Fig. 8
A di-leucine motif as an alternative endocytosis signal in Notch. A Drosophila N and human N1 share a conserved di-leucine motif in its intracellular domain. B–E Endocytosis of N-HA, NLL2AA-HA, NK2R-HA and NK2R-LL2AA-HA. The N constructs were expressed for 14.5 h under control of hh-GAL4 tub-GAL80 ts. Each picture shows a Z-projection of the A/B axis (upper panel) and a focal plane of the apical plasma membrane (lower panel). Areas of magnification are highlighted by white boxes. Localisation of the transgenic constructs was indicated by HA antibody staining. Replacement of the di-leucine motif resulted in increased accumulation of NLL2AA-HA and NK2R-LL2AA-HA in the apical plasma membrane, indicated by the strong honeycomb structure (lower panels). Despite the increased membrane localisation, NLL2AA-HA and NK2R-LL2AA-HA were present in vesicles (upper panels). F To quantify the impact of the LL2AA mutant on endocytosis, the HA fluorescence intensity of the apical plasma membrane was related to the total HA fluorescence intensity area along the A/B axis, without the plasma membrane signal (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 (****)). The replacement of the di-leucine motif significantly increased the membrane fraction of N-HA, as well as NK2R-HA, indicating that the di-leucine motif mediates N endocytosis in a ubi-independent manner. G The meaning of the di-leucine motif for the activity of N-HA and NK2R-HA. The variants were expressed for 14.5 h under control of hh-GAL4 tub-GAL80 ts. For the quantification the activity, the fluorescence intensity of NRE-GFP in the posterior compartment was normalised to the anterior compartment (wt: n = 9 / N-HA: n = 7 / NK2R-HA: n = 11 / NLL2AA-HA: n = 8 / NK2R-LL2AA-HA: n = 7 wing imaginal discs, significance test: one-way ANOVA p ≥ 0.05 (n.s.) / p ≤ 0.05 (*) / p ≤ 0.01 (**) / p ≤ 0.001 (***) / p ≤ 0.0001 (****)). Despite the observed reduced endocytosis, the di-leucine motif had no significant impact on the activity of N-HA and NK2R-HA