View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus Tomohiro Tamura*t, Istvin Nagy**, Andrei Lupast, Friedrich Lottspeicht, Zdenka Cejkat, Geert Schoofs*, Keiji Tanaka§, Rene De Mot* and Wolfgang Baumeistert tMax-Planck-lnstitute for Biochemistry, D-82152 Martinsried, Germany. F.A. Janssens Laboratory of Genetics, Catholic University of Leuven, B-3001 Heverlee, Belgium. §Institute for Enzyme Research, University of Tokushima, Tokushima 770, Japan. Background: The 26S proteasome is the central protease chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in of the ubiquitin-dependent pathway of protein degrada- the presence or absence of 0.05 % SDS. Purified prepara- tion. The proteolytic core of the complex is formed by tions reveal the existence of four subunits, two of the the 20S proteasome, a cylinder-shaped particle that in a-type and two of the 3-type, the genes for which we archaebacteria contains two different subunits ( and 3) have cloned and sequenced. Electron micrographs show and in eukaryotes contains fourteen different subunits that the complex has the four-ringed, cylinder-shaped (seven of the a-type and seven of the 3 -type). appearance typical of proteasomes. Results: We have purified a 20S proteasome complex Conclusions: The recent description of the first eubac- from the nocardioform actinomycete Rhodococcus sp. strain terial ubiquitin, and our discovery of a eubacterial pro- NI86/21. The complex has an apparent relative mol- teasome show that the ubiquitin pathway of protein ecular mass of 690 kD, and efficiently degrades the degradation is ancestral and common to all forms of life. Current Biology 1995, 5:766-774 Background proteasome at 3.4 A resolution [15,16] and the identifica- tion of its catalytic nucleophile, a threonine residue Proteasomes are ubiquitous multisubunit proteases of (PThrl) of the 13 subunit [16-18]. eukaryotic organisms. The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein Recently, a scan of the GenBank database revealed the degradation [1-4] and plays a key role in many cellular existence of several proteins from eubacteria with processes including the cell cycle [5,6], transcriptional sequences that show significant similarity to those of regulation [7,8] and antigen presentation [9]. The protein proteasome -type subunits [19]. Although most of them is formed by two, asymmetric, 19S cap complexes occur in an operon with a ClpX-related ATPase, and attached to the ends of a barrel-shaped 20S particle [10]. may form structures more similar to the Clp protease The 20S particle, generally referred to as the multi- than to proteasomes, one protein, from Mycobacterium catalytic proteinase or 20S proteasome, consists of four leprae, is significantly more similar to the Thermoplasma seven-membered rings that contain fourteen related but subunit and may form a true proteasome. In this article, different subunits [11,12]. These fall into two families, we report the discovery, purification and characteriza- with oa-type subunits forming the outer rings and 13-type tion of a 20S proteasome complex from a nocardioform subunits the inner rings of the complex. actinomycete, Rhodococcus sp. strain NI86/21, that is closely related to Mycobacterium. This discovery extends Until their discovery in the archaebacterium Thermo- the occurrence of proteasomes to the third kingdom plasma acidophilum [13], proteasomes were thought to and shows that the proteasome is an ancestral particle of occur exclusively in eukaryotes. Following this discovery, universal distribution. searches were made for proteasomes in other archaebac- teria and in eubacteria without success [14], leading to speculation that a Thermoplasma-like organism may have Results been the precursor of the eukaryotic cytosol. Although structurally indistinguishable from eukaryotic protea- Cloning and sequencing of an operon containing somes at a resolution of 2 nm, the proteasome of Thermo- proteasome-related genes plasma is much simpler, being formed of only two During the analysis of the thc gene cluster of Rhodococcus subunits, a and [3 [13]. This simplicity has allowed the sp. strain NI86/21, which is required for degradation of determination of the structure of the Thermoplasma thiocarbamate herbicides and the s-triazine herbicide *The first two authors contributed equally to this work. Correspondence to: W. Baumeister. E-mail address: [email protected] 766 © Current Biology 1995, Vol 5 No 7 Characterization of a eubacterial proteasome Tamura et al. RESEARCH PAPER 767 Characterization of a eubacterial proteasome Tamura et al. RESEARCH PAPER 767 atrazine [20,21], four open reading frames (ORFs) were chromatography on Sepharose 6B (Fig. 2a) and tested identified downstream of thcR (Fig. la). One of the the eluted fractions for Suc-Leu-Leu-Val-Tyr-AMC- encoded proteins, PrcB, is clearly related to proteasomal hydrolyzing activity in the presence and absence of 3-type subunits, and a second, PrcA, shows a similarity 0.05 % SDS. Two peaks of