UEV-1 and Glutamate Receptors Figure 2. UEV-1 is a member of the E2 ubiquitin-conjugating enzyme variant family. (A) The intron/exon structure of uev-1 based on sequenced cDNAs is shown in the top panel. Gray boxes indicate exonic coding sequences. The arrow indicates the start of transcription. The site of the od10 molecular lesion is indicated. Predicted alpha helices and beta sheets, based on alignment with human MMS2, are indicated [99]. The purple line indicates the sequences that are removed by the ok2610 deletion. The bottom panel shows the amino acid alignment of C. elegans (C.e.) uev-1 with its two putative homologs in humans (H.s.), UBE2V1/UEV1 and UBE2V2/MMS2, along with Mms2 from yeast (S.c.). Black highlighting indicates identity, and gray indicates similarity. The locations of the F8 residue (mutated in our binding constructs) and the residue altered in od10 to nonsense are indicated. Predicted alpha helices and beta sheets are indicated below the aligned sequence. (B–E) GLR-1::GFP fluorescence in the ventral cord from (B) wild type, (C) uev-1(od10) homozygotes, (D) uev-1(od10) containing the Pglr-1::uev-1(+) transgene, and (E) uev-1(ok2610) homozygotes. The mean number of (F) GLR-1::GFP puncta and (G) GLR-1::GFP accretions per 100 micron of ventral cord length is indicated for the given genotypes. Cell autonomous expression of wild-type uev-1 via the glr-1 promoter is sufficient to rescue uev-1 mutants. Bar, 5 microns. Error bars are SEM. N = 20–30 animals for each genotype. **P,0.01 by ANOVA with Dunnett’s multiple comparison to wild type. doi:10.1371/journal.pone.0014291.g002 molecular nature of these alleles, we sequenced genomic DNA from both uev-1 mutants. The od10 mutation alters the UEV-1 protein sequence from an arginine to an opal stop codon at amino acid 70, while the ok2610 deletion removes an approximately 500 base pair region that includes the first two exons of uev-1 and the beginning of the third (Figure 2A and Figure S1). We hypothesized that the phenotype observed in od10 homozygotes was likely due to a complete or severe loss of UEV-1 function, as half of the C-terminus of the protein is predicted to be absent. To confirm that the uev-1(od10) phenotype is due to loss of UEV-1 function, we also examined GLR-1 localization in uev-1(ok2610) and found that it was virtually identical in appearance, number of puncta, and number of accretions observed in uev-1(od10) mutants (Figure 2C, E, F, and G). Since uev-1(ok2610) mutants are deleted for DNA encoding the N-terminus of UEV-1, as well as the promoter and start of transcription, we conclude that both od10 and ok2610 are likely to be alleles of uev-1. UEV-1 is broadly expressed and affects motoneuron development In addition to the GLR-1 mislocalization phenotype, we observed that uev-1 mutants have a locomotion defect, flexing their bodies more than wild-type nematodes as they move, suggesting that UEV-1 might function in multiple neuron types. To determine where UEV-1 might function, we created a transgenic reporter containing 2 kilobases of upstream uev-1 promoter sequences and the entire uev-1 transcription unit fused in frame to GFP. We introduced the resulting Puev-1::uev-1::gfp reporter into the C. elegans germline, and observed that UEV-1 is broadly expressed in the pharynx, neurons, muscle cells, vulva, embryos, intestine, and in the anus/tail (Figure 3A–F). We found strong expression in the distal tip cell (Figure 3B), which is important in development for gonad morphology [65], but upon examination we found that the gonads of uev-1 mutant worms were normal in shape (data not shown). We also found expression in the motoneurons that line the ventral cord (Figure 3B, D, F). We noted that the UEV-1::GFP chimeric protein is localized diffusely throughout the cytosol of most cells, but with some enrichment in the