V100 exerts an acidification-independent function on early endosomes. (A–C″) High resolution section of WT (A) and v100 mutant (B and C) 1-d adult photoreceptor terminals expressing pHluorin (green) and immunolabeled for CSP or Syx7/Avl (magenta). Note that pHluorin forms accumulations that exclude CSP (arrowheads) but partially colocalize with Syx7 (arrows) in the mutant. (D–F) Levels of pHluorin accumulation in live adult photoreceptor terminals at equal pH for the genotypes indicated. (G and G′) Negatively marked v100 mutant clone (magenta, WT terminals). Both mutant and control (ctrl) clones equally overexpress pHluorin and v100^R755A. Note that v100^R755A expression only in the mutant terminals leads to pHluorin accumulations. The dotted line approximates the clonal boundary. (H–M) Live confocal scans of P + 20% eye discs before and after 10-min incubation with the v-ATPase inhibitor bafilomycin 1A. (H and I) Control before and after bafilomycin treatment. (J and K) v100 mutant before and after bafilomycin. (L and M) v100 mutant with v100^R755A rescue. (N) Quantification of fluorescence increases after bafilomycin treatment shows that pHluorin accumulates in compartments that are acidified in a v-ATPase–dependent manner. Error bars indicate SEM. Bars, 10 µm.