Fig. 6

Dx and Su(dx) induce ubiquitination-independent endocytosis of N. A Ubiquitination-assay of N-HA and N^K2R-HA. The variants were co-expressed with Dx or Su(dx) and Flag-tagged Ubiquitin (Flag-Ubi) in S2 cells and then immuno-precipitated with an HA antibody and analysed by Western blotting. N-HA and N^K2R-HA bands were revealed by HA antibody staining. Ubiquitin was revealed by anti-Flag antibody staining. The results show that N-HA, but not N^K2R-HA, was ubiquitinated by Dx, as well as Su(dx), indicating that Dx and Su(dx) ubiquitinate the ICD of N on Ks. B–J Dx and Su(dx) mediated endocytosis of N, N-HA and N^K2R-HA. N-HA and N^K2R-HA were co-expressed with Dx or Su(dx) for 14.5 h under control of hh-GAL4 tub-GAL80ts. Each box shows a Z-projection of (upper panel) and a focal plane at the apical plasma membrane (insert) of late third instar larvae wing imaginal discs. B–D As a control, endogenous YFP-N was monitored in wild-type discs and disc overexpression of Dx (C) and Su(dx) (D). In wild-type cells, N localised at the subapical membrane domain, generating the characteristic honeycomb pattern observed in the corresponding optical plane. C Expression of Dx induced strong endocytosis of N from the apical membrane, indicated by the loss of the apical honeycomb pattern (insert) and increased vesicle formation (upper panel). D In contrast to Dx, Su(dx) had no obvious effect on the subcellular localisation of N. E–G Co-expression of N-HA with Dx or Su(dx). N-HA was predominantly localised at the apical plasma membrane if expressed alone. F, G Co-expression of N-HA with Dx, or Su(dx), resulted in strong decrease of the apical N-HA membrane fraction, indicating that Dx and Su(dx) can induce endocytosis of N-HA from the plasma membrane. H Likewise, N^K2R-HA strongly accumulated at the apical plasma membrane upon its expression. I, J Like in the case of N-HA, co-expression with Dx or Su(dx) induced a strong decrease of the N^K2R-HA membrane fraction, indicating that Dx and Su(dx) can induce endocytosis of N^K2R-HA in a ubiquitin-independent manner. K Quantification of Dx and Su(dx) induced endocytosis of N-HA and N^K2R-HA. For quantification, the HA fluorescence intensity of the apical plasma membrane was compared to that over the apical-basal axis without the plasma membrane (for each genotype n = 3 wing imaginal discs were analysed, significance test: one-way ANOVA p ≥ 0.05 (n.s.) / p ≤ 0.05 (*) / p ≤ 0.01 (**) / p ≤ 0.001 (***) / p ≤ 0.0001 (****)). Dx and Su(dx) can significantly reduce the membrane fraction of N-HA and N^K2R-HA, indicating that Dx and Su(dx) can induce endocytosis of N-HA and N^K2R-HA. Compared to Dx, the ability of Su(dx) to induce endocytosis of N-HA and N^K2R-HA was reduced