UEV-1 and Glutamate Receptors Figure 7. UEV-1-dependent trafficking of GLR-1 is independent of clathrin-mediated endocytosis. (A-H) GLR-1::GFP ventral cord fluorescence is shown for the indicated genotypes: (A) wild type, (B) uev-1, (C) itsn-1, (D) itsn-1 uev-1, (E) lin-10, (F) lin-10 uev-1, (G) rab-10, and (H) rab- 10 uev-1. The uev-1(ok2610) allele was used in these experiments. Mean (I, K) size and (J, L) number of GLR-1::GFP accretions observed along the ventral cord of the indicated genotypes. Bar, 5 microns. Error bars are SEM. N = 20–30 animals for each genotype. ***P,0.001, **P,0.01, *P,0.05 by ANOVA with Bonferroni multiple comparison tests. For (K) and (L), only comparisons between double mutants and their corresponding single mutants are shown for clarity. doi:10.1371/journal.pone.0014291.g007 GLR-1 ubiquitination occurs at four critical lysines on the intracellular tail of the receptor, and expression of GLR- 1(4KR)::GFP, a mutant version in which each of the four lysine residues has been changed to arginine to preclude direct receptor ubiquitination, results in an increase in the number and size of GLR-1 puncta along the ventral cord because of decreased endocytosis (Figure 8E) [21]. There is also a slight increase in the number of accretions relative to wild type (Figure 8I). We introduced the Pglr-1::glr-1(4kr)::gfp transgene into uev-1 mutants to determine whether the phenotype of uev-1 was directly dependent on receptor ubiquitination. We found that GLR-1(4KR)::GFP, like wild-type GLR-1::GFP, accumulates in accretions in uev-1 mutants (Figure 8F, I, J), although with some reduction in number. Furthermore, expression of GLR-1(4KR)::GFP in uev-1 mutants Figure 8. UEV-1 and ubiquitin-mediated turnover of GLR-1. (A–D) GLR-1::GFP ventral cord fluorescence in (A) wild type, (B) uev-1(od10) mutants, (C) wild type that overexpress free ubiquitin, and (D) uev-1(od10) mutants that overexpress free ubiquitin. (E, F) GLR-1(4KR)::GFP ventral cord fluorescence in (E) wild type and (F) uev-1(od10) mutants. The mean (G, I) number and (H, J) size of GLR-1::GFP accretions is plotted for the indicated genotypes. In (I–J), ‘‘GLR-1(+)’’ indicates the presence of the wild-type GLR-1::GFP-expressing transgene, whereas ‘‘GLR-1(4KR)’’ indicates the presence of the GLR-1(4KR)::GFP-expressing transgene; GLR-1(4KR)::GFP cannot be ubiquitinated. Bar, 5 microns. Error bars are SEM. N = 20–30 animals for each genotype. ***P,0.001 by ANOVA with Bonferroni multiple comparison tests. doi:10.1371/journal.pone.0014291.g008 PLoS ONE | www.plosone.org 12 December 2010 | Volume 5 | Issue 12 | e14291