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, ␣, ␤, and ␥, 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 REG␣. Most are monomers that produce functional proteasome activators when mixed with REG␤ subunits. Five REG␣ mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REG␣ heptamer shows that this region forms a loop at the base of each REG␣ subunit. One mutation in this loop (N146Y) yields a REG␣ heptamer that binds the proteasome as tightly as wild-type REG␣ but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REG␤ (N135Y) and REG␥ (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.A major mechanism for controlling viral infections involves cytotoxic T lymphocytes that recognize viral peptides presented on the cell surface by major histocompatability complex class I molecules and lyse the infected cells (1, 2). There is considerable evidence that some presented peptides, at least, are produced by the proteasome (3, 4). Crystal structures of Thermoplasma and yeast proteasomes reveal that they are cylindrical protein complexes, composed of four stacked rings (5, 6). The two end rings consist of catalytically inactive ␣-type subunits, whereas the two inner rings are composed of ␤-type subunits, some of which are catalytically active (7). The protease active sites are located within an inner chamber that is virtually sealed from the particles surface (5, 6). Thus, it seems clear that substrate entry to the sites of peptide bond hydrolysis must be tightly regulated.Two proteasome activators have been identified so far. The proteasome can either associate with a 19S regulatory complex to form the 26S protease, which is capable of degrading intact proteins (8-11), or the proteasome can bind an 11S activator called REG or PA28. This association greatly enhances fluorogenic peptide hydrolysis by the proteasome (12, 13). As isolated from human red blood cells, REG is a hexameric or heptameric ring formed from two homologous subunits, REG␣ and REG␤; these two subunits are, in turn, homologous to KI antigen or REG␥. cDNAs for all three proteins have been expressed in Escherichia coli, and each recombinant protein is capable of activating the proteasome in vitro (14).A variety of evidence suggests that REG is involved in antige...

The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated ␣, ␤, and ␥, 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 REG␣⌬i, REG␤⌬i, and REG␥⌬i. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REG␣⌬i and REG␥⌬i formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REG␤⌬i exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REG␤ protein. REG␤⌬i was able to form hetero-oligomers with a single site, monomeric REG␣ mutant and with REG␣⌬i. At low concentrations, the REG␣⌬i/REG␤⌬i hetero-oligomers stimulated the proteasome less than REG␣/REG␤ oligomers formed from wild-type subunits, and the reduced activation by REG␣⌬i/REG␤⌬i was due to removal of the REG␤ insert, not the REG␣ insert. These studies demonstrate that the REG␣ and REG␥ inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REG␤ insert reduces binding of this subunit and REG␣/REG␤ 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.The proteasome is a major proteolytic organelle in the cytosol and nucleus of eukaryotic cells (1-3). The enzyme is a cylindrical structure containing 28 subunits arranged as four rings of seven subunits each. The two end rings are composed of catalytically inactive subunits belonging to the ␣ subunit family based on homology to proteasome subunits from the archebacterium, Thermoplasma. The two central rings are composed of members of the ␤ subunit family (4 -6), some of which are proteolytically active (7). Crystal structures of Thermoplasma and yeast proteasomes reveal that the ␤ subunits form a central proteolytic chamber far from the particle's surface and that entry to this central chamber is greatly restricted (8, 9).By itself, the proteasome does not degrade intact proteins. Association with a 19 S regulatory complex converts the proteasome to the 26 S protease, an energy-dependent enzyme capable of degrading intact proteins (10 -13), including those marked by ubiquitin (14). The proteasome also binds an 11 S protein complex, which we have termed REG and others have named PA28 (15-17). REG binding to the proteasome can stimulate peptide hydrolysis as much as 100-fold. Human red blood cell REG is composed of two ϳ30 KDa subunits, REG␣ and REG␤. These two proteins are closely related to each other and to a third protein, REG␥ (18). The three proteins show extensive sequence similarities, except for a region of 15-32 amino a...

The proteasome 11 S regulator (REG) consists of two homologous subunits, REG␣ and REG␤. Each subunit is capable of activating the proteasome, and when combined, they form superactive REG␣/REG␤ complexes. We have previously shown that a highly conserved loop in the REG␣ crystal structure is critical for proteasome activation. We now show that hetero-oligomers formed from REG␣ activation loop mutants and wild-type REG␤ or vice versa are partially active. By contrast, heterooligomers bearing mutations in the activation loops of REG␣ and REG␤ subunits are inactive, demonstrating that both ␣ and ␤ subunits contribute to proteasome activation. We have also characterized REG proteins with mutations near or at their C termini. Partially active REG␣(Y249C) and REG␣(M247V) and an inactive REG␣ subunit bearing five additional C-terminal amino acids formed active hetero-oligomers with REG␤. REG␤ subunits lacking 1, 2, or 9 C-terminal amino acids did not bind or activate the proteasome, but each of these mutants formed partially active hetero-oligomers with the monomer REG␣(N50Y). However, hetero-oligomers formed from REG subunits lacking the last 14 amino acids were unable to bind the proteasome. Thus, C-terminal regions of both ␣ and ␤ subunits are required for hetero-oligomers to bind the proteasome.The proteasome is a large proteolytic enzyme found in all three kingdoms, the archeae, prokaryotes, and eukaryotes (1-7). In higher eukaryotes, the proteasome is believed to play an important role in a variety of cellular processes, including cell cycle progression (8, 9), control of gene expression (10), and antigen presentation (11-13). The proteasome from the archaebacterium Thermoplasma acidophilum is formed from 14 identical ␣ subunits and 14 identical ␤ subunits. To date, 17 distinct subunits have been identified in proteasomes from higher eukaryotes. All can be classified into ␣ or ␤ families based on their homology to the ␣ or ␤ subunits of the Thermoplasma proteasome (13). The quaternary structures of yeast and Thermoplasma proteasomes are quite similar, as revealed by electron microscopy (14 -16) and confirmed by x-ray crystallography (17, 18). The enzymes are composed of four stacked rings with seven subunits in each ring. The two inner rings are formed from ␤ subunits, and the two outer rings consist of ␣ subunits. The proteolytic active sites are located in an inner chamber of the proteasome, with the N-terminal threonines on the ␤ subunits acting as nucleophiles (17)(18)(19). These active sites are not readily accessible from the cytosol, because only two 13-Å pores are present in the ␣ rings of the archaebacterial proteasome, and even these small pores are absent in the crystallized yeast proteasome (17,18). Therefore, some mechanisms must exist to promote substrate access to the proteasome's active sites.Several proteins have been found to bind the proteasome (20). Of these, the 19 S regulatory complex (21-24) and 11 S regulator (REG) 1 , or PA28 (25,26), are best characterized. The 19 S regulatory complex cons...