Suppression of retinal degeneration by two novel ERAD ubiquitin E3 ligases SORDD1/2 in Drosophila
Fig 2
The E3 ubiquitin ligases SORDD1/2 reduce the UPR by promoting the degradation of Rh1G69D.
(a) Fluorescence images of eye imaginal discs. The ER stress markers, Xbp1-EGFP and ATF4-mCherry (control, GMR-gla4 UAS-Xbp1 ATF4-mCherry/+), were induced by the expression of Rh1G69D–Myc (Rh1G69D–Myc, GMR-gal4 UAS-Xbp1 ATF4-mCherry UAS-Rh1G69D–Myc/+). Expressing SORDD1 (Rh1G69D–Myc+sordd1, GMR-gla4 UAS-Xbp1 ATF4-mCherry UAS-Rh1G69D–Myc/+;UAS-sordd1/+) or SORDD2 (Rh1G69D–Myc+sordd2, GMR-gla4 UAS-Xbp1 ATF4-mCherry UAS-Rh1G69D–Myc/+;UAS-sordd2/+) reduced Rh1G69D–Myc levels as well as Xbp1-EGFP and ATF4-mCherry signals. In contrast, expressing CG8141 (Rh1G69D–Myc+CG8141, GMR-gla4 UAS-Xbp1 ATF4-mCherry UAS-Rh1G69D–Myc/+;UAS-CG8141/+) did not affect levels of Rh1G69D–Myc, Xbp1-EGFP, or ATF4-mCherry. The Rh1G69D–Myc epitope is labeled white; spliced Xbp1-GFP is labeled green; mCherry under control of the ATF4 promoter/5’ UTR is labeled red. Scale bar, 50 μm. (b) Quantification of relative fluorescence from a; error bars indicate SEM (n = 4); ns, not significant; ****p<0.0001 (one-way ANOVA, Sidak's multiple comparisons test). (c) Western blot analysis shows the level of Rh1G69D–Myc is reduced by expressing SORDD1 and SORDD2 in pupa eyes. Note Rh1G69D can form a dimer. (d) Quantification of b, error bars indicate SEM; n = 3, **p<0.01, ***p<0.001 (one-way ANOVA, Sidak's multiple comparisons test). (e) E3 ubiquitin ligase activity of SORDD1, SORDD2 and HRD1 is essential for suppression of Rh1G69D induced cell degeneration. SORDD1 (sordd1C165S), SORDD2 (sordd2C165S) and HRD1 (hrd1C327S) are inactivated by mutating the conserved Cys on the active site of the RING domain to Ser. Scale bar, 100 μm. (f) Ubiquitination of Rh1G69D by SORDD1 in vivo. Head lysate of Rh1G69D–Myc (GMR-gla4 UAS-Rh1G69D–Myc/rpn12RNAi) flies co-expressing SORDD1 (GMR-gla4 UAS-Rh1G69D–Myc/rpn12RNAi;UAS-sordd1/+) or SORDD1C165S (GMR-gla4 UAS-Rh1G69D–Myc/rpn12RNAi;UAS-sordd1C165S/+) were immunoprecipitated with anti-Myc antibody, and stained against Myc (left) or ubiquitin (right). The proteasomal degradation of Rh1G69D was inhibited by knocking down the proteasome component Rpn12 (rpn12RNAi) [54]. (g) Quantification of relative ubiquitination levels of Rh1G69D–Myc from f, error bars indicate SEM; n = 3, ns, not significant, ****p<0.0001 (one-way ANOVA, Sidak's multiple comparisons test).