PA700, an ATP-dependent activator of the 20 S proteasome, is an ATPase containing multiple members of a nucleotide-binding protein family.

PA700 is a 700,000-dalton multisubunit protein that activates multiple proteolytic activities of the 20 S proteasome by a mechanism dependent upon ATP hydrolysis (Ma, C.-P., Vu, J.H., Proske, R.J., Slaughter, C.A., and DeMartino, G.N. (1994) J. Biol. Chem. 269, 3539-3547). In order to determine the identities of and structural relationships among the subunits of PA700, individual PA700 subunits were isolated by a combination of reverse phase high performance liquid chromatography (HPLC) and SDS-polyacrylamide gel electrophoresis. Seven of the 16 subunits of PA700 so isolated were subjected to solid phase protease digestion followed by reverse phase HPLC. Selected peptides from each protein were sequenced by automated Edman degradation. Comparison of the resulting amino acid sequences with those in current data bases indicated that three of the subunits represented novel proteins, whereas four subunits were homologous to previously describe proteins. Three subunits of the latter group were, in turn, homologous to one another and are members of a large family of proteins containing a consensus sequence for ATP binding. Purified PA700 demonstrated ATPase activity. Treatment of PA700 with alkylating agents, such as N-ethylmaleimide, inhibited with similar kinetics both proteasome activation and ATPase activity, suggesting that these two activities are functionally linked. Thus, PA700 is composed of multiple members of a protein family that may function in the ATP-dependent regulation of proteasome activity.

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 and specific proteasome regulators, crude soluble lysates of red blood cells were fractionated by gel filtration chromatography and by velocity sedimentation centrifugation. The fractionated lysates were then tested for the relative distribution of proteasome activity, proteasome protein, and protein of a known proteasome activator, PA28. At least two proteasome complexes containing PA28 were identified. One of these complexes had an apparent molecular weight of approximately 1,750,000, and appeared to have much more proteasome activity than could be accounted 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 red blood cells. A proteasome activator with an apparent molecular weight of approximately 700,000 was identified, purified, and characterized. This activator, termed PA700, greatly stimulated the peptidase activities of the proteasome in an ATP-dependent fashion. PA700 was composed 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 that 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.

Purification and characterization of a protein inhibitor of the 20S proteasome (macropain).

An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.