Abstract
The proteasome 11 S regulator (REG) consists of two homologous subunits, REGalpha and REGbeta. Each subunit is capable of activating the proteasome, and when combined, they form superactive REGalpha/REGbeta complexes. We have previously shown that a highly conserved loop in the REGalpha crystal structure is critical for proteasome activation. We now show that hetero-oligomers formed from REGalpha activation loop mutants and wild-type REGbeta or vice versa are partially active. By contrast, hetero-oligomers bearing mutations in the activation loops of REGalpha and REGbeta subunits are inactive, demonstrating that both alpha and beta subunits contribute to proteasome activation. We have also characterized REG proteins with mutations near or at their C termini. Partially active REGalpha(Y249C) and REGalpha(M247V) and an inactive REGalpha subunit bearing five additional C-terminal amino acids formed active hetero-oligomers with REGbeta. REGbeta 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 REGalpha(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 alpha and beta subunits are required for hetero-oligomers to bind the proteasome.
Highlights
The proteasome is a large proteolytic enzyme found in all three kingdoms, the archeae, prokaryotes, and eukaryotes [1,2,3,4,5,6,7]
We have shown that amino acids Arg141 to Gly149 in REG␣ are critical for proteasome activation, and this region forms a loop in the REG␣ crystal structure [35]
In contrast to the conclusions reached by DeMartino and colleagues that REG serves only to modulate REG␣ activity [37], we have found that in the 11 S REG both ␣ and  subunits contribute to proteasome activation
Summary
Materials—The fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-MCA (LLVY-MCA) was purchased from Sigma. REG␣241␥8, a REG␣ variant bearing the eight C-terminal amino acids of REG␥, was produced by the Kunkel site-directed mutagenesis method This method was used to obtain the REG mutants described in these studies. The extinction coefficient of each REG␣ variant was calculated based on its amino acid composition according to the method described by Gill and Von Hippel [39] The concentrations of those selected REG␣s determined by both methods agreed well with each other within the experimental errors. REG␣, REG, and their variants (e.g. mutants and REG␣/REG hetero-oligomers) were incubated with proteasomes tethered to an enzyme-linked immunosorbent assay plate. Analysis of REG-to-REG␣ Ratio in Hetero-oligomers Using HPLC— The apparent molar ratio of REG␣ and REG subunits in heterooligomers was determined by HPLC as described [38]
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