Fig. 2. Degradation of albino TYR(C89R) can be reconstituted with semi-permeabilized melanocytes. (A) TYR or TYR(C89R) mRNAs were translated in the presence of Tyr(C85S) or wild-type Tyr SP-melanocytes (membranes), or microsomes (MS). Protein synthesis was terminated by the addition of cycloheximide and MS or SP-melanocytes were isolated. Isolated membranes were resuspended in an Energy Regeneration System (ERS) in Untreated rabbit Reticulocyte Lysate (URL), and incubated at 37°C for the indicated time. TYR and ^UTYR indicate translocated and untranslocated tyrosinase, respectively. (B) TYR(C89R) mRNA was translated in RRL in the presence of Tyr(C85S) SP cells and either directly incubated with either URL or KHM buffer devoid of proteasomes. All the samples were incubated at 37°C for the indicated time and treated as above. (C) Proteins from Tyr(C85S) cell lysates (lane 1) or SP-melanocytes incubated in the presence of buffer (lane 2) or URL (lane 3) were separated by SDS-PAGE, and subjected to immunoblotting with antibodies to the -subunit of the proteasome.