Retrograde trafficking of TYRP1 to the Golgi is specifically increased in BLOC-2⁻ melanocytes. (a–c) WT melan-Ink4a or BLOC-2⁻ cells transiently expressing TYRP1-EGFP and GM130-mCh to mark the Golgi, were pretreated with CHX for 60 min at 37°C and then imaged by spinning disk microscopy (t = 0). GFP fluorescence within the Golgi region labeled by GM130-mCh was bleached at frame 5 (t = 5), and recovery of TYRP1-EGFP fluorescence intensity was quantified over time by normalizing to prebleach GFP intensity of the same region. (a and b) Representative images for GM130-mCh and TYRP1-EGFP from t = 0, t = 5, and t = 300 (300 s) for WT (a) and BLOC-2⁻ cells (b). Bleached region is indicated by the dotted white line in TYRP1-EGFP images. Bars, 10 µm. (c) Quantification of fluorescence recovery. Rel., relative; fl., fluorescence. Values represent the means ± SD of GFP intensity traces of 15 cells of each type. (d and e) WT (melan-Ink4a), “rescued” BLOC-2^R (melan-coa:hHPS3), or melan-ru:HPS6, or BLOC-2–deficient BLOC-2⁻ (melan-coa), BLOC-2^−C, or melan-ru cells on coverslips were incubated with AF488-conjugated CTxB on ice for 1 h and then at 20°C for 15 min to allow for internalization. Cells were washed once and incubated at 37°C for the indicated times, fixed, labeled with the anti-giantin antibody and analyzed by IFM. (d) Representative CTxB fluorescence images of BLOC-2⁻ and BLOC-2^R cells at the 0 and 90 min time points (t = 0, t = 90) showing appearance of CTxB in the Golgi (giantin staining; red outline) by 90 min. Bar, 10 µm. (e) Fluorescence intensity of AF488-CTxB in the giantin-positive region was quantified and plotted over time for all cell types. Values (means ± SD) for all BLOC-2–deficient lines are significantly different (P < 0.01 at 60 min and P < 0.001 at 120 and 240 min) from their paired BLOC-2–sufficient lines.