functional expression and molecular characterization of atubc2-1, a novel ubiquitin-conjugating...

Plant Molecular Biology 23: 387-396, 1993. © 1993 Kluwer Academic Publishers. Printed in Belgium. 387

Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana

Dieter Bartling, Peter Rehling and Elmar W.Weiler Lehrstuhl fiir Pflanzenphysiologie, Ruhr-Universitgit Bochum, Postfach 102148, D(W)-4630 Bochum, Germany

Received 1 April 1993; accepted in revised form 12 July 1993

Key words:Arabidopsis thaliana, ubiquitin carrier protein, ubiquitin-conjugating enzyme, ubiquitin-dependent proteolysis

Abstract

The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana. Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L. Sullivan and R.D. Vierstra, Proc. Natl. Acad. Sci. USA 86 (1989) 9861-9865). The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa. The protein encoded by AtUBC2-1 is shown to accept ~25I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions. It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme.

Introduction

In eukaryotic cells, one of the major protein degradation pathways is provided by the ubiquitin- dependent system of proteolysis (for reviews, see [ 12, 16, 21, 22]), where ubiquitin is transferred, during a two- or three-step enzymatic reaction, to target proteins. From the analysis of mutants, especially in Saccharomyces eerevisiae, it was proposed that the ubiquitin-system plays a vital regu- latory role in the turnover of transcriptional

regulators [ 17 ], proteins of DNA repair [20], cell cycle control [13], peroxisome biogenesis [41] and the removal of incorrectly folded proteins under normal and in particular under stress conditions [33, 23]. Ubiquitin is a highly conserved small protein present in all eukaryotic cells [12, 16]. During the enzymatic ubiquitination cycle, its carboxy-terminal glycine residue is activated by ATP to form an adenylate which then serves to donate the ubiquitin moiety to a thiol group of the ubiquitin-activating enzyme (El).

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X68306.

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From the E1 complex, ubiquitin is further transferred to a specific cysteine of one of several ubiquitin-conjugating enzymes (UBC or E2-type enzymes, sometimes also called ubiquitin carrier proteins). These E2 enzymes attach ubiquitin, in the case of class I E2 enzymes, via a ubiquitin protein ligase (E3) or directly (class II E2 enzymes) to lysyl e-amino groups of the target protein. Ubiquitin may be linked to proteins as monomer, dimer or in the form of multiubiquitin units [ 12, 27]. The recognition of target proteins for ubiquitination largely depends on their N- termini (N-end rule) [2, 38]. This pathway was first described for rabbit reticulocytes [10, 16], later for yeast [20] and wheat [14]. In yeast and animal tissues, polyubiquitinated proteins are then degraded by a 26S proteasome, a tighly regu- lated, ATP-dependent protease complex [ 16, 31 ].

So far, ten genes encoding ubiquitin-conjugating enzymes (UBC 1-10, for review see [21, 22]) have been cloned from S. cerevisiae and shown to serve quite different cellular roles. Different E2 genes have been cloned from plants, but very little is known about their function, regulation and substrate specificity with respect to target proteins. The hundred-fold faster proteolytic degradation of the far-red-absorbing form of the pho- toreceptor phytochrome (P730), compared to the red-absorbing form (P660), has been proposed as due to ubiquitin-dependent proteolysis of P730 [ 19]. In plants, little is known about the compar- timentation of the ubiquitin-dependent proteolysis system. In addition to a nuclear and cytoplas- mic pathway, the chloroplast may be a site of ubiquitin-dependent proteolysis [39]. Due to functional and subcellular compartmentation, a quite large number of E2 enzymes and conse- quently genes is to be expected.

The small genome size of Arabidopsis thaliana (Brassicaceae), the availability of transformation systems and the low complexity of gene families in this species may help understand functions of the ubiquitin-conjugation system in higher plants during development, environmental changes, stress and senescence. Recently [36], the first ubiquitin-conjugating enzyme (AtUBC1) was reported from A. thaliana. AtUBC1 shows 82~o

identical amino acids to the wheat UBC1 and 63~o identical amino acids to the S. cerevisiae DNA repair gene RAD6 (UBC2) [35]. In this work, we report the molecular characterization, bacterial expression and proof of function of a novel ubiquitin-conjugating enzyme (E2) desig- nated, using the nomenclature established for A. thaliana [36], as AtUBC2-1. AtUBC2-1 is part of a small gene family of two members which seem to be quite different from the AtUBC 1 gene. AtUBC2-1 gene is therefore a good candidate to investigate one of the several functions as well as the selectivity ofubiquitin-dependent processes in plants.

Materials and methods

Plant material and cDNA screening

Arabidopsis thaliana (L.) Heynh. plants were grown in short days (8 h) at 24 °C (day) and 20 °C (night) at a relative humidity of 70-90~o and 90 W m 2 irradiation, cDNA from poly(A)- rich RNA, prepared from leaves of six-week old plants, was unidirectionally cloned into the 2ZAPII vector [34] and packed into phage in vitro according to the manufacturer's instruction (Stratagene, La Jolla, CA) with 2 x 10 6 recombi- nants per/~g cDNA. The cDNA encoding the ubiquitin-conjugating enzyme was identified by subtractive immunoscreening procedures as previously described [4, 5].

DNA sequencing and sequence analysis

Sequencing of the originally isolated clone PM42 encoding AtUBC2-1 and of multiple overlapping restriction fragments subcloned into Bluescript (pB SC-SK) (Stratagene) was carried out on both strands as described in the protocol of Sequenase (USB, Cleveland, OH) with (c~-35S)-dATP (Amersham Buchler, FRG). Nucleotide sequences were analysed using the DNASIS program (Pharmacia, Freiburg, FRG). The deduced amino acid sequence was compared with the EMBL/SwissProt databases using HUSAR

Fastp. Amino acid sequences were aligned with the Clustal 5 program [ 15].

Southern hybridization

DNA from Arabidopsis thaliana was digested with either restriction enzyme Hind III or Eco RI, subjected to electrophoresis in a 0 .8~ agarose gel and blotted onto Hybond-N membranes (Amer- sham Buchler, FRG) according to the 'Southern' procedure [ 1 ]. Prehybridization at 64 ° C for 4 h and hybridization at 64 °C for 16h was per- formed with random-primed Biotin-16-dUTP probes (Boehringer, FRG) in a solution containing 1 mM EDTA, 7~o SDS, 0.25 M Na2HPO4, 5% dextran sulphate (Sigma, F R G ) a n d 100 #g/ ml sonicated salmon sperm DNA. Filters were washed accbrding to the Southern-Light protocol (version M, Tropix, USA), developed with alka- line phosphatase-conjugated streptavidin and di- sodium 3-(4-methoxyspiro{ 1,2-dioxetane-3,2'- (5'-chloro)tricyclo [ 3.3.1.13,7 ] dec an }-4-yl)phenyl phosphate (CSPD) (Tropix) as a substrate and exposed to Kodak XAR X-ray films for 3 h. To identify genomic DNA fragments corresponding to the cDNA PM42, hybridization was also carried out with a 5'-terminal Eco RI and a 3'- terminal Eco RI/Xho I subfragment of cDNA PM42 as a biotinylated probe.

Construction of protein expression clones and expression of ubiquitin-conjugating enzyme (E2) in E. coli

A fragment o fcDNA PM42, generated by Hae III (bp position 30) and Kpn I (pBSC-SK, multiple cloning site) was ligated into pBSK-SK Sma I/ Kpn I restriction sites and transformed into E. coli DH5~ cells [1] resulting in the clone 1577-10. To isolate protein inclusion bodies for protein purification, a Hae III/Xho I fragment of cDNA PM42 was ligated into Sma I/Xho I re- stricted pEXP3 vector [29] and transformed into E. coli W3110 [7] resulting in the clone 1638-13.

For the expression ofplasmid 1577-10 in E. coli

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DH5a, 4ml of LB medium [1] was inoculated with 50 gl of a stationary 1577-10 preculture with 40 #g/ml ampicillin in 2TY medium (16 g Bacto trypton, 10 g yeast extract, 5 g/l Nac1). The culture was shaken at 220 rpm and 37 ° C for 1 h and then adjusted to a concentration of 10mM isopropyl-fi-D-thiogalactopyranoside (IPTG). The culture was maintained under these conditions for further 4 h. The cells were then har- vested by centrifugation and incubated in 175 mM Tris-HC1 pH7.5, 2mg/ml lysozyme (Sigma, FRG) for 15 min, frozen twice at -20 °C and sonicated for 10 s. Cell debris was sedimentated at 12000xg for 15 min at 4 °C and the supernatant used as a crude extract for further studies. The expression of 1638-13 in E. coli W3110 followed the same procedure with the exception that a final concentration of 0.2 mM IPTG was used to induce the tac-promotor in the main culture. After harvesting the cells by centrifugation, protein from inclusion bodies and soluble protein was obtained by the procedure of [ 18]. Protein in sodium dodecyl sulphate (SDS)-containing gel loading buffer was separated on SDS-PAGE according to Laemmli [24], or on Tricine-SDS gels [30] containing 6 M urea. In order to produce anti-E2 antibodies, the protein solubilized from inclusion bodies resulting from the bacterial expression of clone 1638-13 was separated on SDS- PAGE. The band representing the overexpressed polypeptide was excised and the protein re- covered in a Biotrap electroelution chamber (Schleicher & Sch/all, FRG) with 25 mM Tris, 190 mM glycine, 0.025 ~o (w/v) S D S. The protein was precipitated with 4 volumes of acetone, dis- solved in a small volume of 10 mM K2HPO4/ NaH2PO4 pH 7.5 containing 150mM NaC1. Antisera were generated in six-week old Balb/c mice by immunization with 50/~g E2-fusion protein, mixed with an equal volume of Alumn-Inject (Pierce, Rockford, USA).

Labelling of 125I-ubiquitin and preparation (?f ubiquitin-activating enzyme (El)from wheat

Ubiquitin was labeled with 125| by a modification of the chloramine-T method previously described

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[9]. A mixture of 1.5 mg bovine ubiquitin (Sigma, FRG) in 50 #1 of 200 mM potassium phosphate buffer pH 7.5 (buffer A) was incubated with 2 mCi Na125I and 10 #1 chloramine-T (4 mg/ml buffer A) for 40 s. The reaction was terminated by the addition of 10 #1 Na2S205 (4 mg/ml in buffer A). Free 1251 was removed by column chromatography on Bio-Gel P-6 DG (Bio Rad, Richmond, USA).

Ubiquitin-activating protein (El) was isolated from wheat germ (Triticum vulgare) by affinity chromatography as described [14]. Following elution from the ubiquitin-Sepharose column [ 10, 14], in 500/A fractions of 50 mM Tris-HC1 pH 9.0, 10 mM dithiothreitol (DTT), the protein content was estimated by the Coomassie blue method [6] and purity was checked by SDS- PAOE.

Assay for ubiquitin-E1 and -E2 thiol ester binding

Formation of E 1- and E2-thiol ester adducts with ubiquitin was assayed essentially as described [40]. Conjugation of 125I-ubiquitin to E1 proteins was assayed in 25 #1 total reaction volume containing 1.6 #g 125I-ubiquitin (1.06/~Ci/#g protein), 0.4/~g E1 protein (10 #1 of the peak fractions from the ubiquitin-Sepharose column chromatography) 50 mM Tris-HC1 pH 7.6, 0.2 mM ATP, 0.5 mM MgC12, 1 mM creatine phosphate, 1 U creatine kinase for 4 min at 30 °C. The reaction was terminated by the addition of one volume of 4~o SDS gel loading buffer (0.3 M Tris/HC1 pH 6.8, 20~o glycerine, 8 M urea, 0.4 mg/ml bro- mophenol blue). El-dependent ubiquitination of E2 protein was assayed using 0.4 #g E1 protein and 20/~g bacterial protein extract in 25 #1 total reaction volume containing 1/~g 125I-ubiquitin in 50 mM Tris-HC1 pH 7.6, 0.2 mM ATP, 0.5 mM MgC12, 1 mM creatine phosphate, 1 U creatine kinase. After incubation for 4 min at 30 ° C, the reactions were terminated with one volume of SDS sample buffer with 8 M urea as described above. Proteins were separated by SDS-PAGE [24], stained with Coomassie blue, dried and the gels were then subjected to autoradiography.

Results and discussion

Isolation and characterization of a cDNA from Ara- bidopsis thaliana with homology to ubiquitin- conjugating enzymes

In a more general approach, we have, from an Arabidopsis thaliana cDNA expression library maintained in 2ZAP, selected by immunoscreening a cDNA collection overrepresenting plasma membrane associated proteins. The preselected cDNAs are all characterized by sequencing. This system has been described in detail [4, 26]. The cDNA described here (PM42) originated as part of this cDNA collection. The PM42 cDNA was characterized by partial restriction enzyme map- ping (Fig. 1A) and DNA sequencing (Fig. 1B). The obtained nucleotide sequence showed 50.2 ~o identity within the coding region to the A. thaliana AtUBC1 and 48.5~o identity to the WHUBC1, whereas AtUBC1 and WHUBC1 share 82~ nucleotide sequence identity [36].

The cDNA PM42 sequence is ligated by an Eco RI adaptor (Fig. 1B) to the multiple cloning site downstream of the lacZ gene product to produce a fusion protein product upon transcription and translation in E. coli. However, a 'weak' TAA stop codon is located 9 bp downstream of the adaptor. The first methionine codon is at position 78. This probably represents the eukaryotic start codon to give a deduced sequence of 161 amino acid corresponding to a protein of 18.24 kDa molecular mass. This amino acid sequence shares 36.2~o identical (plus 37.0~o conserved) amino acids with AtUBC1 [36] and WHUBC1 [27], 22.4~o identical (plus 48.6~o conserved) amino acids with WHUBC4 [35] and 31.5~ identical (plus 37.8~o conserved) amino acids with WHUBC7 [27]. A more detailed sequence analysis in comparison to the most re- leated yeast E2-type proteins is presented in Fig. 2. The length of the amino acid sequence indicates that the PM42-encoded protein consists of a ca. 160 amino acid conserved 'core region' which is typical for class I E2-type proteins [22] and lacks additional N- and/or C-terminal tracts extending from the core. Two highly conserved

391

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I GGAATTCGGCACGAGAGACA AAGTA AGGAGGCCAGATAGCAGACCCGTCTCTCTCCGTCT

61 CCGTCTCCGT(2TCCGCCATG.%CCAGCTCTTCCGCTCCTTCACGCAAGGCTTTA AGCAAGA

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121 TCGCGTGTAATAGGTT(;CAGAAAGAGCTTTCTGAGTGGCAATTGAATCCCCCCTCTGGAT

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181 TTCGTCACAAAGTCACTGATAATCTTCAAA AATGGACAATAGATGTTACCGGAGCTCCAG

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341 GGACGCTTTACGCTAATG AGACTTATCAGCTTCAGGTTGAATTTCCTGAACATTACCCTA

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301 TGGAAGCACCCCAGGTAGTGTTTGTTTCTCCGGCACCTTCACATCCACATATTTACAGCA

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361 ATGGACATATTTGTTTAGATATTCTATATGACTCATGGTCACCAGCAATGACAGTGAATT

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42 [ CAGTCTGCI~TCAGCI~TTCTTTCGP, TGCTATCIiAGTTCACCCGCAAAGCAACGCCCTGCGG

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481 ATAACGATCGTTATGTGAAGAACTGTAAGAATGGGAGGTCTCCTAAGGAGACGAGGTGGT

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601 ATGATGACATTTAA AA ATGTCAAAACCTAACTTTTTGTCTCACTCTTACTGTGGAATATG

661 ICAAGTGAAGAGCTCTGAATTATATAAGAGAACACTGAGAGCCGGAATCTACATTTCGTA

721 CTGAACATGTGCACAAAAAGATTAATIAATGCTAAACAATAAGTTGTCATTAATGCCAAT

781 AGTTATTACACAAAAAAAAAAAAAAAAAACTCGAG

Fig. 1. A (top). Partial restriction map of the eDNA PM42 and sequencing strategy as indicated by arrows. B (bottom). cDNA sequence and the deduced amino acid sequence of PM42 encoded ubiquitin-conjugating enzyme (E2) from A. thaliana. The DNA sequence given spans the putative coding region with the amino acid sequence aligned below the DNA sequence, the 5'- and Y-non-coding regions and the regions of the oligo-dT/Xho 1 primer and Eco RI adaptor used in eDNA synthesis (underlined). Asterisks indicate a stop codon in the 5' region in frame with the assumed translational start codon and a stop codon at the end of the only long open reading frame.

motifs establish the E2-1ike structure, the first being the domain around the active cysteine-99. The thiol group of this cysteine is required in AtUBC1 [36], W H U B C 4 [36], yeast UBC1 [37] and yeast UBC10 [41] for thiol ester formation with El-activated ubiquitin. A second highly con-

IO 20 30 40 FM42 MTSSSA PSRKALS KI ACNRLQKELSEWQLNPPSGFR|IKVT .... DNLQKW ATUBCI M - STPARK ........ RLMRDFKRLQQDpPAGIS---GAPQDNNIMLW WIIU [4C ! M- - - STPA~K ........ RLMRDFKRLQQDP PAGI S - - -GAPHDNN ITLW WHUBC4 M- -SSPSKR ........ REMDLMKLMMSD ...... YKVDMINDG -MHEF WIIUBC7 M- - -ATAPAR ........ R A SSSR $SS ESRTTPSMGFQLGFVDDSN~VFEW UBC2 M- - -STPARR ........ RLMRDFK~KEDA PPGVS - - -AS P[* PDN%'HV%~ UBCI M- - -SRA- -K ........ R IMKEIQAVKDDPAAH IT- - LEFVSESDIHHL UBC5 M- - -SSS- -K ........ [~ IAKELSDLGRDPPASCS - -AG PVGD -DLYIIW consensus S m D W

50 60 70 80 90 rM42 T I DVTGA PGTLYANETYQLQVEF PEHY PMEA FQWFVS PA pSH pH I Y S - N ATUBC1 NAV I FG PDDTPWDOGTFKLS LQFS EDY PNK P PTVRF~SP~M - FH pN I yA - D WIIU 8(/I NAV I FG PDDT PWDC, GT F K LTLQFTE D¥ PNK PPT~FVSRM - FH PN I YA - D WHUBC4 FVHFHGPKDSIYQGGVWKVRVELTEAYPYKSPSIGFTNKI - YH PNVDEMS WHUBC7 QVTI IGPPETLYDGGYFNAINSFPQNYPNS pPTVRFTSEM-WII pNVyp- D UBC2 NAMI IGPADTpYEDGTFRLLLEFDEEYPNK PPHVKFLSEM - FHpNVYA- N III+CI KGTFLG PPGTPYEGGKFWDI EV PMEYPFK PPKMQFDTKV -Y{I pN ISSVT UB('5 QAS I MG PSDS PYAGGVFFLS I HF pTDy PFKp PKVNFTTK I -YIiPNINS-S consensus OP T GG F F YP KPP V F HPN

1OO 110 120 130 rM42 GHICLDIL ........ YD .... SWS PAMTVNSVCI S I LSMLSSS PAKQRp ATUBC i GSICLDII~ ............ N(~4S P I YDVAA I L - TS I QS LLCD PN PNS p WIIUBCI GSICLDILQ ............ NQWSPIyDVAAIL-TSIQSLLCDPNPNSP W~IUBC4 GSVCLDVIN ............ QTWS PMFDLVN I FEVFL ~LLLYPN PSDP W}[UBC7 GRVC I S I HP~DDPNGYELASERWTPVHTVES I V - LS I ISMLSS PNDESP UBC2 GEICLDILQ ............ NI~WTPTYDVAS I L-TS IQSLFNDPNPAS p 1113<21 GAICLDI LK ............ NAWSPVITLKSAL - I $ LQAL LQS pE F~NDP UBC5 GNICLDILK ............ DQWS PALTLS KVL - LS I CS LLTDAN PDDP consensus (I ICLDIL WSP S LL PNP P

140 150 160 PM42 ADND- - R YV~NCKNGRS PK ETRWWF HDDKV ATUBC 1 ANS EAARMY SESKR EYNRRVR DWEQSWTAD WHUBC I ANS EAAP~Y SENKREYNRI(VR EWEQSWTAD WIIUBC4 LNGEAAS LPDMRDKNAY ENRVKEYC ER YAK PED . . . . WHUBC7 ANI EAAKDWR EKQDE FKKI~VR RAVRGK SQEM L UI8C2 At~V EAATLFK DII K SQYVKRVKETVEM SWEDDM .... UBC 1 QDAEVAQHYLRDR E S FN MTAA LWTR LY A S [~TS .... UBC% LV PE I AQ I Y ~TDKAKY EATAKEWTK KYAV COnSensus E A K

Fig. 2. Amino acid sequence comparison of the deduced amino acid sequences of five plant ubiquitin-conjugating enzymes and three yeast ubiquitin-conjugating enzymes with clostest homology to plants (PM42, this work; ATUBCI [36]; WHUBC1, wheat UBCI [36]; WHUBC4, wheat UBC4 [35]; WHUBC7, wheat UBC7 [27]; UBC2, yeast UBC2 [20]; UBCI, yeast UBC1 [32]; UBC5, yeast UBC5 [31]. Wheat UBC4, yeast UBC2 and yeast UBC1 are not completely listed in the C-terminal end as indicated by dots. Spaces are set for highest homology and are indicated by vertical lines. A consensus (lower line) was determined if 6 (normal type) or all (bold type) out of 8 sequences showed an identical amino acid residue at that position in the sequence.

served domain N-terminal to the active site cysteine in E2 proteins is the 'Pro-X-X-Pro-Pro' motif [11] at amino acid position 74. In PM42 and WHUBC4, proline-77 is exchanged against alanine or serine, respectively. This suggests structural differences in this part of the protein which is, according to the X-ray structural analysis of AtUBC1 [11], composed of an antiparal- lel fl-sheet structure with the prolines constituting the turn between the two fl-sheet strands. From the X-ray analysis of AtUBC1, it can further be proposed that the only hydrophobic domain of the PM42 gene product (data not shown) is located around the active cysteine. It was suggested [27] that the amino acid insertion of wheat UBC7 is important for transferring ubiquitin to itself.

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resulting in multiubiquitin formation. This insertion is lacking in the PM42 sequence which means that the PM42-encoded protein is expected to transfer monoubiquitin moieties as other E2-type 'core' enzymes. A special feature of the PM42- encoded protein is the additional sequence of eight amino acids between position 10 and 19 near the N-terminus. This sequence interrupts a common motif of three basic residues (position 9, 10 and 19) present at the N-terminus of several other E2 types [32, 36]. From the analysis of sequential N-terminal deletion mutants of AtUBC1, a function of this common N-terminal amino acid motif in the rate of thiol ester formation [36] by binding E1 proteins [ 11 ] was suggested. It will be very interesting to see if differences in kinetic and/or enzyme specificity towards El-type proteins can be obtained for the PM42-encoded enzyme.

Genome analysis indicates a small gene family

Three different gene fragments of cDNA PM42 were isolated and labelled for Southern analysis of genomic DNA fragments from A. thaliana. Using 5'- and 3'-unique restriction sites of the multiple cloning site, the whole cDNA sequence (810 bp as shown in Fig. 1B plus 28 bp out of the multiple cloning site of pBSC) was isolated (probe 1). Two bands were labelled when using Hind III-digested fragments of genomic DNA (Fig. 3). Digestion of genomic DNA with Eco RI resulted in four labelled bands. Two of these hy- bridized specifically with a 5'-terminal 414 bp Eco RI fragment of PM42 (probe 2 in Fig. 1B, bp 2 to 416) and a 3'-terminal 393 bp Eco RI/ Xho I fragment (probe 3 in Fig. 1B, bp 417 to 810) due to an internal Eco RI site in the cDNA PM42. The Southern experiment thus indicates a second closely related member within this gene family. The second gene detected in Southern analysis cannot be due to the formerly described AtUBC1 [35] gene, where sequence identity (50.2~o) is scattered and extends over a maximum of 7 bp in one stretch. The Southern analysis thus suggests the presence, in A. thaliana genomic DNA, of at least two different small gene families of E2-type

Fig. 3. Southern blot hybridization of A. thaliana restriction enzyme-digested genomic DNA. Single-letter notation in the top indicates the restriction enzymes used for genomic DNA digestion (B/X, Bam Hl/Xho I; H, Hind III; E, Eco RI). The probe used was a 837 Bp Bam HI/Kpn I fragment containing the whole cDNA PM42 sequence (probe 1), or a 5'-terminal 414 bp Eco RI fragment (probe 2), or a 3'-terminal 393 bp EcoRI/XhoI fragment (probe 3). Arrows indicate PM42 cDNA homologous genomic Eco RI fragments. Mobility of molecular size markers is indicated in kb. As a control, 50 pg of the PM42 cDNA Bam HI/Xho I fragment (B/X) was used.

enzymes (namely AtUBC1 [35] and AtUBC2, this study). Members of these families may serve different functions in cellular metabolism. We thus propose to label the UBC encoded by PM42 as AtUBC2-1 to designate it being the first cloned member of the AtUBC2 subfamily of E2 enzymes. According to this terminology, it would be appro- priate to designate AtUBC1 [35] as AtUBCI-1. By transforming the complete PM42 cDNA and the Eco RI/Xho I subfragment in antisense orien- tation, using an Agrobacterium-mediated vector system, into A. thaliana, we hope to regenerate phenotypic mutants impaired in the function of AtUBC2-1.

Expression of AtUBC2-1 from its cDNA in E. coli

In order to remove the 5'-terminal TAA-stop codon upstream of the coding region, a deletion construct was generated by cloning a 780 bp Hae III/Xho I fragment in frame with the coding re-

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gion of the 8 amino acid N-terminal end of T4 lysozyme under control of plac/ptac promotors in the plasmid vector pEXP2. Protein overexpres- sion in bacteria harbouring this construct (clone 1638-13) resulted in an additional protein which was obtained from inclusion bodies and gave an apparent molecular mass of 28 kDa on SDS- PAGE (Fig. 4A). This finding was at variance with the expected molecular mass of the fusion protein of 189 amino acids (21.3 kDa). Using 6 M urea-containing denaturating gels, however, the overexpressed fusion protein gave an apparent molecular mass of 22 kDa, in agreement with the expected value (Fig. 4B). The atypical migration of the protein in standard SDS-PAGE might be due to the globular and very compact structure [ 11 ] of E2-type enzymes. An antiserum was raised in mice against the purified, overexpressed protein (Fig. 4C). This serum clearly recognized the AtUBC2-1 protein from inclusion bodies (Fig. 4D) while the signal was less pronounced in the soluble protein fraction from bacterial lysates, in agreement with the results of protein staining (cf. Fig. 4C). The serum will be used in further

studies on the expression and localization of AtUBC2-1 in A. thaliana.

AtUBC2-1 shows El-mediated complex formation with ubiquitin

The confirmation that cDNA PM42 encoded a functional ubiquitin-conjugating enzyme was provided by proof of complex formation between 125I-ubiquitin and bacterially expressed AtUBC2-1 in the presence of wheat ubiquitin- activating enzyme (El).

Three El-type proteins from wheat, using a standard purification protocol [ 14] can be eluted from a ubiquitin-Sepharose column and are visu- alized as two bands of 117kDa or 123 plus 126 kDa (not resolved) molecular mass on SDS PAGE (Fig. 5A, lanes 2, 3 and 4) [14]. The E1 enzyme could be loaded with lzSI-ubiquitin in an assay containing an ATP-regenerating system with creatine phosphate and creatine kinase. The 125I-ubiquitin loaded El-preparation is shown in Fig. 5B. For this gel, mercaptoethanol in the

Fig. 4. SDS-PAGE of proteins expressed in E. coli W3110 (A,B), purification of the ubiquitin-conjugating enzyme (C) and immunoblot against the purified protein (D). A. IPTG (10 mM) induction of the ubiquitin-conjugating enzyme from plasmid 1638-13 in E. coli W3110 cells. Lane 1, protein extract from inclusion bodies in standard SDS-PAGE [24]; lane 2, uninduced control; lane 3, IPTG-induced control clone carrying a nitrilase cDNA [5]; lane 4, same clone in the absence of IPTG. B. Protein as in A, lane 2, but separated on urea gels according to Schgtgger and von Jagow [30]. The overexpressed polypeptide (open circle) exhibits an apparent molecular mass of 22 kDa in this system. C. The ubiquitin-conjugating enzyme fusion protein can be found in the presence of IPTG only in proteins from inclusion bodies (lane 2) less in the soluble protein supernatant from 1638-13 cells (lane 1) and be isolated from SDS-PAGE (lane 3). D. Immunoblot showing soluble proteins from the control clone used in A, lane 3 (lane 1), and clone 1638-13 (lane 2). Lane 3, proteins solubilized from inclusion bodies of clone 1638-13. Molecular masses of marker proteins are given in kDa.

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Fig. 5. Preparation of ubiquitin-activating enzyme (E 1, parts A, B) and loading of ubiquitin-conjugating enzyme (E2, part C) with 125I-ubiquitin. Proteins of fractions 3, 4, 5 and 6 eluted from a ubiquitin-Sepharose column [ 14] were subjected to SDS-PAGE and stained with Coomassie blue (A, lane 1, 2, 3 and 4; the closed circles indicate the position of E1 proteins on the gels) or aliquots corresponding to 0.4/~g El protein were incubated with 1.6/~g ~25I-ubiquitin (1.7/~Ci) as described under Materials and methods prior to SDS-PAGE and autoradiography (B, lanes 1 to 4 which correspond to lanes 1 to 4 in A). The smear between 43 and 67 kDa presumably represents endogenous wheat ubiquitin acceptors (e.g. E2 proteins) eluting from the affinity column [ 14]. C. Ubiquitination ofA. thaliana E2 enzyme fusion proteins with 125I-ubiquitin in protein extracts containing only E1 (lane 1) or protein from clone 1577-10 in the absence (lane 2) or in the presence of E1 proteins (lane 3) or from clone 1638-13 in the absence (lane 4) or in the presence of E1 proteins (lane 5), To avoid contamination with wheat germ E2 proteins, only E1 fractions with the purity shown in lane 4 (part A, B) were used. Molecular masses of marker proteins are given in kDa.

sample buffer was replaced with 4 M urea to pre- serve thiol ester linkages. In the presence of mercaptoethanol in the sample buffer, the complex between ~25I-ubiquitin and the E1 proteins is not preserved, indicating a thiol ester linkage between the two (not shown).

Ubiquitin transfer from El-activated 1251- ubiquitin was tested with protein extracts from two bacterial strains harboring constructs of PM42 which resulted in the expression of fusion proteins of different molecular mass. The first construct (clone 1638-13, vide supra) gives rise to a 28 amino acid N-terminal extension in the fusion protein (total calculated molecular mass 21.3kDa) and the second construct (clone 1577-10, containing a 796 bp Hae III/Kpn I fragment of cDNA PM42 fused to the lacZ N- terminal end in pBSC under control of the plac promotor) results in a fusion protein extension of 46 amino acids and a calculated molecular mass of the fusion protein of 22.9kDa. When El- activated 125I-ubiquitin was added to soluble protein fractions from either of the two clones ex- pressing AtUBC2-1, label was transferred from the E1 adduct to a new location which showed an apparent molecular mass of ca. 36 kDa (Fig. 5C,

lane 3) or 34 kDa (Fig. 5C, lane 5). These were the expected locations as well as the expected differences in location of the two monoubiquiti- nated AtUBC2-1 fusion proteins in the gel system used (vide supra). We conclude that AtUBC2-1 is a functional ubiquitin acceptor from ubiquitinated El. The data furthermore show that the recognition of the heterologous wheat E 1-type activating enzyme is not abolished by the N-terminal 8 amino acid insertion of AtUBC2-1 (shown in Fig. 2 at positions 10 to 19).

Next, the conditions for unloading of ubiquitinated AtUBC2-1 were analysed. Samples of 1251- ubiquitinated AtUBC2-1 were supplied to SDS- PAGE under non-reducing (4 M urea) as well as under reducing (5 ~o 2-mercaptoethanol or 5 mM DTT) conditions. Most of the E2-type proteins so far described show a rapid degradation of the thiol ester with ubiquitin under reducing conditions. However, such conjugates are usually not easily cleaved in the cell's reducing environment. The replacement of urea by 5 ~o 2-mercaptoethanol or 5 mM DTT in the sample buffer and 30 min incubation at 100 °C gave no significant decrease in 125I-ubiquitin labelling of AtUBC2-1 (data not shown). A decrease of 20 -30~ was obtained

Fig. 6. Deubiquitination by lysine of ~25I-ubiquitinated AtUBC2-1 expressed from clone 1638-13. Lanes 1 and 2 show time-dependent auto-deubiquitination of AtUBC2-1 and lanes 3 to 6 show loss of 125I-ubiquitin in the presence of 100 mM lysine during the times indicated. Assays were terminated by the addition of a 4 M urea, proteins separated by SDS-PAGE and autoradiographed. Free 12SI-ubiquitin is indicated by Ub.

when the urea was replaced with 5 mM cysteine (data not shown). This result shows that the ubiquitin-AtUBC2-1 bond, once formed, is not readily accessible to extraneous thiols. This was taken as evidence for a high structural integrity of the ubiquitin-AtUBC2-1 product formed in vitro under the conditions employed. In vivo, ubiquitin is transferred from the E2 complex to e-amino groups of lysyl side chains of its target protein(s). This reaction can be analysed in vitro even in the absence of an E3-1igase when using high concen- trations of lysine [28]. Such an experiment is shown in Fig. 6, While in the absence of lysine, a slow disappearance of the 125I-ubiquitin- AtUBC2-1 complex due to degradation is ob- served, in the presence of 100 mM lysine, the signal was rapidly abolished (Fig. 6).

25I-ubiquitin loading and unloading of AtUBC2-1 confirmed the function of this protein as a ubiquitin-conjugating enzyme with a conserved transfer mechanism which functions even in the heterologous wheat-E1 and A. thaliana-E2 in vitro assay used in our study.

Up to now no direct evidence exists that AtUBC2-1 is located at the plasma membrane. In yeast, UBC6 is an integral membrane protein

395

[21, 22] with functions in the secretory pathway. No strong homology to yeast UBC6, nor a membrane anchor sequence, nor palmitoylation sites could be located in the amino acid sequence of AtUBC2-1 deduced from the cDNA. AtUBC2-1 is to be classified as a soluble protein from in- spection of the primary sequence as well as from its derived secondary structure. It is therefore likely that AtUBC2-1, which we detected as a member of an immunoselected cDNA library overrepresenting plasma membrane associated proteins, is attached to components of this com- partment through interactions other than lipo- philic ones (e.g., via the cytoskeleton [3]). The antisera raised against AtUBC2-1 will be a use- ful tool to further studies of the subcellular location of AtUBC2 proteins.

Acknowledgements

This work was supported by the Deutsche For- schungsgemeinschaft, Bonn to E.W. and D.B. and Fonds der Chemischen Industrie, Frankfurt (literature provision) to E.W.

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functional expression and molecular characterization of atubc2-1, a novel ubiquitin-conjugating...

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