An ATP-stabilized inhibitor of the proteasome is a component of the 1500-kDa ubiquitin conjugate-degrading complex.
Proteins conjugated to ubiquitin are degraded by a 26S (1500-kDa) proteolytic complex that, in reticulocyte extracts, can be formed by the association of three factors: CF-1, CF-2, and CF-3. One of these factors, CF-3, has been shown to be the proteasome, a 650-kDa multicatalytic protease complex. We have purified a 250-kDa inhibitor of the proteasome and shown that it corresponds to CF-2. In the presence or absence of ATP, this factor inhibited hydrolysis by the proteasome of both fluorogenic tetrapeptides and protein substrates. When the inhibitor, proteasome, and CF-1 were incubated together in the presence of ATP and Mg2+, degradation of ubiquitin-125I-lysozyme occurred. Both the inhibitory activity and the ability to reconstitute ubiquitin-125I-lysozyme degradation were very labile at 42 degrees C, but both activities were stabilized by ATP or a nonhydrolyzable ATP analog. SDS/PAGE indicated that the 250-kDa inhibitor fraction contained a major subunit of 40 kDa (plus some minor bands). The 125I-labeled inhibitor and purified proteasome formed a complex. When CF-1, ATP, and Mg2+ were also present, the 125I-labeled inhibitor along with the proteasome formed a complex of 1500 kDa. The inhibitor (CF-2) thus appears to be an ATP-binding component that regulates proteolysis within the 1500-kDa complex.
Driscoll
J
J
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, MA 02115.
Frydman
J
J
Goldberg
A L
AL
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D014452:Ubiquitins / Q000378:metabolism*
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D011506:Proteins; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex