Proteasome Activator 11S REG or PA28: Recombinant REGR/REG Hetero-oligomers Are Heptamers 1 Zhiguo Zhang, ‡,§ Andrew Krutchinsky, §,| Scott Endicott, ‡ Claudio Realini, ‡ Martin Rechsteiner,* ,‡ and Kenneth G. Standing | Department of Biochemistry, UniVersity of Utah, 50 North Medical DriVe, Salt Lake City, Utah 84132, and Department of Physics, UniVersity of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada ReceiVed January 8, 1999; ReVised Manuscript ReceiVed March 4, 1999 ABSTRACT: The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REGR and REG. Recombinant REGR forms a heptamer, whereas recombinant REG is a monomer. When mixed with REG, a monomeric REGR mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG to REGR(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REGR(N50Y)/REG hetero-oligomer being a heptamer composed of three R and four  subunits. Chemical cross-linking of the R/ oligomers revealed the presence of REGR-REG and REG-REG dimers, but REGR-REGR dimers were not detected. The mass of the REGR(N50Y)/REG hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 ( 40 Da in good agreement with the theoretical mass of 194 856 Da for an R34 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent /R molar ratio of ∼1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly R34 heptamers, with trace amounts of R43 heptamers also present. On the other hand, the mass spectrum contained a mixture of R7, R61, R52, and R43 heptamers when the REG/REGR ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REGR and REG subunits. On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely R34 or a mixture of R34 and R43 species. The proteasome, first described by Wilk and Orlowski in bovine pituitary extracts (1), is found in bacteria, archaea, and eukaryotes (2). Recent structural studies on the protea- some from the archaebacterium Thermoplasma acidophilum and the yeast Saccharomyces cereVisiae indicate that the structure of proteasomes from all species is quite similar (3, 4). Proteasomes are barrel-shaped molecules assembled from four stacked rings. In the Thermoplasma proteasome, each end ring consists of seven identical R subunits, whereas 14 identical  subunits form the two inner rings. In eukaryotes, the two R rings are composed of seven distinct R subunits, and each of the two inner rings also contains seven different  subunits. The 19S regulatory complex and 11S regulator (REG) 1 are protein complexes that bind the end rings of the proteasome and activate its peptidase activities (5). The 19S regulatory complex consists of 18 distinct subunits, and it associates with the proteasome in an ATP-dependent reaction to form the 26S proteasome. This large energy-dependent protease not only removes abnormal proteins, it also controls a variety of important cellular processes such as cell cycle progression and gene expression by degrading important regulatory proteins (2, 5, 6-8). The 11S REG is a molecule composed of two homologous subunits, REGR and REG, that are reported to form a six- or seven-membered ring (9- 12). Its association with the proteasome dramatically activates the proteasome to hydrolyze fluorogenic peptide substrates in vitro, although 11S REG does not promote the degradation of intact proteins (13, 14). The 11S REG is being extensively studied since it appears to play an important role in Class I antigen presentation (15, 16), and it provides an excellent opportunity to understand how the proteasome can be activated. For instance, it has been shown that recombinant REGR and REG subunits can, by themselves, activate the proteasome to a very similar extent (11). However, when mixed, the two subunits preferentially form hetero-oligomers that activate the pro- teasome to a greater extent than either subunit alone (11). Solution of the REGR crystal structure demonstrates that recombinant REGR forms a heptamer (17). A highly conserved stretch of 10 amino acids in REGR and REG is critical for proteasome activation (18), and this region forms a loop on the presumed proteasome binding surface of each REGR subunit in the crystallized heptamer (17). Removal of the 25-30 residue, homolog-specific inserts from REGR † These studies were supported by Grant GM37009 from the National Institutes of Health and by grants from the Lucille P. Markey Charitable Trust and the American Cancer Society to M.R., and by grants from the NSERC (Canada) to K.G.S. * To whom correspondence should be addressed. Phone: 801-585- 3128. Fax: 801-581-7959. ‡ University of Utah. § These authors contributed equally to this work. | University of Manitoba. 1 DEAE, diethylaminoethyl; DTT, dithiothreitol; ESI-TOF MS, Electrospray ionization time-of-flight mass spectrometry; HPLC, high- performance liquid chromatography; PMSF, phenylmethanesulfonyl fluoride; REG, 11S proteasome activator; SDS, sodium dodecyl sulfate. 5651 Biochemistry 1999, 38, 5651-5658 10.1021/bi990056+ CCC: $18.00 © 1999 American Chemical Society Published on Web 04/08/1999