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. 2020 Jul 28;9:e55596. doi: 10.7554/eLife.55596

Figure 6. ER recruitment of NGLY1 is required for BMP4 deglycosylation and signaling in MEFs and Dpp signaling in Drosophila embryonic midgut.

(A) Western blots with V5, HA and pSMAD1/5 antibodies on cell lysates from Ngly1–/– cells expressing BMP4HA-Myc alone or with human NGLY1V5-His6 (wild-type or VCP-binding mutants). Wild-type NGLY1 efficiently deglycosylates BMP4 (downward shift of the HA+ band) and induces robust BMP signaling (pSMAD1/5), but NGLY1 with mutations in its VCP-binding sites fail to deglycosylate BMP4 and induce BMP signaling. (B) Projection views of confocal image stacks of stage 14 embryos with indicated genotypes stained for pMad. Expression of wild-type human NGLY1 in mesoderm rescues BMP signaling in the midgut region (arrowheads), but the VCP-binding mutant versions of human NGLY1 do not rescue this phenotype. (C) Coomassie Brilliant Blue (CBB) stain of recombinant wild-type and VCP binding mutant (N41P and G79A/F80A) NGLY1V5-His6 proteins generated in wheat germ cell-free system and deglycosylation activity assays on these proteins. Note that the VCP-binding mutants are stably expressed (arrowhead) and do not decrease NGLY1’s enzymatic activity. One-way ANOVA with Tukey's multiple comparisons test was used. Mean ± s.d. **p<0.01; NS, not significant. (D) Western blot of HA-tagged BMP4 from cell lysates of WT and Ngly1–/– MEFs treated with bortezomib (BTZ, 10 nM for 6 hr). (E) Quantification of deglycosylated HA-tagged BMP4 expression. n = 3 biologically independent samples. Two-way ANOVA test with Tukey's multiple comparisons test was used for statistical analysis. Mean ± s.d. ***p<0.001. These results indicate that deglycosylated BMP4 is degraded by proteasome as result of ER-to-cytosol retrotranslocation. The proteasome impairment of BMP4 degradation does not block BMP signaling.

Figure 6—source data 1. Raw data and statistical analysis for panels C and E.

Figure 6.

Figure 6—figure supplement 1. Transgenic expression of human NGLY1 in Drosophila wing discs.

Figure 6—figure supplement 1.

Shown are third instar wing imaginal discs that express wild-type human NGLY1 and NGLY1 with mutations in VCP-binding sites (N41P and G79A/F80A) using the dpp-GAL4 driver. NGLY1 is marked in red; the dpp expression domain is marked in green by using UAS-CD8::GFP in the background. n = 5 for each genotype. Scale bar, 25 μm. Note that VCP-binding mutant versions of NGLY1 are expressed in vivo but show a pattern different from that of wild-type NGLY1.