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The Proteasome from Thermoplasma Acidophilum Is Neither a Cysteine Nor a Serine Protease

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The Proteasome from Thermoplasma Acidophilum Is Neither a Cysteine Nor a Serine Protease FEBS 15108 FEBS Letters 359 (1995) 173-178 The proteasome from Thermoplasma acidophilum is neither a cysteine nor a serine protease Erika Seemiiller, Andrei Lupas, Frank Ziihl, Peter Zwickl, Wolfgang Baumeister* Max-Planck-Institut fiir Biochemie, D-82152 Martinsried, Germany Received 22 December 1994; revised version received 10 January 1995 mum of the proteasome tends to be slightly basic, making it Abstract The 20 S proteasome, found in eukaryotes and in the unlikely that the enzyme is an aspartic protease [6]. Similarly, archaebacterium Thermoplasma acldophilum, forms the prote- no inhibition is observable by up to 10 mM EDTA, by other olytic core of the 26 S proteasome which is the central protease chelators, or by phosphoramidon, making it unlikely that the of the non-lysosomal protein degradation pathway. Inhibitor stud- ies have indicated that the 20 S proteasome may be an unusual proteasome is a metal protease [6,7]. Inhibition experiments type of cysteine or serine protease and a recent study of the with various cysteine and serine protease inhibitors have Thermoplasma [3 subunit has indicated that it carries the prote- yielded ambiguous results and have mostly been successful at olytic activity. We have attempted to obtain information on the high or very high inhibitor concentrations [5-9], generally in- nature of the active site by mutating the only cysteine, both hibiting only one of the different hydrolytic activities and some- histidines and two completely conserved aspartates in the times concurrently activating others [5]. On the basis of these archaebacterial complex as well as all serines of the/3 subunit, experiments, the proteasome has been variously described as a without decreasing the catalytic activity of the enzyme to any serine or cysteine protease, with most recent opinions favoring significant extent. Indeed, mutation of the conserved aspartate in an unusual type of serine protease [5 8]. the/3 subunit increased the activity of the proteasome threefold. The proteasome of Thermoplasma is much simpler than the We conclude that the proteasome is not a cysteine or serine eukaryotic proteasomes and has only a single, chymotrypsin- protease. like activity, although experiments with denatured haemo- Key words: Proteasome; Proteolysis; Archaebacterium; globin and insulin ft-chain show that the enzyme will hydrolyze Thermoplasma acidophilum virtually any peptide bond [10]. Both subunits of the Thermo- plasma proteasome have been cloned and overexpressed in Escherichia coli [11], opening the system up to easy genetic manipulation. In addition, since the Thermoplasma proteasome 1. Introduction contains only one type of active site, mutations are considerably simpler to interpret than in eukaryotic proteasomes. We have The 26 S proteasome is the central protease of the ubiquitin- therefore focused on this system in order to elucidate the nature dependent pathway of protein degradation and has recently of the proteasome active site. Although we have mutated resi- been shown to play an important role in MHC class I-linked dues in both c~ and/3 subunits, we have focused on the ft antigen display. The core of the elongated 45 nm molecule is subunits because these carry the active site: when expressed formed by the multicatalytic proteinase or 20 S proteasome, a separately, cc subunits assemble into 7-membered rings but re- barrel-shaped complex consisting of four seven-membered main inactive, while ft subunits have a low but significant pro- rings (reviewed in [1 3]). The rings are formed by 14 related but teolytic activity even though they only form disordered aggre- different subunits that fall into two families, o-type subunits gates [12]. forming the outer and ft-type subunits the inner rings of the In this communication we show that no histidine or cysteine complex. 20 S proteasomes, ubiquitous in eukaryotes, have also residues are involved in the catalytic mechanism of the Thermo- been found in the archaebacterium Thermoplasma acidophilum, plasma proteasome. In addition we also exclude all serine resi- where the complex is made up of only two proteins, ~ and ft, dues of the ft subunit as well as several conserved serines of the that have given their names to the eukaryotic subunit families. subunit and the universally conserved aspartate (~D84, The role of the 26 S proteasome as the central enzyme of the ftD51). Indeed, mutation of the latter in theft subunit increases non-lysosomal protein degradation pathway has generated the activity of the complex at least threefold. considerable interest in the activity and regulation of the 20 S core complex. The latter was initially characterized from bovine pituitary as a multicatalytic protease with chymotrypsin-like, 2. Materials and methods trypsin-like and peptidylglutamyl-peptide hydrolase activities 2.1. Materials [4], and has more recently been proposed to contain up to five Enzymes for DNA restriction and modification were obtained from different proteolytic components on the basis of inhibitor stud- New England Biolabs, Stratagene and USB. Oligonucleotides for in- ies [5]. Yet, despite intensive biochemical efforts, the nature of verse PCR mutagenesis were synthesized on an Applied Biosystems the proteasome active-site(s) remains unknown. The pH opti- 380A DNA synthesizer. The synthetic fluorescent peptide Suc-Leu- Leu-VaI-Tyr-AMC (7-amido-4-methylcoumarin) was obtained from Bachem, Heidelberg. For screening and propagation of plasmids, we used the £ cob strain XL1-Blue [13]; for expression of wild-type and mutant proteasomes E. coli BL21 (DE3) [ 14]. All transformations were *Corresponding author. Fax: (49) (89) 857 82641. performed by electroporation using an E. coli Pulser from Bio-Rad E-mail: [email protected] Laboratories. 0014-5793/95/$9.50 © 1995 Federation of European Biochemical Societies. All rights reserved. SSDI 0014-5793(95)00036-4 174 F~ Seemiiller et al./FEBS Letters 359 (1995) 173 178 2.2. Construction of mutants directly ligated. Incorporation of nucleotide exchanges was confirmed Mutations were introduced by inverse PCR mutagenesis. Two plas- by DNA sequencing. mids, pT7-5-,8-e and pT7-5-flA-~ [11], that contain the genes encoding the ¢z and fl subunits of the T. acidophilum proteasome, were used as 2.3. Preparation of