Figure 5.
Further evidence that LLnL enhances maturation of tyrosinase, leading to translocation to prememalnosomes. (A) Autoradiogram of radioactive proteins immunoprecipitated with tyrosinase antibodies (lanes 1–21) or control rabbit IgG (lane 22). Cells were pulsed with 35S for 15 min and harvested immediately (0) or after incubation for the indicated periods of time in regular medium. LLnL was either absent (−) or present (+) during the incubation–chase period. (B) LLnL promotes tyrosinase–calreticulin complexing. Western blot of calreticulin-immune precipitates probed with anti-tyrosinase mAb. Lysates for immunoprecipitation were prepared from cells treated with DMSO (−) or LLnL (50 μM for 4 h) (+). (C) LLnL enhances translocation of tyrosinase into premelanosomes. Electron micrographs depict dihydroxy l-phenyl alanine-(tyrosinase) reactivity in untreated (a, d) or LLnL-treated (50 μM for 4.5 h; b, c, and e) amelanotic YUSAC2 melanoma cells. Solid arrowheads indicate electron-opaque reaction product. Insets d and e show unreactive (open arrow) and reactive (solid arrowhead) premelanosomes. Dopa-reactive, trans-Golgi elements (small arrowheads) in an LLnL-treated cell in c. n = nucleus. (Bars = 1 μm in a, b, and c and 0.5 μm in d and e.)