Properties of the Proteasome Activator Subunit PA28α and its Des-Tyrosyl Analog

Abstract

The proteasome activator protein PA28 or 11 S regulator may play an important role in facilitating the generation of peptides for presentation by the MHC class I system. PA28 is composed of two homologous subunits termed α and β. Removal of the carboxyl terminal tyrosine of the α subunit of PA28 abolishes activity (X. Songet al.,1997,J. Biol. Chem.272, 27994–28000). To explore the structural basis of this effect the des-tyrosyl analog of PA28α prepared by site-directed mutagenesis and PA28α were expressed at high levels in a baculovirus system and purified by FPLC. Des-tyrosyl-PA28α neither stimulated the proteasome nor competed with PA28α for binding to the proteasome. Hydrophobic interaction chromatography revealed that the hydrophobicity of the mutant protein was considerably greater than PA28α. When the mutant protein was chromatographed on a calibrated Superose 6 column a mixture of approximately 25% oligomer and 75% monomer was found. The oligomer weakly stimulated the proteasome but this molecule was labile. Very low concentrations of SDS (0.005%) dissociated PA28α and abolished its stimulatory activity. It is concluded that the lack of activity of des-tyrosyl-PA28α is due to conformational changes resulting in dissociation and that the oligomeric form of PA28α is required for activation.