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 dependen t 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 amo ng 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 subu nits of PA700 so isolated were subjected to solid phase protease diges tion followed by reverse phase HPLC. Selected peptides from each prote in were sequenced by automated Edman degradation. Comparison of the re sulting amino acid sequences with those in current data bases indicate d that three of the subunits represented novel proteins, whereas four subunits were homologous to previously described proteins. Three subun its of the latter group were, in turn, homologous to one another and a re members of a large family of proteins containing a consensus sequen ce for ATP binding. Purified PA700 demonstrated ATPase activity. Treat ment of PA700 with alkylating agents, such as N-ethylmaleimide, inhibi ted with similar kinetics both proteasome activation and ATPase activi ty, suggesting that these two activities are functionally Linked. Thus , PA700 is composed of multiple members of a protein family that may f unction in the ATP-dependent regulation of proteasome activity.