Late events in the assembly of 20S proteasomes.
Electron microscopy and STEM mass measurements have been used to characterize late intermediates in the assembly pathway of wildtype and mutant Rhodococcus proteasomes. A proteolytically inactive and processing-incompetent mutant, betaK33A, 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 cavity and most of the two antechambers. It is further shown that in wildtype Rhodococcus proteasomes the propeptides are degraded in a processive manner undergoing multiple cleavages before the products are discharged and the inner cavities are cleared. It appears that the docking of two half-proteasomes, i.e., preholoproteasome formation, is sufficient to trigger autocleavage of the Gly-1/Thr1 bond necessary for active site formation and the subsequent degradation of the propeptides.
Max-Planck Institut für Biochemie, Martinsried, D-82152, Germany.
J
J
Mayr
E
E
Seemüller
S A
SA
Müller
A
A
Engel
W
W
Baumeister
D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000648:ultrastructure*; D046911:Macromolecular Substances; D017348:Microscopy, Electron, Scanning Transmission; D008958:Models, Molecular; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000737:chemistry / Q000648:ultrastructure*; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000648:ultrastructure; D012240:Rhodococcus / Q000201:enzymology*
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex