Suppression of retinal degeneration by two novel ERAD ubiquitin E3 ligases SORDD1/2 in Drosophila
Fig 4
SORDD1/2 and Hrd1 function redundantly in promoting Rh1G69D degradation.
(a) Western blot shows Rh1G69D-GFP accumulation in sordd1/21; hrd11 double mutants (ey-flp sordd1/21;FRT82B ninaE-Rh1G69D-GFP hrd11/FRT82B GMR-hid CL), compared to sordd1/21 (sordd1/21;ninaE-Rh1G69D-GFP) and hrd11 (ey-flp;FRT82B ninaE-Rh1G69D-GFP hrd11/FRT82B GMR-hid CL) mutant alone. Rh1G69D-GFP is driven by an endogenous ninaE promoter. The higher molecular weight of Rh1G69D aggregates could be seen at the top of the lanes. (b) Quantification of a, error bars indicate SEM; n = 3, ns, not significant, ****p<0.0001 (ANOVA, Sidak's multiple comparisons test). (c) Quantification of the percentage of the deep pseudopupil in control (ey-flp;FRT82B), sordd1/21, hrd11 (ey-flp;FRT82B hrd11/FRT82B GMR-hid CL) and sordd1/21;hrd11 (ey-flp sordd1/21;FRT82B hrd11/FRT82B GMR-hid CL) flies at 0, 5, 10, 15 and 20 days old. At least ~100 flies in 3 groups were measured for each genotype. Error bars indicate SEM. (d) TEM images of 1-day-old and 20-day-old adult eye tangential sections. Genotypes are as indicated on the left side of the panels. R, rhabdomere. Scale bar, 2 μm. The sordd1/21;hrd11 double mutant flies show severe degeneration of photoreceptor cells at age of 20 days. All flies are in white eye background, and raised under 12 h light /12 h dark cycles. (e) Western blot analysis of Rh1, TRP, and INAD in control, sordd1/21, hrd11, and sordd1/21;hrd11 flies at 3 days old. (f) Quantification of relative protein levels of Rh1, TRP, and INAD in e; error bars indicate SEM (n = 3); ***p<0.001 (one-way ANOVA, Sidak's multiple comparisons test). (g) Proposed model of SORDD1/2 in the degradation of misfolded Rh1G69D parallel to HRD1 complex.