Proteasome activation by REG molecules lacking homolog-specific inserts.
The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated alpha, beta, and gamma, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant proteins REGalphaDeltai, REGbetaDeltai, and REGgammaDeltai. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REGalphaDeltai and REGgammaDeltai formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REGbetaDeltai exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein. REGbetaDeltai was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalphaDeltai. At low concentrations, the REGalphaDeltai/REGbetaDeltai hetero-oligomers stimulated the proteasome less than REGalpha/REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalphaDeltai/REGbetaDeltai was due to removal of the REGbeta insert, not the REGalpha insert. These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha/REGbeta oligomers to proteasomes. On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome. We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.
Zhang
Z
Z
Department of Biochemistry, University of Utah, Salt Lake City, Utah 84132, USA.
Realini
C
C
Clawson
A
A
Endicott
S
S
Rechsteiner
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D051847:Lithostathine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D046988:Proteasome Endopeptidase Complex; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D051847:Lithostathine; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; C497617:REG1A protein, human; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex