Figure 4.

Sulfate incorporation precedes endoproteolytic processing. (A) Continuous labeling. AtT-20 cells were labeled with [³⁵S]sulfate for 5, 20, or 60 min. Cells were extracted with detergent, and the samples were immunoprecipitated with anti-ACTH antibodies. The labeled proteins in the immune precipitates were analyzed by SDS-PAGE and PhosphorImager. (B) Pulse–chase analysis. Cells were pulse-labeled with [³⁵S]sulfate for 5 min and then chased for the times indicated. At the end of each chase, labeled POMC products in the cells were extracted, immunoprecipitated, and analyzed as in A. (C) Effect of a 20°C incubation on proteolytic processing; duplicate samples are shown. Lanes 1 and 2, cells pulse-labeled as in B (lane 1) were shifted to 20°C for 2 h before extraction. Under this condition, little processing occurred during the 20°C incubation period. Lanes 3 and 4, pulse-labeled cells were first chased at 37°C for 15 min as in B (lane 2) before being shifted to 20°C for 2 h. The 15-min chase allowed labeled POMC to exit the sulfation compartment, and subsequent incubation at 20°C resulted in the production of mature ACTH. Thus, processing requires POMC exit from the TGN, and the prohormone convertase is not inactivated at 20°C.