Mol Gen Genet (1991) 230:81-90

© Springer-Vertag 1991

Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes Jane H. Gould 1, Martin R. Hartley 1, Philip C. Welsh 3, Deborah K. Hoshizaki 2, Arthur Frankel 3, Lynne M. Roberts 1, and J. Michael Lord 1

1 Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, West Midlands, England 2 Department of Biochemistry, College of Medicine, University of Illinois, Chicago, IL 60612, USA 3 Florida Hospital Cancer and Leukemia Research Center, Suite 100, 616 E. Altamonte Drive, Altamonte Springs, FL 32701, USA

Received April 4, 1991

Summary. Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains ana- lysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alter- ations within the RTA-encoding DNA causing produc- tion of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modifica- tion in vitro suggesting that the strains contain alter- ations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Al- though serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modify- ing activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a pro- portion of the ribosomes become modified. We demon- strate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activi- ty compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA cataly- sis and of further understanding the precise mechanism of action of RTA.

Key words: Ricin - Toxin - Mutant - Saccharomyces cerevisiae - Expression

Offprint requests to ." J. Gould

Introduction

Ricin is a heterodimeric protein toxin found in the seeds of the castor oil plant, Ricinus communis. It consists of a 32 kDa toxic A chain (RTA) linked by a disulphide bond to a 33 kDa galactose-binding B chain (RTB) (Olsnes and Pihl 1982). Binding of RTB to exposed ga- lactose residues on mammalian cell surfaces leads to in- ternalization of whole ricin followed by release of RTA from an intracellular compartment into the cytoplasm, where it inhibits protein synthesis (Olsnes and Sandvig 1988). RTA is an N-glycosidase and inactivates eukar- yotic 60S ribosomal subunits by removing a single aden- ine residue located in a highly conserved sequence near the 3' end of 28S or 26S rRNA (A-4324 in rat liver 28S rRNA) (Endo et al. 1987). Depurinated 28S and 26S rRNA is susceptible to amine-catalysed hydrolysis by reagents such as aniline, which release a fragment of approximately 400 bases from the 3' end of the rRNA (Endo et al. 1987). This fragment can be easily detected on denaturing agarose gels and its appearance is diag- nostic of RTA activity. We have taken advantage of the sensitivity of this assay to demonstrate that yeast transformants induced to express RTA produce active toxin.

Most eukaryotic ribosomes are susceptible to the N- glycosidase activity of RTA, although ribosomes from different species vary in their degree of sensitivity (Stirpe and Hughes 1989). For example, wheat germ ribosomes are relatively resistant to RTA but are highly sensitive to another RTA-like toxin, dianthin 32 (from Dianthus caryophyllus leaves). Since all eukaryotic ribosomes ana- lysed to date contain the conserved 28S/26S rRNA tar- get sequence of RTA (Chan et al. 1983), the varying sensitivities most likely reflect differences in other

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rRNA-associated components such as ribosomal pro- teins or translation factors. Recently Chinese hamster ovary (CHO) mutant cell lines with ribosomes exhibiting increased resistance to RTA have been isolated (Sallustio and Stanley 1990), demonstrating that it is possible to alter ribosomes and maintain their function while de- creasing their sensitivity to RTA.

Inhibition of protein synthesis by RTA is associated with, and possibly caused by, conformational changes in the 60S ribosomal subunit due to cleavage of the N- glycosidic bond at A-4324 in rat liver 28S rRNA (Terao et al. 1988), resulting in inhibition of EF2-dependent translocation (Olsnes and Sandvig 1988). This is consis- tent with the findings in Escherichia coli that EF-Tu and EF-G bind to the region of 23S rRNA that is homolo- gous to the conserved RTA target loop of eukaryotic 28S rRNA (Moazed et al. 1988; Tapprich and Dahlberg 1990).

The exact mechanism by which RTA cleaves the N- glycosidic bond of A-4324 is not known. X-ray crystallo- graphic data from whole ricin show RTA to have a prominent cleft which is assumed to be the active site (Montfort et al. 1987). To understand further the mecha- nism of action of RTA, Frankel et al. (1989) selected mutations in yeast that inactivate RTA. The nine RTA mutants analysed all had alterations in amino acid resi- dues located in the proposed active site cleft. The results described in this paper describe a serine (203) to aspara- gine change outside the proposed active site which re- duces RTA activity and suggests that alteration of amino acids outside this region can also affect the catalytic activity of RTA.

Materials and methods

General. E. coli strain RK1400, (Thr- leuB6 Thi- thyA trpCll17 Str r r- m-, a gift from R.D. Kolodner, Har- vard Medical School), was used for plasmid propaga- tion. Plasmids were prepared by the alkaline lysis or boiling methods (Maniatis et al. 1982) and purified on caesium chloride gradients if required for yeast transfor- mation. Restriction and DNA modification enzymes were from Amersham or BRL and were used according to the manufacturers' recommendations. DNA probes were prepared by the random priming method (Feinberg and Vogelstein 1983, 1984) using hexanucleotides sup- plied by Pharmacia. DNA sequences were determined directly from plasmids by the chain termination method of Sanger et al. (1977), using a US Biochemical Corpora- tion kit and RTA-specific oligonucleotide primers syn- thesized on an Applied Biosystems 380B D N A synthe- sizer. Yeast strain JRY188 (~leu2 ura3 trpl his4) was grown in YPD medium (Sherman et al. 1986) and trans- formed to uracil prototrophy by the spheroplast method (Hinnen et al. 1978). Transformants were grown in syn- thetic medium lacking uracil (URA DO) supplemented with 2% ethanol, 2% glucose or 2% galactose, or on YPD-agar plates (Sherman et al. 1986).

