Figure 6: RAB6 controls the trafficking of melanosomal cargoes.

(a) WB of control or RAB6-depleted MNT-1 cell lysates probed for RAB6, TYRP2, MART-1 or Tubulin. (b) Quantification of the relative expression levels of MART-1 or TYRP2 in control or RAB6 siRNAs-treated cells. Data are normalized to control and presented as mean±s.e.m. (n=7, TYRP2; n=3, MART-1). (c) WB of PNS (1%) and melanosomes-enriched fraction (5%, Mel) isolated from control- or RAB6-depleted MNT-1 cells and probed for RAB6 (*) and GAPDH (left panel) or TYRP1, TYRP2 and MART-1 (right panel). Note the increase in TYRP2 and MART-1 levels in PNS (black asterisk) and corresponding decrease in Mel fractions (white asterisk). (d) Quantification of the expression level of MART-1 and TYRP2 in melanosomes-enriched fractions of RAB6-depleted melanocytes relative to controls. Data are normalized presented as mean±s.e.m. of three independent experiments. (e,f) Immunogold labelling on ultrathin cryosections of GFP-RAB6 (d) or GFP-TYRP2 (e) expressing MNT-1 cells co-stained for GFP (PAG-15 nm) and MART-1 (PAG-10 nm) (arrows). Black arrows point co-distribution of MART-1 and TYRP2 on similar vesicles as in (d), and white arrows on Golgi apparatus (GA). (g) IFM on control- or RAB6-depleted NHEM stained for endogenous MART-1 and TGN46. (h) Quantification of the MART-1 fluorescent intensity enrichment in the Golgi area in control- or RAB6-depleted NHEM or MNT-1 cells (n) presented as mean±s.e.m., (NHEM, n=51–63, MNT-1, n=22–24, per respective condition, three independent experiments). (i) Working model illustrating the possible role of RAB6 and ELKS in the Golgi to melanosomes pathway. Note that other alternative routes cannot be excluded. Scale bars: 500 nm (d, e), 20 μm (f). M.W. in kDa, two-tailed, Unpaired t test (b,c,g). *P<0.05; **P<0.01; ****P<0.0001.