Relative Functions of the α and β Subunits of the Proteasome Activator, PA28

Abstract

PA28 is a 180,000-dalton protein that activates hydrolysis of small nonubiquitinated peptides by the 20 S proteasome. PA28 is composed of two homologous subunits, ␣ and ␤, arranged in alternating positions in a ringshaped oligomer with a likely stoichiometry of (␣␤) 3 . Our previous work demonstrated that the carboxyl terminus of the ␣ subunit was necessary for PA28 to bind to and activate the proteasome. The goals of this work were to define the exact structural basis for this effect and to determine the relative roles of the ␣ and ␤ subunits in proteasome activation. Each subunit and various mutants of the ␣ subunit were expressed in Escherichia coli and purified. PA28␣ stimulated the proteasome, but had a much greater K act than native heteromeric PA28. In contrast, PA28␤ was unable to stimulate the proteasome. Mutants of the ␣ subunit in which the carboxyl-terminal tyrosine residue was deleted or substituted with charged amino acids could neither bind to nor activate the proteasome. However, substitution of the carboxyl-terminal tyrosine with other amino acids resulted in proteins which could stimulate the proteasome to various extents. Tryptophan mutants stimulated the proteasome as well as did native PA28, whereas serine or phenylalanine mutants stimulated the proteasome much poorer than did wild type PA28␣. Deletion of the "KEKE" motif, a 28-amino acid domain near the amino terminus of PA28␣, had no effect on proteasome stimulatory activity. Hetero-oligomeric PA28 proteins were reconstituted from isolated wild type and mutant subunits. PA28 reconstituted from wild type subunits had structural and functional properties that were indistinguishable from those of the native hetero-oligomeric protein. PA28 molecules reconstituted from inactive ␣ subunits and wild type ␤ subunits remained inactive. However, PA28 molecules reconstituted from suboptimally active ␣ mutants and wild type ␤ subunits had the same activity as native heteromeric PA28. These results indicate that the ␤ subunit modulates PA28 activity, perhaps by influencing the affinity of PA28 for the proteasome.

The proteasome: a protein‐destroying machine

“…Realini et al (1997) reported that recombinant REG␤ and REG␥ (equivalent to PA28␤ and PA28␥, respectively) are capable of stimulating the peptidase activities of the 20S proteasome. Contrary to these results, Song et al (1997) reported that PA28␤ alone has no detectable effect on the peptidase activities of the 20S proteasome but that it appreciably reduces the K m without affecting the V max for the PA28␣-activated proteasome, suggesting an important functional interaction between PA28␣ and PA28␤. The reason for these conflicting results is not known.…”

Proteasome Activation by REG Molecules Lacking Homolog-specific Inserts

“…4 clearly rule out a requirement for REG␣ and REG␤ inserts in hetero-oligomer formation. While this manuscript was in review, a study from DeMartino's laboratory (29) reported that deletion of the insert from rat REG␣ does not affect its activity, a finding that agrees with results presented here. However, Song et al (29) found reduced activation by REG␣/REG␤ hetero-oligomers formed from rat REG␣ molecules lacking inserts, whereas removal of the human REG␣ insert did not affect proteasome activation by REG␣/ REG␤ (Fig.…”

“…Therefore, we do not believe that the activity observed for human REG␤ is an experimental artifact. The discrepancy between our findings and those of Song et al (29) could reflect differing properties of rat and human REG␤ molecules or differences in assay conditions. Regarding the latter possibility, we note that the assay of Song et al uses REG␤ concentrations 10-fold lower than those employed by us.…”

“…4). Song et al (29) also reported that recombinant rat REG␤ is inactive. The results presented here (Fig.…”

“…5C). Song et al (29) speculate that decreased activation resulted from impaired interactions between REG␣ and REG␤ upon removal of the REG␣ inserts. Although we cannot rule out the possibility that the inserts facilitate association of these two subunits, they are clearly not required for human REG␣ to oligomerize with REG␤ (see Fig.…”