Reconstitution of proteasome activator PA28 from isolated subunits: optimal activity is associated with an α,β‐heteromultimer

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...

Section: Proteasome Activation Bysupporting

confidence: 80%

“…Thus, we call this region the activation loop. There are two reports that REG␤ is inactive (36,37), and it has recently been proposed that REG␤ functions only to modulate the activity of REG␣ (37). However, several publications from our laboratory have shown that REG␤, by itself, activates the proteasome (34,35,38).…”

mentioning

confidence: 99%

The Proteasome Activator 11 S Regulator or PA28

Zhang

Clawson

Rechsteiner

1998

“…4 show REG␤ active at 42°C, other REG␤ preparations were markedly inactivated at 35°C. Accordingly, enzyme reactions incubated at 37°C may not detect REG␤ activity, and this is the reason that we assayed the recombinant REG homologs at room temperature rather than at 37°C as in previous studies (18,53). In conclusion, we are confident that REG␤ activates the proteasome, but its activity is more difficult to detect than that of REG␣ or REG␥.…”

Section: Discussionmentioning

confidence: 82%

“…The finding that REG␤ is a proteasome activator might be considered unexpected for two reasons. First, there are two reports that REG␤ does not activate the proteasome (2,53). Second, we have produced 31 monomeric mutants of REG␣ that cannot stimulate peptide hydrolysis by the proteasome.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Recombinant REGα, REGβ, and REGγ Proteasome Activators

Realini

Jensen

Zhang

et al. 1997

164

199

Full-length cDNAs for three human proteasome activator subunits, called REG␣, REG␤, and REG␥, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant ␣ or ␥ subunits form heptameric species; recombinant ␤ subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REG␣ and REG␤. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant ␣ and ␤ subunits bind each other with high affinity, and the REG␣/␤ heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-␤-nitroanilide (LLE-␤NA) more than REG␣ or REG␤ alone. Using filter binding and gel filtration assays, recombinant REG␥ subunits were shown to bind themselves but not ␣ or ␤ subunits. REG␥ differs from REG␣ and REG␤ in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-␤NA by the proteasome. REG␥ binds the proteasome with higher affinity than REG␣ or REG␤ yet with lower affinity than complexes containing both REG␣ and REG␤. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.

“…Thus, a limited structural domain of one of the two subunits appeared to be required for binding of PA28 to the proteasome and resultant proteasome activation. Further support for a key role of the ␣ subunit in PA28 function was obtained in studies showing that the isolated ␣ subunit, prepared as a recombinant protein or electrophoretically separated from denatured native PA28, could activate the proteasome although not as efficiently as the native protein containing both ␣ and ␤ subunits (34,36,39,40). Thus, we hypothesized that the ␤ subunit might act to mediate function of the ␣ subunit, perhaps by increasing the affinity of PA28 for the proteasome.…”

mentioning

confidence: 99%

Relative Functions of the α and β Subunits of the Proteasome Activator, PA28

Song

Kampen

Slaughter

et al. 1997

PA28 is a 180,000-dalton protein that activates hydrolysis of small nonubiquitinated peptides by the 20 S proteasome. PA28 is composed of two homologous subunits, ␣ and ␤, arranged in alternating positions in a ringshaped oligomer with a likely stoichiometry of (␣␤) 3 . Our previous work demonstrated that the carboxyl terminus of the ␣ subunit was necessary for PA28 to bind to and activate the proteasome. The goals of this work were to define the exact structural basis for this effect and to determine the relative roles of the ␣ and ␤ subunits in proteasome activation. Each subunit and various mutants of the ␣ subunit were expressed in Escherichia coli and purified. PA28␣ stimulated the proteasome, but had a much greater K act than native heteromeric PA28. In contrast, PA28␤ was unable to stimulate the proteasome. Mutants of the ␣ subunit in which the carboxyl-terminal tyrosine residue was deleted or substituted with charged amino acids could neither bind to nor activate the proteasome. However, substitution of the carboxyl-terminal tyrosine with other amino acids resulted in proteins which could stimulate the proteasome to various extents. Tryptophan mutants stimulated the proteasome as well as did native PA28, whereas serine or phenylalanine mutants stimulated the proteasome much poorer than did wild type PA28␣. Deletion of the "KEKE" motif, a 28-amino acid domain near the amino terminus of PA28␣, had no effect on proteasome stimulatory activity. Hetero-oligomeric PA28 proteins were reconstituted from isolated wild type and mutant subunits. PA28 reconstituted from wild type subunits had structural and functional properties that were indistinguishable from those of the native hetero-oligomeric protein. PA28 molecules reconstituted from inactive ␣ subunits and wild type ␤ subunits remained inactive. However, PA28 molecules reconstituted from suboptimally active ␣ mutants and wild type ␤ subunits had the same activity as native heteromeric PA28. These results indicate that the ␤ subunit modulates PA28 activity, perhaps by influencing the affinity of PA28 for the proteasome.