VARP localizes to melanosomes by interaction with both VAMP7 and RAB38. (a–g) WT melan-Ink4a cells were transiently transfected with mCh-STX13 (red) and either WT VARP-GFP (green, a) or site-directed mutants (green, b–e) with interfering mutations in the binding sites for VAMP7 (-VAMP7, b), RAB38 (-RAB38, c), both VAMP7 and RAB38 (-V7, RAB38, d), or the VPS29 retromer subunit (-retromer, e). Cells were fixed 48 h after transfection and analyzed by deconvolution FM; bar, 10 µm. Insets are boxed regions magnified five times. Bar, 2 µm. BF images are pseudocolored blue in merged images. Arrowheads show VARP-GFP puncta adjacent to melanosomes visualized by BF, and arrows point to VARP-GFP puncta adjacent to mCh-STX13-labeled endosomes. (f) WT or mutant VARP-GFP puncta that were associated with melanosomes (visualized by BF; black bars) or with endosomes (labeled by mCh-STX13; gray bars) were quantified as mean ± SD from 10 cells per VARP variant representing three independent experiments. (g) P-values for pairwise comparisons of melanosome-associated VARP puncta in f. For endosome-associated VARP, only -V7 & R38 (P < 0.05 vs. WT) and -retromer (P < 0.0001 vs. all others) showed significant differences.