UEV-1 and Glutamate Receptors Figure 9. UEV-1 interacts with UBC-13 to regulate GLR-1. (A) Top left panel shows a Coomasie-stained SDS-PAGE for bacterially produced GST and GST::UBC-13 used in the pull down assay. Top right panel shows a Western blot using anti-GFP antibodies to detect GFP::UEV-1 or GFP::UEV- 1(F8A) pulled down from COS7 lysates using either GST or GST::UBC-13 bound to beads. ‘‘Input’’ indicates 10% of the lysate used in the binding reaction. Arrow indicates the specific GFP::UEV-1 protein. Arrowheads indicate non-specific bands in the lysate that are pulled down by GST::UBC-13 and detected on the Western blot. Bottom panel shows growth on media either selecting for interaction (–Leu –His –Trp) or allowing growth without selection (–His –Trp) for 10 fold serial dilutions of yeast cultures co-expressing the indicated bait and prey plasmids. Similar results were found in 3 independent experiments. (B) Western blots for K63-linked polyubiquitinated proteins or actin as a loading control. Purified tetra-ubiquitin (Ub4) is present on the SDS-PAGE in either the K63-linked form (which runs at around 25 kDa) or the K48-linked form (which runs at around 30 kDa). Coomasie-stained SDS-PAGE for each tetra-ubiquitin protein is also shown. Similar results were found in 5 independent experiments. (C) GLR-1::GFP fluorescence from either cell bodies (left hand panels) or neurites around the retrovesicular region (right hand panels) are shown for the indicated genotypes. Like uev-1 mutants, ubc-13(tm3546) mutants accumulate GLR-1::GFP in their cell bodies and at proximal regions along their ventral cord neurites. Bar, 5 microns. The (D) number of GLR-1::GFP puncta, (E) number of GLR-1::GFP accretions, and (F) size of GLR-1::GFP accretions are indicated for the given genotypes. *P,0.05, **P,0.01, ***P,0.001 by ANOVA with Dunnett’s comparison to wild type. Error bars are SEM. N = 20–30 animals for each genotype. doi:10.1371/journal.pone.0014291.g009 However, mutations in known ESCRT complex genes do not appear to affect GLR-1 trafficking at a gross level. In addition, UEV-1 does not appear to be localized to endosomes, as would be expected for a Vps23 homolog and ESCRT complex protein. Finally, the overall levels of GLR-1 are not different in uev-1 mutants compared with wild type, suggesting that the GLR-1 accretions observed in uev-1 mutants are not simply a result of elevated GLR-1 protein levels, but instead are due to a change in GLR-1 trafficking and subcellular localization. Another possibility is that UEV-1 forms a heterodimer with UBC-13 [38] to catalyze the formation of K63-linked polyubi- quitin chains. In other organisms, K63-linked polyubiquitination is important in several processes, including lysosomal targeting [27,30,40,41], regulating protein inclusions [42,43], and protein transport [45]. Consistent with this possible function, RNAi screens have uncovered a role for UEV-1 and UBC-13 in regulating polyglutamine inclusions in C. elegans [60]. Importantly, we find that a deletion mutation in ubc-13 that results in an early nonsense codon (and therefore is likely a null; Figure S5) causes the same GLR-1 trafficking defects as observed in uev-1 mutants, supporting the idea that a UEV-1/UBC-13 heterodimer complex is regulating GLR-1 trafficking. We find that the levels of K63- linked polyubiquitin are not grossly different in uev-1 mutants compared to the levels found in wild type, suggesting that UEV-1 is not needed for most K63-linked ubiquitination events in nematodes. C. elegans does not contain other homologs of Uev1/ Mms2 in its genome. However, the ubiquitin conjugating enzyme UBC-1 contains a short N-terminal amino acid stretch that is similar to that of UEV-1, and UBC-1 can interact with UBC-13 in a yeast two-hybrid assay [59,92]. UBC-13 can also interact with the E3 ubiquitin ligase NHL-1, and together UBC-13, UBC-1, and NHL-1 can form polyubiquitin conjugates in vitro [59]. Thus, one possible explanation is that UBC-13 conducts most K63- linked