V-ATPase function is required for apical membrane protein delivery. (A– D ) Identification of aa24.2^pd1209 as a splice site mutation in atp6ap1b of the V-ATPase complex. SNPtrack linkage analysis (A) and positional cloning (B) of the aa24.2^pd1209 mutation affecting the splice donor site of exon 9 of atp6ap1b (C). Rec, recombinants; TM, transmembrane. (D) RT-PCR showing decay of the atp6ap1b mutated transcript. (E) Illustration of the V-ATPase complex. (F)aa24.2^pd1209 mutants show decreased labeling of acidic organelles; aberrant vacuoles are not acidified (arrows). Larvae were gavaged with LysoTracker and processed within 30 min for confocal microscopy. n = 8 mutants in three independent experiments. (G and H) All V-ATPase subunits analyzed showed variable defects in apical membrane protein trafficking and sorting. (G) Genetic analysis of p75-GFP localization in V-ATPase subunit mutants. Plus signs indicate relative penetrance for each phenotype. (H) p75-GFP localization in different V₀ and V₁ sector V-ATPase mutants. WT larvae treated with 250 nM bafilomycin for 16-h phenocopy p75-GFP localization of V-ATPase mutants. Arrows point to vacuolar accumulation; arrowheads show basolateral missorting. n > 10 larvae of each genotype/treatment from at least two independent experiments. Scale bars are 10 µm. (I and J) p75-GFP localization in the pronephros of aa24.2^pd1209 mutants and bafilomycin-treated larvae. n > 3 larvae of each genotype/treatment from at least two independent experiments. Scale bars are 5 µm.