RELATIVE FUNCTIONS OF THE ALPHA-SUBUNIT AND BETA-SUBUNIT OF THE PROTEASOME ACTIVATOR, PA28

PA28 is a 180,000-dalton protein that activates hydrolysis of small no nubiquitinated peptides by the 20 S proteasome. PA28 is composed of tw o homologous subunits, alpha and beta arranged in alternating position s in a ring-shaped oligomer with a likely stoichiometry of (alpha beta )(3). Our previous work demonstrated that the carboxyl terminus of the alpha subunit was necessary for PA28 to bind to and activate the prot easome. The goals of this work were to define the exact structural bas is for this effect and to determine the relative roles of the alpha an d beta subunits in proteasome activation. Each subunit and various mut ants of the alpha subunit were expressed in Escherichia coli and purif ied. PA28 alpha stimulated the proteasome, but had a much greater K-ac t than native heteromeric PA28. In contrast, PA28 beta was unable to s timulate the proteasome. Mutants of the alpha subunit in which the car boxyl-terminal tyrosine residue was deleted or substituted with charge d amino acids could neither bind to nor activate the proteasome. Howev er, substitution of the carboxyl-terminal tyrosine with other amino ac ids resulted in proteins which could stimulate the proteasome to vario us extents. Tryptophan mutants stimulated the proteasome as well as di d native PA28, whereas serine or phenylalanine mutants stimulated the proteasome much poorer than did wild type PA28 alpha. Deletion of the ''KEKE'' motif, a 28-amino acid domain near the amino terminus of PA28 alpha, had no effect on proteasome stimulatory activity. Hetero-oligo meric PA28 proteins were reconstituted from isolated wild type and mut ant subunits. PA28 reconstituted from wild type subunits had structura l and functional properties that were indistinguishable from those of the native hetero-oligomeric protein. PA28 molecules reconstituted fro m inactive a subunits and wild type beta subunits remained inactive. H owever, PA28 molecules reconstituted from suboptimally active alpha mu tants and wild type beta subunits had the same activity as native hete romeric PA28. These results indicate that the beta subunit modulates P A28 activity, perhaps by influencing the affinity of PA28 for the prot easome.