Late events in the assembly of 20S proteasomes

Electron microscopy and STEM mass measurements have been used to characteri ze late intermediates in the assembly pathway of wildtype and mutant Rhodoc occus proteasomes. A proteolytically inactive and processing-incompetent mu tant, beta K33A, allowed a short-lived late intermediate of the pathway to be captured, the preholoproteasome. In this fully assembled 20S complex the 14 propeptides with an aggregate mass of 100 kDa fill the whole central ca vity and most of the two antechambers. It is further shown that in wildtype Rhodococcus proteasomes the propeptides are degraded in a processive manne r undergoing multiple cleavages before the products are discharged and the inner cavities are cleared. It appears that the docking of two half-proteas omes, i.e., preholoproteasome formation, is sufficient to trigger autocleav age of the Gly-1/Thr1 bond necessary for active site formation and the subs equent degradation of the propeptides. (C) 1998 Academic Press.