Luminal acidification is required for distinct steps of apical membrane protein sorting and trafficking. (A) Schematic (adapted and reprinted with permission from the Journal of Cell Biology; Poëa-Guyon et al., 2013) of the mechanisms of action and luminal pH kinetics of V-ATPase inhibitors versus ionophores. (B) Experimental strategy. Immediately upon TGN release, larvae were gavaged with DMSO, 500 nM bafilomycin, 25 µM monensin, or 100 mM NH4Cl, immersed in the same compound, and processed for imaging 1 h later. (C) Ionophore treatment results in apical-basolateral missorting (arrowheads), while bafilomycin treatment leads to subapical accumulation (arrows) after a 1 h TGN release. n ≥ 5 larvae per condition in two independent experiments. Scale bars are 10 µm. (D) Experimental strategy. The intestines of larvae were pretreated with bafilomycin by gavage before p75-GFP induction. After induction, larvae were immersed in bafilomycin and processed for imaging at time points indicated. (E) p75-GFP exhibits apical-basolateral missorting when bafilomycin is applied before its expression and sorting. Arrowheads point to basolateral missorting; arrows point to apical accumulation. n ≥ 8 larvae per condition in two independent experiments. Scale bars are 10 µm.