nucleus (Figure 3D–F). To examine UEV-1 subcellular localization specifically in the command interneurons, we generated a transgene containing the glr-1 promoter sequences driving sequences encoding the fluores- cent protein mCherry fused to the complete UEV-1 reading frame sequences. We introduced the Pglr-1::mCherry::uev-1 transgene into nematodes expressing GLR-1::GFP, and observed that mCher- ry::UEV-1 protein is enriched in neuronal nuclei (Figure 3G-I). As observed in the cytosol for UEV-1::GFP, we find that mCher- ry::UEV-1 is diffusely distributed throughout ventral cord neurites (Figure 3J–L). Given the locomotion defect and the motoneuron expression, we hypothesized that UEV-1 might play a role in regulating motoneuron synapse formation. We examined the subcellular localization of SNB-1 (synaptobrevin) using a transgene, juIs1[Punc- 25::SNB-1::GFP], that expresses a SNB-1::GFP chimeric protein in the GABAergic motor neurons [66]. In wild-type animals, SNB- 1::GFP is localized to large neuromuscular junction (NMJ) boutons regularly spaced along both the dorsal and ventral cords (Figure 4A) [66]. In uev-1(od10) mutants, we observed a decreased number of SNB-1::GFP NMJ boutons and an irregularity in interbouton spacing, similar to the phenotype observed in rpm-1 mutants (Figure 4B, C) [67]. We quantified the number of dorsal cord SNB-1::GFP boutons in uev-1(od10) mutants, and found it to be significantly less than that seen in wild-type animals, although not as severely depressed as in rpm-1 nulls (Figure 4E). RPM-1 is an E3 ubiquitin ligase that directly ubiquitinates the DLK-1 kinase, which is part of the PMK-3/p38 MAPK signaling pathway. RPM-1 is thought to regulate presynaptic bouton formation by repressing PMK-3 activity [68]. RPM-1 also acts in the command interneurons to regulate GLR-1 endocytosis by repressing PMK-3 activity [56]. Indeed, both the motoneuron NMJ phenotype and the interneuron GLR-1-trafficking pheno- type observed in rpm-1 mutants are suppressed by loss of function mutations in pmk-3 [56,68]. We tested if either the NMJ bouton defect or the GLR-1 accumulation defect is suppressed in animals mutated for both pmk-3 and uev-1, but detected no suppression of either defect (Figure 4D–F). We conclude that UEV-1 can regulate either presynaptic or postsynaptic differentiation depending on the specific neuron type, similar to what has been observed for RPM- 1. However, the regulatory targets of UEV-1 are likely to be distinct from those of RPM-1. UEV-1 regulates GLR-1 trafficking independent of the ERAD pathway One possible explanation for the accumulation of GLR-1 in uev- 1 mutants could be that UEV-1 contributes to the Endoplasmic Reticulum Associated Protein Degradation (ERAD) pathway. This pathway targets misfolded proteins in the ER for ubiquitination, export into the cytoplasm, and subsequent degradation by the 26S proteasome [69–71]. Consistent with this possibility, we also observed that the cell bodies of uev-1 mutants have higher levels of GLR-1::GFP than those observed in wild-type animals (Figure 5A, C, K). A closely related pathway, the Unfolded Protein Response (UPR), regulates the expression of ER resident chaperones that assist in dealing with ER stress [72,73]. GLR-1 requires proper UPR signaling, even in the absence of stress, to exit the ER [74]. In mutants for the UPR gene ire-1, GLR-1::GFP becomes trapped in the ER, visible as a ‘‘donut’’ of fluorescence (Figure 5E) surrounding neuronal nuclei, while very little GLR-1 is found in puncta along the ventral nerve cord (Figure 5F) [74]. In addition, the level of GLR-1 in the cell bodies of ire-1 mutants is lower than that in wild-type cell bodies, suggesting that much of the ER- trapped receptor in ire-1 mutants is removed by ERAD [74,75]. PLoS ONE | www.plosone.org 5 December 2010 | Volume 5 | Issue 12 | e14291