaptic proteins, it is unlikely that the defects observed in uev-1 mutants are due to an elevation in glr-1 transcription. UEV-1 encodes a member of the UEV family of proteins The uev-1 gene contains three exons that encode a 139 amino acid protein in the UEV family of proteins. UEV proteins are well conserved across species as diverse as yeast (S. cerevisiae), nematodes (C. elegans), and humans (Figure 2A) [31–34]. We identified od10 as an allele of uev-1 by genetic mapping and transformation rescue using the F39B2 cosmid, which contains uev-1 under its own PLoS ONE | www.plosone.org 2 December 2010 | Volume 5 | Issue 12 | e14291