Fig. 1. Tyr and Tyr(C85S) are proteasome substrates. (A) Wild-type Tyr or mutant Tyr(C85S) mouse melanocytes were pulsed with [³⁵S]Met/Cys for 30 minutes and chased for the indicated times. Lysates were immunoprecipitated with anti-tyrosinase antibodies. Arrowheads and arrows indicate high-mannose and complexed forms of tyrosinase, respectively. The plot displays the quantification of five separate experiments. (B) Tyr(C85S) melanocytes were treated as in A, except half of the samples were incubated with 25 µM lactacystin (LCT) during the starvation, pulse, and chase periods. The plot displays the quantification of four separate experiments. (C) Tyr(C85S) melanocytes were incubated for 4 hours in the presence of various inhibitors: DMSO, 50 µM Leu-Leu nor-Leucinal (LLnL), 25 µM LCT, 50 µM MG132, or 50 µM E64 as indicated. Cell lysates were then either directly subjected to immunoblotting using anti-tyrosinase antibodies, or fractured and ultracentrifuged to separate the cytosol (S) from the membrane (P). Unglycosylated tyrosinase is indicated by a double arrowhead.