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. 2018 Jul 1;29(13):1640–1651. doi: 10.1091/mbc.E17-09-0546

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© 2018 Zhao et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 3: — Vha68-1 is required for Rh1 deglycosylation and pH homeostasis of secretory vesicles. (A) Rh1 vesicles and nSyb secretory vesicles largely colocalized. Eyes from control (ninaE-nSyb-mCherry-pHluorin/+) and vha68-1¹ (ey-flp; vha68-1¹ FRT40A/GMR-hid CL FRT40A; ninaE-nSyb-mCherry-pHluorin/+) flies expressing nSyb-mCherry-pHluorin were labeled with antibodies against Rh1 (green) and RFP (red). Images with high magnification are shown in right panels. (B) Secretory vesicle pH was increased in vha68-1¹ photoreceptors. Live confocal imaging of dissected ommatidia from control and vha68-1¹ flies expressing nSyb-mCherry-pHluorin. The pHluorin fluorescence was brighter in vha68-1¹ flies compared with controls, which indicates an increase in pH. (C) Quantification of fluorescence ratios in control and vha68-1¹ flies. Four eyes from different flies and 20 ommatidia from each eye were quantified. Error bars represent SD; ***, p < 0.001 (Student’s unpaired t test). (D) The increase in Rh1 MW is due to a failure of deglycosylation. Head extracts prepared from 1-d-old vha68-1¹ or vha100-1² flies were digested with Endo H or PNGase F at 37°C for 4 h before being labeled with antibodies against Rh1. (E) Rh1 MW was reverted to wild type upon expression of vha68-1, vha68-2, or vha68-3 in photoreceptor cells. Western blot analysis of head extracts from 1-d-old wild-type, vha68-1¹, vha68-1¹;ninaE-vha68-1, vha68-1¹;ninaE-vha68-2, and vha68-1¹;ninaE-vha68-3 flies that were labeled with antibodies against Rh1. One-day-old flies were used. (F) Vha68-1 functions downstream of dMPPE. Head extracts were prepared from 1-d-old wild-type, vha68-1¹, dmppe^e02905, vha68-1¹ dmppe^e02905(ey-flp rh1-GFP;vha68-1¹ FRT40A dmppe^e02905/GMR-hid CL FRT40A dmppe^e02905), and ninaA²flies and labeled with antibodies against Rh1. (G) Similar Rh1 MW in vha68-1¹ and α-Man-IIa^G4901 flies. Head extracts were prepared from 1-d-old flies and labeled with antibodies against Rh1. (H) Tangential resin-embedded sections of compound eyes from ∼1-d-old wild-type, vha68-1¹, and α-Man-IIa^G4901 flies were labeled using antibodies against Rh1 (green). Rh1 vesicles did not accumulate in α-Man-IIa^G4901 flies, which showed deglycosylation defects. (I) Light-sensitivity analysis of ∼1 d posteclosion flies. ERG amplitudes at each intensity are normalized to ERG amplitudes at maximum light intensity (10^–1, ∼300 lux). n = 7; data are presented as mean ± SD; ***, p < 0.001 (Student’s unpaired t test).