Figure 7. 
Differential requirements of luminal acidification for sorting and trafficking of membrane proteins. (A) Experimental strategy. After transgene induction, larvae were treated with DMSO, 750 nM bafilomycin, or 25 µM monensin and then subjected to a TGN block and 2-h release. (B) Illustrations of different classes of polarized membrane proteins. C, C-terminus; glyc, glycan; N, N-terminus; TMD, transmembrane domain. (C–F) Under control conditions (DMSO), each membrane protein is enriched at the cell surface after a 2-h TGN release. Scale bars are 10 µm. (G–J) After bafilomycin treatment, apical membrane proteins p75-GFP, CD36-GFP, and NaPi-2b-intGFP accumulate subapically (arrows), while basolateral membrane protein MICA-GFP does not accumulate comparably. Scale bars are 10 µm. (K–N) After monensin treatment, the O-glycosylated apical membrane protein p75-GFP is basolaterally missorted (arrowheads), the N-glycosylated apical membrane proteins CD36-GFP and NaPi-2b-intGFP accumulate subapically (arrows), and the N-glycosylated basolateral membrane protein MICA-GFP is relatively unaffected. Scale bars are 10 µm. (O) Quantification of intracellular accumulation expressed as a ratio of mean apical membrane intensity to cytoplasm intensity. Data were normalized to DMSO controls, and each data point is an individual larva. n ≥ 3 larvae per condition representing ≥20 cells. Error bars are SD. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Baf., bafilomycin; EC, extracellular; IC, intracellular; Mon., monensin; Norm., normal. Two-way ANOVA with Tukey’s multiple comparison test.

Differential requirements of luminal acidification for sorting and trafficking of membrane proteins. (A) Experimental strategy. After transgene induction, larvae were treated with DMSO, 750 nM bafilomycin, or 25 µM monensin and then subjected to a TGN block and 2-h release. (B) Illustrations of different classes of polarized membrane proteins. C, C-terminus; glyc, glycan; N, N-terminus; TMD, transmembrane domain. (C–F) Under control conditions (DMSO), each membrane protein is enriched at the cell surface after a 2-h TGN release. Scale bars are 10 µm. (G–J) After bafilomycin treatment, apical membrane proteins p75-GFP, CD36-GFP, and NaPi-2b-intGFP accumulate subapically (arrows), while basolateral membrane protein MICA-GFP does not accumulate comparably. Scale bars are 10 µm. (K–N) After monensin treatment, the O-glycosylated apical membrane protein p75-GFP is basolaterally missorted (arrowheads), the N-glycosylated apical membrane proteins CD36-GFP and NaPi-2b-intGFP accumulate subapically (arrows), and the N-glycosylated basolateral membrane protein MICA-GFP is relatively unaffected. Scale bars are 10 µm. (O) Quantification of intracellular accumulation expressed as a ratio of mean apical membrane intensity to cytoplasm intensity. Data were normalized to DMSO controls, and each data point is an individual larva. n ≥ 3 larvae per condition representing ≥20 cells. Error bars are SD. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05. Baf., bafilomycin; EC, extracellular; IC, intracellular; Mon., monensin; Norm., normal. Two-way ANOVA with Tukey’s multiple comparison test.

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