Properties of the proteasome activator subunit PA28 alpha and its des-tyrosyl analog.
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 alpha and beta. Removal of the carboxyl terminal tyrosine of the alpha subunit of PA28 abolishes activity (X. Song et al., 1997, J. Biol. Chem. 272, 27994-28000). To explore the structural basis of this effect the des-tyrosyl analog of PA28alpha prepared by site-directed mutagenesis and PA28alpha were expressed at high levels in a baculovirus system and purified by FPLC. Des-tyrosyl-PA28alpha neither stimulated the proteasome nor competed with PA28alpha for binding to the proteasome. Hydrophobic interaction chromatography revealed that the hydrophobicity of the mutant protein was considerably greater than PA28alpha. 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 PA28alpha and abolished its stimulatory activity. It is concluded that the lack of activity of des-tyrosyl-PA28alpha is due to conformational changes resulting in dissociation and that the oligomeric form of PA28alpha is required for activation.
Wilk
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Department of Pharmacology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York, 10029, USA. S_Wilk@smtplink.mssm.edu
Chen
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Magnusson
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D009124:Muscle Proteins; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D018411:Spodoptera; D014443:Tyrosine / Q000235:genetics*
D009097:Multienzyme Complexes; D009124:Muscle Proteins; C085047:PSME1 protein, human; D011506:Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases