IDENTIFICATION, PURIFICATION, AND CHARACTERIZATION OF A HIGH-MOLECULAR-WEIGHT, ATP-DEPENDENT ACTIVATOR (PA700) OF THE 20-S PROTEASOME

In order to identify protein complexes consisting of the proteasome an d specific proteasome regulators, crude soluble lysates of red blood c ells were fractionated by gel filtration chromatography and by velocit y sedimentation centrifugation. The fractionated lysates were then tes ted for the relative distribution of proteasome activity, proteasome p rotein, and protein of a known proteasome activator, PA28. At least tw o proteasome complexes containing PA28 were identified. One of these c omplexes had an apparent molecular weight of approximately 1,750,000, and appeared to have much more proteasome activity than could be accou nted for by its relative concentrations of proteasome and PA28 protein . We hypothesized that this complex contained another activator of the proteasome, and we sought to purify this activator from extracts of r ed blood cells. A proteasome activator with an apparent molecular weig ht of approximately 700,000 was identified, purified, and characterize d. This activator, termed PA700, greatly stimulated the peptidase acti vities of the proteasome in an ATP-dependent fashion. PA700 was compos ed of about 16 polypeptides ranging in molecular weight from 20,000 to 100,000. The ATP-dependent activation of the proteasome by PA700 was closely linked to the formation of a high molecular weight complex tha t required no additional ATP for activated proteolysis. These results indicate that PA700 is a regulatory protein of the proteasome and is a component of at least one high molecular weight proteasome-containing complex occurring in cell extracts.