activity, eluting at apparent of unclear statistical significance to or-type subunits. molecular masses of 500-700 kD and 200-300 kD, were Database searches revealed a region with a strikingly sim- obtained in the absence of SDS. In the presence of SDS, ilar gene organization in the genome of the related the low molecular weight fraction lost all activity, whereas nocardioform actinomycete Mycobacterium leprae (Collab- the high molecular weight fraction showed no change of orative Research Inc., GenBank accession U00017; Fig. activity, indicating that it may contain proteasomes. We 1c). The similarity between the regions is highly signifi- cant at the protein level (61-81 % sequence identity), the main difference being a large amino-terminal deletion in Rhodococcus Orf6 relative to Mycobacterium C1_172. At the DNA level, similarities include a putative ribosome- binding site found in front of the start codons of orf7 (AAGGAGG) and oX (AGGAGG), the unusual GUG start codon of prcB, and the apparent translational coupling of the of7-prcB-prcA and ofx-prcB-C3_260 genes, as inferred from the two-base overlaps of the stop and start codons of the gene pairs orf7/odX-prcB and prcB-prcA/C3_260 (data not shown). Purification and biochemical characterization of a 20S proteasome complex In order to isolate the proteasome complex from Rhodococcus, we tested the hydrolyzing activity of crude cell-extract fractions using the substrate Suc-Leu-Leu- Val-Tyr-AMC, which is a typical fluorigenic substrate for chymotrypsin-like activity. Previous studies had shown that low concentrations of SDS do not affect the chymotryptic activity of proteasomes from Thermoplasma (unpublished data) and indeed activate the chymotryptic activity of most eukaryotic proteasomes [22,23]. We sub- jected crude cell extracts of Rhodococcus to gel filtration Fig. 2. Identification of an SDS-resistant, high molecular weight Fig. 1. Gene organization of (a) the DNA region downstream of protease in Rhodococcus sp. strain N186/21. (a) Crude cell thcR in Rhodococcus sp. strain N186/21, (b) the homologous extracts (163 mg) were chromatographed on a Sepharose 6B col- region in the same strain, and (c) the equivalent region in umn as described in Materials and methods, and total protein Mycobacterium leprae cosmid B2126 (GenBank accession No. was detected by absorbance at 280 nm (red line). The column U00017). The black arrowheads indicate the position of a poten- fractions were assayed for the hydrolysis of the synthetic peptide tial transcription terminator (stem-loop structure with Suc-Leu-Leu-Val-Tyr-AMC in the presence (white circles) or AG =-160 kJ). The sequence of of61, up to the Sacl site, was absence (grey circles) of 0.05 % SDS. (b) Tricine-SDS-PAGE determined previously 20]. The hybridization probes used for analysis and (c) peptidase activity of fractions obtained by Super- identification of overlapping fragments are shown as black bars. ose 6 FPLC column chromatography. This column represents the One major scale division represents 500 bp. Only restriction sites last step in the purification procedure. The column was cali- mentioned in the text are shown. The sequences of the 3 751 bp brated with the markers thymoglobulin (669 kD), apoferritin BamHI-Bglll fragment (accession No. U26421) and the 3 554 bp (443 kD) and alcohol dehydrogenase (150 kD). In panel (b), a BstXI-Pstl fragment (accession No. U26422) have been submitted lane carrying Thermoplasma caand [3 proteins (T.A.) has been to GenBank. added for comparison. 768 Current Biology 1995, Vol 5 No 7 therefore subjected this fraction to further purification were determined by Edman degradation. Internal using a sequence of conventional chromatography steps. sequence information was obtained by digesting each band in situ using Lys-C, separating the products by high The putative Rlhodococcus proteasome was purified to over pressure liquid chromatography (HPLC), and subjecting 95 % homogeneity by the method outlined in Table 1. them to Edman degradation. The 31 kD and 29 kD bands The overall purification was approximately 440-fold, and were amino-terminally blocked. Two internal peptide the yield was 46 %. The native molecular mass of the parti- sequences from the 31 kD band were found to be identi- cle was estimated to be approximately 690 kD by fast pro- cal to sequences encoded by the prcA gene, whereas four tein liquid chromatography (FPLC) (Fig. 2c). The substrate internal sequences from the 29 kD band, covering about a specificity of the particle was similar to that of the archae- third of the protein, were found to be related but non- bacterial proteasome [24] and showed high activity against identical to the prcA gene product (Fig. 3). We concluded the chymotryptic substrates Suc-Leu-Leu-Val-Tyr-AMC, that the two proteins did not arise from one precursor by Z-Gly-Gly-Leu-AMC and Suc-Ala-Ala-Phe-AMC, posttranslational processing, but were the products of dif- but no tryptic or peptidyl-glutamyl-peptidase activities ferent genes.