Relative functions of the α and β subunits of the proteasome activator, PA28

Xiaoling Song, Jan Von Kampen, Clive A. Slaughter, George N. DeMartino

Research output: Contribution to journalArticle

64 Scopus citations

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, α and β, arranged in alternating positions in a ring- shaped oligomer with a likely stoichiometry of (αβ)3. Our previous work demonstrated that the carboxyl terminus of the α 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 α and β subunits in proteasome activation. Each subunit and various mutants of the α subunit were expressed in Escherichia coli and purified. PA28α stimulated the proteasome, but had a much greater K(act) than native heteromeric PA28. In contrast, PA28β was unable to stimulate the proteasome. Mutants of the α 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 PA28α. Deletion of the 'KEKE' motif, a 28-amino acid domain near the amino terminus of PA28α, 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 α subunits and wild type β subunits remained inactive. However, PA28 molecules reconstituted from suboptimally active α mutants and wild type β subunits had the same activity as native heteromeric PA28. These results indicate that the β subunit modulates PA28 activity, perhaps by influencing the affinity of PA28 for the proteasome.

Original languageEnglish (US)
Pages (from-to)27994-28000
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number44
DOIs
StatePublished - Oct 31 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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