does not suppress the low reversal rate observed in uev-1 mutants containing wild-type GLR-1::GFP (data not shown). Taken together, these data indicate that (1) preventing the ubiquitination of GLR-1 tails cannot mimic the uev-1 mutant phenotype, and (2) the ubiquitination of GLR-1 itself does not seem to alter the internalized accumulation of receptors in uev-1 mutants dramat- ically. Thus, UEV-1 probably regulates the ubiquitination of other proteins that in turn regulate GLR-1 trafficking. UEV-1 might function by regulating the K63-linked ubiquitination state of one or more substrates UEV-1 is similar to other UEV proteins in a variety of organisms (Figure 2A), including the ESCRT protein Vps23 [33,85]. ESCRT proteins are important for routing ubiquitinated receptors from the endosome to multivesicular bodies and lysosomes, and Vps23 is a subunit of ESCRT-1, which binds ubiquitinated receptors in the early endosome [36]. We reasoned that if UEV-1 is working as part of the ESCRT complex, then mutants for homologs of other ESCRT complex genes or related vesicular trafficking proteins might have a GLR-1::GFP localiza- tion phenotype similar to that seen in uev-1. However, when we introduced Pglr-1::glr-1::gfp into several of these mutants, including alx-1 [86] and stam-1 [87,88], we found that GLR-1:GFP localization was similar to that found in wild type (data not shown). UEV proteins in other organisms can function by forming heterodimers with Ubc13 so as to catalyze the formation of K63- linked polyubiquitin chains. We performed two different assays to test whether C. elegans UEV-1 and UBC-13 can physically interact. First, we expressed either GST or GST::UBC-13 in bacteria, and then tested the ability of these proteins when bound to glutathione agarose beads to pull down GFP-tagged UEV-1 from lysates of transfected COS7 cells. We found that GST::UBC-13 specifically pulls down GFP::UEV-1 (Figure 9A). The binding between Mms2 (a yeast homolog of UEV-1) and Ubc13 can be disrupted by mutating the eighth residue (Phenylalanine) of Mms2 to Alanine [89]. We introduced this change into the corresponding residue of GFP::UEV-1, and found that it abolished the interaction with GST::UBC-13 (Figure 9A). Similar results were observed in five different pull down experiments. Second, we introduced UBC-13 and UEV-1 into bait and prey vectors for yeast two-hybrid, using growth on –Leu plates as an indicator of an interaction. We found that wild-type UEV-1, but not UEV-1 with the F8A mutation, interacted with UBC-13 regardless of which protein was bait and which was prey (Figure 9A). Our results indicate that C. elegans UEV-1 and UBC-13 can interact in a similar fashion as their homologs in other species do. Based on the subtle phenotypes and overall viability and fertility of uev-1 mutants, we reasoned that UEV-1 might have a smaller, more selective set of targets for K63-linked ubiquitination than would UBC-13, and that other proteins might function with UBC- 13 to conduct most K63-linked ubiquitination events in nema- todes. To test this possibility, we generated lysates of well-fed, mixed stage nematodes from either wild type or uev-1 mutants, and then separated the proteins from these lysates by SDS-PAGE. We used an antibody specific for K63-linked polyubiquitin chains to probe these lysates on Western blots [90]. As controls for specificity, we used SDS-PAGE to separate pure K63-linked and K48-linked tetraubiquitin proteins on the same gels. The anti- K63-linked ubiquitin antibody detected multiple bands of various sizes and quantities (Figure 9B). Interestingly, we found the same pattern of K63-linked ubiquitinated proteins in both wild-type and uev-1-mutant lysates. Importantly, the antibody detected K63- linked but not K48-linked tetraubiquitin control proteins, supporting its specificity. Similar results were found in five UEV-1 and Glutamate Receptors independent immunoblots. Our results indicate that UEV-1 does not affect the overall levels of K63-linked polyubiquitination in C. elegans. A viable deletion mutant for C. elegans ubc-13 has recently been identified (Figure S5; [91]). To directly test whether UEV-1 might regulate GLR-1 trafficking by working with UBC-13, we introduced the Pglr-1::glr-1::gfp transgene into ubc-13 mutants. Similar to uev-1 mutants, ubc-13 mutants accumulate GLR- 1::GFP in their neuron cell bodies and proximal neurites (Figure 9C-F). In addition, double mutants for uev-1 and ubc-13 show quantitatively and qualitatively similar phenotypes compared to both single mutants (Figure 9C-F and data not shown), suggesting that UEV-1 and UBC-13 might work as a heterodimer to regulate GLR-1 trafficking, most likely by mediating K63-linked polyubiquitination. Given that we did not observe changes in global levels of K63-linked polyubiquitinated proteins in uev-1 mutants, we speculate that UEV-1 controls the ubiquitination of a small number of specific substrates (present at low enough levels to escape detection by our anti-K63 Western analysis), and that one or more of these substrates has a key role in regulating GLR-1 trafficking out of RAB-10-containing endosomes. Discussion 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 autono- mously in GLR-1-expressing interneurons by expression of a uev-1 cDNA. Our evidence indicates that UEV-1 likely does not function as part of the ERAD pathway, but in determining this we uncovered a strong spatial preference for where GLR-1 accretions form along the ventral cord of C. elegans in uev-1 mutants. 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 found in other species are thought to function by binding to Ubc13, with the resulting heterodimers forming K63-linked ubiquitin chains. We find that whereas UEV- 1 can interact with C. elegans UBC-13, global levels of K63-linked ubiquitination seem unaffected in uev-1 mutants. Nevertheless, UBC-13, like UEV-1, is required to regulate GLR-1 trafficking. Our results suggest that UEV-1 and UBC-13 could regulate a small subset of K63-linked ubiquitination events in nematodes, at least one of which is critical in regulating GLR-1 trafficking. UEV-1 might work in conjunction with UBC-13 in C. elegans to regulate the K63-linked ubiquitination state of one or more substrates UEV-1 is similar in sequence to other proteins in the highly conserved UEV family, which are similar in sequence and structure to the ubiquitin E2 conjugating enzymes, but lack the catalytic cysteine [31–35]. UEV family members have been implicated both in generating K63-linked ubiquitin chains, as well as in the recognition and binding of previously ubiquitinated substrate proteins. For example, UEV-1 is similar to TSG101, itself a homolog of the yeast ESCRT protein Vps23 [33,85]. ESCRT proteins function to route ubiquitinated receptors from the early endosome to multivesicular bodies and eventually to lysosomes, and Vps23 is a subunit of ESCRT-1, which binds ubiquitinated receptors in the early endosome [36]. Therefore, one possible mechanism of action for UEV-1 in regulating GLR-1 trafficking could be by functioning as part of the ESCRT complex. PLoS ONE | www.plosone.org 13 December 2010 | Volume 5 | Issue 12 | e14291