Identification of an activation region in the proteasome activator REGalpha.
Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, alpha, beta, and gamma, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REGalpha. Most are monomers that produce functional proteasome activators when mixed with REGbeta subunits. Five REGalpha mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REGalpha heptamer shows that this region forms a loop at the base of each REGalpha subunit. One mutation in this loop (N146Y) yields a REGalpha heptamer that binds the proteasome as tightly as wild-type REGalpha but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REGbeta (N135Y) and REGgamma (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.
Zhang
Z
Z
Department of Biochemistry, University of Utah, 50 North Medical Drive, Salt Lake City, UT 84132, USA.
Clawson
A
A
Realini
C
C
Jensen
C C
CC
Knowlton
J R
JR
Hill
C P
CP
Rechsteiner
M
M
D000595:Amino Acid Sequence; D001216:Asparagine; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D016296:Mutagenesis; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D009097:Multienzyme Complexes; D011506:Proteins; D011994:Recombinant Proteins; D001216:Asparagine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex