Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli

Volume 312, number 2,3, 157-160 FEBS 11716 © 1992 Federation of European Biochemical Societies 00145793/92/$5.00

November 1992

Expression of functional Thermoplasma acidophilum proteasomes in

Escherichia coli Peter Zwickl, Friedrich Lottspeieh a n d Wolfgang Baumeister Max-Planek-htstftut fflr Biochemle, Am Klopferspitz 18a, D-8033 Martinsried, Germany Received 21 September 1992 The two genes encoding the constituent subunits of the Thermoplasma ac/dophilum protcasome were expressed in Eseher/ehia coli yielding fully assembled molecules as shown by electron microscopy. The recombinant protcasomes were purified to homogeneity and were shown to have proteolytic activity indistinguishable from proteasomes isolated from T. acldophilum. Protaasome; Gene espressi6n; Arehaebacterium; Thermoplasma acidophihan

1. I N T R O D U C T I O N The proteasome is a high molecular weight (approx. 700 kDa), non-lysosomal multicatalytic proteinase ubiquitous in eukaryotic cells (for review, see [1] and [2]. While all attempts to detect proteasomes in eubacteria have been unsuccesful so far [3,4] proteasomes were recently found to occur in the archaebaeterium, Thermoplasma acidophilum [5]. Unlike eukaryotic proteasomes, which are composed of 10-20 different though related subunits, all in the molecular weight range from 20 to 35 kDa, the T. acidophilum proteasome is made of multiple copies of two subunits only, and fl, with molecular weights of 25.9 and 22.3 kDa, respectively. All the amino acid sequences of proteasoma! subunits from eukaryotes available to date can be related to either the ~- or the/~-subunit of the 7'. acidophilum 'Urproteasome ~ [6]. Also the basic molecular architecture is conserved from T,~ermoplasma to higher eukaryotes; four 7-subunit rings collectively form a cylinder- or barrel-shaped structure [7,8]. The ~-subunits are located in the two outer rings or disks, while the fl-subunits have been mapped to the two juxtaposed iLaer rings by means of immunoelectron microscopy [9]. In this communication we describe the co.expression of the genes encoding the ~- and fl-subunits of the T. acidophilum proteasome in Ezcheriehia coli yielding fully assembled znd functional proteasomes. This proCorrespondenceaddress: W. Baurneister, Max-Planck-lnstitutfar Biochemic, Am Klopferspltz 18a, D.8033 Martinsried, Germany. Fax: (49) (89) 9.578 2641. Abbre~qations: FPLC, fast protein liquid chromatography; NM~, 4methyl-7-eoumarylamide; OD~, optical density measured at 600 nm; pl, isodectric point; RBS, ribosome binding ~ire.

Published by Elsevter Science Publ/shern' B.V.

vides the basis for addressing important questions regarding proteasome assembly and function. 2. MATERIALS AND METHODS 2. I. Clon/ng of the ¢t. and the ~.gene into the expression ~,ectarpTT.$ Starting from the initial pUCl8 clone, which harbours the gene encoding the ,6-subunit of the T. acidophilutn proteasome on an Hindlll fragment, the Dral sites at position - 17 and 654 [6] wel~ used to clone the fl-gene into the Sinai-site of pUCl9. Clones with the ,8-gene in the desired orientation were used for cloning into ti~eexpression vector, pT7-5 ll0], by u ~ of the resl~ctive EcoRI and H/ndlli sites of the pUCi9- and the pT7.5 polylinker; this resulted in the plasmid, pT7-5-fl. The gene encoding the --subunit of the 1". acidophihun p r o t ~ o m e was amplified by a pol~nnerasechain reaction. Two oligonucleotides, one with a flanking Pstl-site (5".A~CTGCAGATTATAGTGGC"I'GGAGG-3') and one with a flanking, H/n,dlllosite (5"-TATAAGCTTGCGATTCAAG-OCGGGG-3')were used to amplify the ~t-genecoding region. These restriction sites were used for cloning the amplified fragment into the Pstl and Htndlli sites of pUCI9. The KpnI. and H&dlll-sites of this plasmid were used to clone the ~-gene into the pT7-5-/~ plasmid. The resulting construct, p'r7-5-fl-at, contained the ~- and fl-gene with their respective putative ribosome binding site (RB$) under the transcriptional control o? the ~10 promoter (Fig. 1). DNA sequencing according to the dideoxynueleotide method [I I] confirmed that the cloning procedures did not change the DNA sequent. K coil BL21(DE3) cell5 [12], which contain the 1"7 .oolymerase gene in their genome under the control of file lacUV5 promoter, were transformed with the plasmid p'l'7.5-fl~. The bacteria were grown in LB-medium to an O D ~ of approximatdy 0.8 and induced with 0.5 mM isopropyl-fl.D-thiogalactopyranoside overnight. 2.2. Purification of the recombinant proteasomes Harvesting of cells from induced overnight cultures, preparation of spheroplasts, sonieation and nucleic acid digestion were performed as described by Lin and Cheng [13]. The lysate was further fractionated by ultraeentrifugation (100,090 ×g, 1.5 h) and the r~ulting supernatant was loaded onto a flepharose 6B column. Subsequent purification was achieved by DEAE-Sephac¢l and hydroxlyapatite chromatography as described previously Ibr the isolation of line prote/a.~omesfrom 1". acldophilum [7]. Fractions of the columr~swere ass.~y~l for proteolytie activity with the fluorigenic peptide subswate, Sue-Leu-Leu-

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pT?-5-1~-~ ~OPromoter,

EcoRI Kpnl

P=t I

Hlndlll

^~

/

%

/

/ \

/

%

/ %

~.I~I~TTGATGTTR T 6

\

/

%

~TGnRP,GAGflTflflCflT6

Fig,. I. Schematic drawing, of the ptasnfid g,T7-5.fl-a u~d for expressing the proteasome in E. colt. Restriction sites for cloning are indicated by vertical arrows. Direction of transcription is h~dieat~l by a horizontal arrow. The nueleotld¢ sequence of the putative RBSs' and the initiation eodons of the two genes are underlined or written in bold type letters, respectively.

VaI-Tyr-NMec. For Western Blot analysis the samples were separated by Trieine-SDS.PAGE, blotted onto nitrocellulose, and probed with antiproteasome antibodies [9]. Two-dimenslonal gel electrophoresis, eleetroblotting and microseqaence analysis were performed as described by Eckerskorn et al. [14].

3. RESULTS AND DISCUSSION We expressed the proteasome of T. acidophilum in E. coR, using the T7 polymerase expression system [10,12], since it was suceesfully applied for the expression of several archaebaeterial ribosomal proteins [15,16]. The ~z- and ,3-gene were subeloned into pT7-5, making use of their own putative ribosome binding sites (RBS) (Fig. 1). Recognition of archaebacterial RBSs' by the E. coli translational machinery has been demonstrated [15,16], particularly for the T. aeidophihon citrate synthase gene .[17,18]. We organized the two genes into an operon yielding pT7-5-fl-~z and demonstrated by Western blot analysis that expression of both proteins in E. coli BL21 (DE3) occurs (Fig. 2B). To investigate whether fully assembled and functional recombinant proteasomes were produced, we applied the purification protocol developed for Thermoplasma cells to induced cultures of E. coli BL21(DE3)/pTT-5-,8-~. After final FPLC/Superose 6 chromatography recombinant proteasomes were purified to homogeneity as judged by SDS-PAGE (Fig. 2A). Electron micrographs of negatively stained purified recombinant proteasomes showed barrel-shaped complexes indistinguishable from T. acidophilum proteasomes (Fig. 3). Assembly of proteolytieally active recombinant proteasomes is demonstrated by their similar rate of degradation of two synthetic substrates, as compared to proteasomes purified from 7". acidophilum (see Table I). In contrast to the ~z-subunit purified from T. acidophilure, the recombinant 0~-subunit was aeeessible to Nterminal protein sequencing, y~.elding an amino acid sequenc¢ (I-MQQGQMAYDRA-I, /wmcn was identical to the sequence derived from the gene [19]. Protein sequencing of the N-terminus of the recombinant fl-subunit gave the same amino acid sequence as determined I

158

~

.

t

I

t

for the T. acMophilvm protein (9-TTI'VGITLKDA-19) [6]. This implies that the processing of the first 8 amino aeids of the fl-subunit takes place in E. coli as it does in T. acidophilum. This processing of the ,S-pro-region is probably effeeted in an autocatalytie reaction, which is dependent on the presence of the 0~-subun~t. This latter conclusion was based on the observation that expression of the fl-subunit alone does not result in the removal of the pro-region (data not shown). As also reported for proteasomes purified from T. acidophilum [6], 2D gel electrophoresis of purified recombinant proteasomes gave rise to multiple protein spots (Fig. 4). N-Terminal protein sequencing of the two major protein spots with approximate pI's of 5.6 and 5.8 gave identical sequences, identifying them unambigously as ~z-subunits differing in their isoeleetrie

A 1

B 2

3

4

5

1

2

3

4

. ..i":.~ • .,..

,~... :~:,

~.~:,.. ~,:. ¢........... .'~ .,.~?, .,:~. ,..: .. ;.. ~ ?

• ..:..

Fig. 2. Tricinc-SDS-PAGE and Western Blot analysis of E, colt BL21(DE3) cell lysates and of purified proteasomes. Panel A shows Tricin~-SDS-PAGE stain,'d with Coomasslc blue G: Lane 1, 5/ag, molecular ma~s standards (carbonic anhydrase, 29 kDa; soybean trypsin inhibitor, 21 kDa; cytoehrome c, 12.5 kD.'t; ltt*.lgtrypsin inhibitor, 6.5 kDa; and insulin, 3.4 kDa. Lanes 2 and 3, ¢ell-lysate of 1PTGinduced E. colt BL21 (DE3) transformed with pT7-5 or pT7-5-/i'-~z, respectively. Lanes 4 and 5, 10/.t B purified rt.'eombinant and native 7'. acidophihmt proteasomes, respectively. Panel B shows Western blot analysis of Trieine-SDS.PAGE probed wilh antibody to T. aetdoohi. lure protcasomes: Lanes 1 and 2, cell lysate of ll"TG-indueed E. eoR BL21 (DE3) transformed with pTT-5 or pT7-5-,fl-a, respectively, Lanes 3 and 4, 10 pg purified recombinant and native 7". acldophilum proteasomes, respectively.

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Table 1 Rate of degradation of synthetic substrates by purified recombinant and native T. acidophihtm protcasomes Substrate

Activity (nmol/mg proteasomes per rain) 37°C

Recombi- Native Recomhinant nant Su¢-Leu-Leu-VaI-Tyr.NMec 0.13 Z-Gly-Gly-Leu-NM¢c 0.20

N

60°C

0.10 0.17

1.01 0.6l

-- 4g,--

Native 0.79 0.50

Activity was determined by incubating 4.1/zg el'protein with 500 pmol ofsubstrate for I h at the given temperature and fluorimetrie measure. ment of enzymatically rclcaaed 4-methyl-7-coumarylamide (NMee) 15].

points; this corroborates our previous conclusions based on amino acid composition analysis [6]. Since it was shown for the Drosophila melanogaster ~t-type subunit PROS28.1 [20] and for two subunits of the bovine multicatalytie proteinase complex (MPC) [21] that they are phosphorylated, and since putative phosphorylation sites exist in the primary sequence of the T. acidophilum ~t-subunit [19], it is quite possible that the isoelectric variants of the ct-subunit purified from I". acidophilum and £. cell result from phosphorylation. Recently it was shown that the eukaryotie proteasome is involved in the ubiquitin-dependent N-end rule pathway of degradation [22,23]. This N-end rule pathway also exists in E. coil and the ATP-dependent pretense, Clp (Ti), is required for degradation of N-end rule substrates [24]. Therefore it will be of interest to investigate whether the arehaebacterial proteasome can complement this function in the null clpA- mutant [24].

Fig. 3. El~tron mierograph of negatively stained purified recombinant T. aeidophilum proteasomes. Bar = 100 rim.

.

C

Fig. 4. 2D gel eleetrophoresis of purified recombinant T. actdophilum proteasomes. Horizontal: isoeleetrle focusing, cathode left and anode right. Vertical: SDS-PAGE. A total of 20 ,ug of protein was applied. The two major protein spots with approximate pl's of 5.6 and 5.8 were identified as a-subtmits by N-terminal protein sequencing. The protein spot with an approximate pl of 6.8 corresponds to the p-subunit.

In conclusion co-expression of the two genes encoding the 1". acidophtlum proteasome in E. cell yields correctly folded and assembled proteolytieally active proteasomes, which do not influence the viability of their host cells. This allows detailed structural and functional investigation to be performed including site directed and deletion mutagenesis. Acknowledgements: The authors wish to thank Mrs. U. Santarius for doin 8 the electron microscopy. A K6pke (GOttingen) for providing E. coli BL21 (DE3) c~lls and for advice, S, Tabor (Boston) for providing pT-7 vectors and instruction manuals and Mrs. A. Erdmann and Mr. R. S¢imet for ¢~perimental help.

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[15] K6pk..~, A.K.E,, Paulke, C. and Gewitz, H.S. (1990) J. Biol. Chem. 265, 6436-6440. [16] K~pkg, A.K.E., Hannemann, F. and Bogckh, T. (1991) Biochim. 73, 647--65:5. [17] Sutherland, K.J., Henneke, C.M., Towner, P., Hough, D.W. and Danson, M.J. (1990) Ear. J, Biochgm, 194, 839-844. [18] Suthgrlaad, K.J., Danson, M.J., Hough, D.W. and Townet, P. (1991) FEBS Lett. 282, 132-134. [19] Zwickl, P., Lottspeich, F., Dahlmann, B, and Baumeister, W.

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(1991) FEBS Lett. 278, 217-221. [20] Haass, C. and Kloetzel, P.M. (1989) Exp. Cell Res. 180, 243-252. [211 Pereira, M.E. and Wilk, S, (1990) Arch. Biochem. Biophys. 283, 68-74. [22] Richter-Ruoff, B., Heinemeyer, W. and Wolf, D.H. (1992) FEBS L~lt, 302, 192-196, [23] Seufert, W, and Jentsch, S. (1992) EMBO J. 11, 3077-3080. [24] Tobias, J,W., Shrader, T,E., Rocap, G. and Varshavsky, A. (1991) Science 254, 1374--1377.