ubiquitination while working with UBC-1 rather than when partnered with UEV-1. We examined GLR-1 localization in mutants containing deletions for ubc-1 and nhl-1, but did not observe defects (data not shown). We also generated uev-1 ubc-1 double mutants and found that they were viable and had similar GLR-1 trafficking defects to uev-1 single mutant animals (data not shown). We speculate that UEV-1 regulates the K63-linked ubiquitination of a small number of substrates, one or more of which is involved in GLR-1 trafficking, although we acknowledge that we cannot rule out the alternative possibility that UEV-1 and UBC-13 regulate GLR-1 trafficking by binding ubiquitinated proteins rather than catalytically modifying their K63 linkages. Does UEV-1 regulate the polyubiquitination of GLR-1 subunits directly? We found that uev-1 mutants contain GLR-1 in accretions and possess the same defects in spontaneous reversal frequency even when the four lysines that are normally ubiquitinated on the tail of GLR-1 are mutated to arginine to prevent their ubiquitination. Preventing direct ubiquitination of GLR-1 does not substantially suppress the uev-1 mutant pheno- type. Thus, it seems unlikely that UEV-1 regulates GLR-1 trafficking via direct polyubiquitination of the receptor itself, although we cannot rule out a subtle role for direct ubiquitination of the receptor. Instead, our data suggests that UEV-1 is ubiquitinating additional factors that in turn regulate GLR-1 trafficking. Several E3 ligases regulate GLR-1 trafficking indirectly by ubiquitinating key regulators of trafficking [56,57]. One of these regulators is the PMK-3/p38 MAPK pathway, which is a downstream target of the E3 ligase RPM-1 in both the command interneurons and the motoneurons [56,68]. Mutants for uev-1 have similar defects to rpm-1 mutants with regard to these different neuron types, and mutations in pmk-3 suppress the defects in rpm-1 mutants in both neuron types. However, mutations in pmk-3 do not suppress the GLR-1 trafficking defects or the motoneuron synaptic differentiation defects in uev-1 mutants, suggesting that p38 MAPK signaling is not the target of UEV-1 activity. Interestingly, we detected UEV-1 enrichment in nuclei. K63- linked ubiquitination of histones has been observed in response to DNA damage [93–95]. While we have not found any evidence to suggest that UEV-1 functions in a DNA damage response pathway, it remains possible that UEV-1 regulates GLR-1 by affecting changes in transcription in the nucleus. We do not see an effect of uev-1 on other reporters expressed by the glr-1 promoter or on glr-1 mRNA levels, so it is unlikely that UEV-1 regulates glr-1 transcription; however, we cannot exclude the possibility that UEV-1 regulates the transcription of yet unknown factors that in turn regulate GLR-1 trafficking. UEV-1 controls the exit of GLR-1 from early endosomes A lower frequency of spontaneous reversals in C. elegans has previously been shown to correlate with less GLR-1 in the postsynaptic membrane of GLR-1 expressing neurons [47–49,51]. We found that the rate of reversals was significantly lower in uev-1 mutants than in wild type, suggesting that a large amount of the receptor is in an intracellular compartment. In addition, we found that in uev-1 mutants there is a significant increase in the colocalization of GLR-1::GFP with mRFP::RAB-10, which is associated with early endosomes, suggesting that UEV-1 regulates the flow of GLR-1 receptors into or out of the early endosome. We also found that mutations or transgenes designed to suppress clathrin-dependent endocytosis were not able to suppress the internal accumulation of GLR-1::GFP seen in uev-1 mutants. In addition, we found that the effect of uev-1 mutations on GLR-1 trafficking is additive when combined with lin-10 mutations (in which the recycling of clathrin-dependent cargo is impaired), suggesting that these two genes regulate GLR-1 trafficking by PLoS ONE | www.plosone.org 15 December 2010 | Volume 5 | Issue 12 | e14291