Figure 3.

Loss of V-ATPase function impairs apical but not basolateral membrane protein delivery. (A–D) Schematic and localization of O- and N-glycosylated apical membrane proteins in aa24.2^pd1209 mutants. Mutants retain apical membrane proteins in aberrant apical vacuoles, marked with Lamp2-RFP. Arrows point to vacuoles; arrowheads to basolateral missorting. n ≥ 7 mutants in at least two independent clutches for A–C; n = 3 mutants for D. Scale bars are 10 µm. C, C-terminus; EC, extracellular; Glyc, glycan; IC, intracellular; N, N-terminus; TMD, transmembrane domain. (E) 0.6-µm depth 2D projection (two optical slices) of p75-GFP/Lamp2-RFP vacuoles (top) and corresponding 7.2-µm depth 3D reconstruction (bottom). p75-GFP shows a tubular appearance in addition to vacuolar retention in aa24.2^pd1209 mutants (arrows). Data are from z-series taken for experiments in A. Scale bars are 2 µm. (F–J) Basolateral membrane proteins are not retained in vacuoles (arrows) upon loss of V-ATPase function. Endogenously expressed E-cadherin-YFP (F) and Na⁺K⁺ ATPase (G) do not accumulate in vacuoles in aa24.2^pd1209 mutants. n > 10 mutants from three independent experiments for F and G. Overexpressed MICA-GFP (H) and Aqp3-GFP (I) do not accumulate intracellularly, as with p75-GFP (J), in larvae treated with 500 nM bafilomycin for 4 h. n ≥ 6 larvae per condition for H–J. Scale bars are 10 µm.