Proteasome activator 11S REG or PA28: Recombinant REG alpha/REG beta hetero-oligomers are heptamers

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta. Recombinant REG alpha form s a heptamer, whereas recombinant REG beta is a monomer. When mixed with RE G beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3. T his apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subu nits, Chemical cross-linking of the alpha/beta oligomers revealed the prese nce of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG a lpha dimers were not detected. The mass of the REG alpha(N50Y)/REG beta het ero-oligomer determined by electrospray ionization time-of-flight mass spec trometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theor etical mass of 194 856 Da for an alpha 3 beta 4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in b acteria cells at an apparent beta/alpha molar ratio of similar to 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4/beta 3 heptamers al so present. On the other hand, the mass spectrum contained a mixture of alp ha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1. Thus, formation of heptamers is an intri nsic property of recombinant REG alpha and REG beta subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 be ta 4 and alpha 4 beta 3 species.