cell extracts for the primary screen templates. ,sA stands for a deletion of 21 5' nucleotides coding for the Cultures of transformed BL21(DE3) cells were grown in SOB me- ,8 pro-region. The PCR reactions and ligations were performed as dium (100,ug/ml ampicillin) to an OD600 of 0.8 and induced with 1 mM described [15], but Pfu polymerase (Stratagene) was used in place of isopropyl-,8-D-thiogalactopyranoside. Cells were harvested 5 h after Taq polymerase, since this enzyme generates blunt ends which can be induction, washed with 10 mM Tris-HC1, 100 mM NaC1, 1 mM EDTA, i0 20 30 40 50 60 70 80 90 Rn C8 Rn-Iota Dm-Pros28 Rn-C9 Rn-C3 Rn-C2 Rn-Zeta Ta~alpha N I0 20 30 40 50 60 Rn C7 MEYLIGIQGP~L~S~RVAASNIVQMKDDHD~KMSEKILLLCV~E~G~TVQFAEYIQKN Rn-C5 RFSPYVFNG~IAGE~FS T~L~E~FSIHTRDSP~CYKLTDKTVIGC~FHG~CLTLTKIIEAR Rn-Cl 0 MSIMSYNGGAV~KGKNC~AI~R_~FGIQAQMVTTDFQ~IFPMGDRLYIGLA~L~T~TVAQRLKFR Rn-3 TQNPMVT~TSVLGV~DC~I~LG~Y~SLARFRIISRIMRVNDSTMLGA~D~YLKQVLGQM Hs-Mec!l ~IAGL ~ GA~T~ATNDSVVADKSCE~IHFIAPKIYCCG~EMTTRMVASK Rn-Lmp2 ~IMAV~ S~S~A~kAVVNRVFD~LSPLHQRIYCAL~S~IADM_AAYQ Rn_Lmp 7 ~TLAF~ SF,A~SYIATIRV~IEINPYLLGTM~~ERLLAKE Ta Beta ~TVGITL TER~VTME~NFIMHKNGK~LFQIDTYTGMTIA~LV~LVRYMKAE A N E i00 ii0 120 130 140 150 160 170 180 Rn C8 Rn-lota Dm-Pros28 Rn-C9 Rn-C3 Rn-C2 Rn-Zeta Ta~alpha V A 7o ~o 9o ~oo no ~o ~3o z4o Rn C7 m~Y~LS~IN~CLRS ....... ~Z~V~LL~HE~-~YU~L~-~~L~LS~ ....... Rn-C5 ~ KHSNNKAMTTG~I~STI~SR~ ......... FF~YVYNI~E~@EEGKG-f~S~PV~SYQ-RDS~K~LQPL~NQVGFKNMQN Rn-Cl 0 ~E~QI~e~L~S~V~4- ........ ~~~I~S~U~C~m~IVVS~C~Q~S~W ....... Rn-3 VIDEE~FGDGHSYSPR~IHSWLTRAMYS~RSK ...... ~LWNTKV~G~YAGGES--F~GYV~AY-EA~SI~T~Y~YLAQPL~REVLEK ..... Hs-Mecll ME~A~ST~REPRVATVTRI~RQT~FRYQ .......... GHVGAS~IVG~LTGP--~GVHPH~SYS-RL~T~I~QDAALA~EDRF ....... Rn_-Lmp2 |E~HG|ELEEPPLVL~|NIVK~ISYKY~ .......... EDLLAH~VA~W~QHE---GGQVYGT~MLIR~IG~TYI~ ....... Rn_Lmp7 CR~Y~RN~ERISVS~[SKL~$~MLQY~ ......... GMGLSMGSM~KKGP-- RL-SGQMFS ....... Ta Beta ~E~P~Q~VNMPIE~V~TL~S~QVK ......... YFf~yMV~LVG~I~-TAP--HVFSIDAA~SV-EDIYAS ....... A A A A A AA A 190 200 210 220 230 Rn C8 -MK~MTCRDVVKEVAKIIYIVHD~---VKDKAF~LELS~ELTKGRHEIVPKD~REEAEKYAKESLKEEDESDDDNM Rn-Iota -KKKFDWTF~QTVETAITCLSTVLSIDFKPSEI~GWT~ENPK--~I~TE~IDAH~VALAERD Dm-Pros28 --R~EVAN|~HGAVKLAIRALLEV-AQSGQNNL~IME~Kp---L~NDVITD~KIIEKEKEEELEKKKQKK Rn-C9 -KEGEMTLKS~LALAVKV~NKTMDVsKLSAEKV~I~TLTRENGKTVI~V~KQK~EQLIKKHEEEEAKAEREKKEKEQREKDK Rn-C3 --N~DLELED|IHTAILT~KESF|-GQMTEDNI~GICNEAG .... ~R~TPT~DY~AIA Rn-C2 -SEFMQCNLDEL~K~GL~LRETLPAEQ~LTTKN~SIGI~KD-L~TIYDD~D~SPF~GLEERPQRKAQPSQAADEPAEKADE~ME~ Rn-Zeta --HKSTTL~IKSSLII~<QVM~-EKLNATNI~L~TVQPgQN---~HMFTKE~LEEVIKDI Ta~alpha --~NLPE~IVTLGIKA~SSL|-EGE~L~P|I!SI~NK---~mIYDQE~K~ 150 160 170 180 190 200 Rn C7 -- T~T I SP~R~E~LRKCLEELQK~F I LNLP TF S~RV~D~D~I HNLENI TF TKRS S Rn-C5 VEHVP~LD~maWDVFIS~EmW~ALRICI~E~IRE~TVW R~D Rn-Cl 0 - -E~D P~H LFE T I SQ~4LN~VDM~AV~GV I~H I~:ET~D K IT T RT LKARMD Rn-3 - -Q~VL SQ~RE~VERCMRVLYY~A~NRFQ~TV~ EK~VE IEGP LSAQTNWD IAHMI S GFE Hs-Mecll - -Q~'QG~LVE~VTAGI LG~G~ACV~KLLRTLS S P TEPVKRS GRYHFVPGTTAVLTQTVKP LTLELVEE TVQAMEVE Rn_-Lmp2 --Kr, DDCR~_TIIT~SIRVIY~VT~DHRVI~ GDELPK~YDE Rn_Lmp7 - -RQDLSP~YD~ARR~IVY~TH~SY~HM-~GWVKVE S TDVSDLLHKYREATL Ta Beta -- S EK~VD~GVD~VI R~I SA~KQ~SA~MID~AV~RKDGYVQLP TDQ IE SRI RKLGL I L A A AA A Fig.
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