A MODEL FOR THE QUATERNARY STRUCTURE OF THE PROTEASOME ACTIVATOR PA28

PA28 is a protein activator of the 20S proteasome. It has a native mol ecular weight of approximately 200,000 and is composed of six 28,000 d alton subunits arranged in a ring-shaped complex, Purified preparation s of PA28 contain two polypeptides, alpha and beta, which are about 50 % identical in primary structure. It has been unclear whether native P A28 consists of two distinct homohexameric proteins or of a single pro tein containing both alpha and beta subunits, To distinguish between t hese possibilities, we prepared antibodies that reacted specifically w ith either the alpha or beta subunit and used these subunit-specific a ntibodies in two types of experiments designed to elucidate PA28 quate rnary structure. In the first experiment, the alpha and beta subunits were completely co-immunoprecipitated by each subunit-specific antibod y, indicating that both subunits were part of a single protein complex , In the second experiment, PA28 was chemically cross-linked using bis (sulfosuccinimidyl)suberate. When the cross-linked products were immun oblotted after SDS-polyacrylamide gel electrophoresis, indistinguishab le patterns were obtained with each subunit-specific antibody, These r esults confirm that the alpha and beta subunits were part of the same protein complex, The pattern of cross linked products also provided in sight as to the relative abundance and arrangement of the subunits wit hin the PA28 complex and indicated that the ring-shaped PA28 hexamer m ay be composed of alternating alpha and beta subunits with a stoichiom etry of (alpha beta)(3). PA28 was inactivated by treatment with carbox ypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl ter minus of the alpha subunit but had very little effect on the beta subu nit, This selective and limited proteolysis prevented binding of both alpha and beta subunits to the proteasome and therefore provides addit ional evidence of the heterodimeric nature of PA28, These results indi cate that a short carboxyl-terminal sequence of the alpha subunit is c ritical for binding of native PA28 to the proteasome. To learn about t he relative functions of the alpha and beta subunits, PA28 alpha was e xpressed in Escherichia coli and purified to homogeneity. Purified PA2 8 alpha stimulated proteasome activity but required 5-10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of P A28 is required for maximal proteasome activation.