Construction of yeast expression vectors and yeast trans- formants. The yeast expression vector, pEMBLyex4

(Fig. 1), containing an inducible GAL-CYC1 hybrid promoter (Guarente et al. 1982) was a gift of J. Murray (University of Cambridge). An integrative vector, pJG2, was derived from this plasmid by deletion of the 2/z origin and insertion of the transcription termination re- gion of the phosphoglycerate kinase (PGK) gene (Fig. 1). RTA-encoding cDNA was cloned into the polylinker SalI site and its orientation determined by restriction enzyme mapping, generating plasmid pJG2RA (Fig. 1). pJG2RA was linearized within the leu2-d or URA3 DNA, by digestion with EcoRI or ApaI, respectively, and yeast was transformed with a mixture of these lin-

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Fig. 1. Construction of the integrative plasmid containing RTA. Plasmid pEMBLyex4 was used as the starting material for con- struction of an RTA expression plasmid. The 2 ~t ORI-STB region of pEMBLyex4 was deleted to prevent autonomous plasmid repli- cation and the transcription termination sequence present in this region was replaced with an equivalent sequence from the PGK gene in pMA91X-R (derived from pMA91; Mellor et al. 1983). pJG2RA encodes cytoplasmically expressed RTA with an addition- al 24 amino acids, derived from part of the preproricin signal se- quence, at its N-terminus (O'Hare et al. 1987)

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earized plasmids and URA3 + transformants selected. Total genomic DNA from selected transformants was prepared by the method of Struhl et al. (1979), cut with EcoRI, separated on a 0.8% agarose gel and analysed by the procedure of Southern (1975). The filter was hy- bridized to a 32p-labelled RTA XhoI-BglII fragment in 50% formamide, 4xSSC, 5 xDenhardts, 100 gg/ml denatured salmon sperm DNA at 37 ° C overnight and washed in 2 x SSC, 0.1% SDS at room temperature for 30 min followed by two sequential washes at 50°C in 0.1 x SSC, 0.1% SDS, 0.1% pyrophosphate for 30rain each (Maniatis et al. 1982). This identified transformants containing single copies of pJG2RA integrated at the leu2 locus (e.g. J20) and others that contained more than one copy, most likely two (e.g. J19; see Fig. 2, lane 3), although the number of copies was not precisely deter- mined. Mutant RTA DNA was recovered by digestion of total yeast genomic DNA with EcoRI and HindIII followed by size fractionation of the DNA on agarose gels. RTA DNA was identified by Southern hybridiza- tion, eluted from the agarose and subcloned into pUC19 using standard procedures.

Growth of transformed yeast strains. Transformants were grown in URA DO-ethanol to a density of approximate- ly 107 cells per ml. Cells were induced to express RTA by resuspension in URA DO supplemented with 2% galactose. Growth rates were monitored by counting cells using a haemocytometer.

Western immunoblo t analysis of R TA- transformed yeast strains. Transformants were induced to express RTA by pregrowth in URA DO-ethanol to approximately 5 x 10 6 cells per ml followed by resuspension in URA DO- galactose and growth at 30 ° C for 9 h. Samples for nega- tive controls were obtained after growth of transfor- mants to approximately 10 7 cells per ml in URA DO- glucose. Cells were harvested, washed with PBS (8 g NaC1, 100 mg KC1, 1.15 g NagHPO4, 200 mg KHzPO4, 100 mg MgC126HzO per litre) and lysed with glass beads (BDH 40 mesh) in PBS containing i mM phenylmethyl- sulphonyl fluoride. The glass beads and cell debris were removed by centrifugation in a Sorvall HS4 rotor at 3000 g, 4°C for 5 rain. The supernatants were trans- ferred to microfuge tubes and centrifuged at 12000g at 4°C for 5 rain. The supernatants were transferred to clean tubes and stored at - 2 0 ° C prior to use. Protein concentrations were determined using the Biorad meth- od. Approximately 100 gg protein was loaded per lane of a 12% SDS-polyacrylamide gel and electrophoresed for 16 h at 10 mA. The proteins were transferred to ni- trocellulose using an Anderman semi-dry blotter and filters probed with affinity purified anti-RTA antibodies (a gift of Dr. A. Hertler, Louisiana State University) as described by Frankel et al. (1989).

Depurination assay for RTA activity. Total RNA was isolated from yeast transformants by lysing the cells with glass beads in Kirby (1968) buffer plus 1 vol. of phenol. The aqueous phase was reextracted with equal volumes of phenol and chloroform and RNA recovered by etha-

nol precipitation. RNA (2 gg) was treated with 20 lal I M aniline-acetic acid, pH 4.5 (d'Alessio 1982) at 60 ° C for 2 rain. The RNA was recovered by ethanol precipita- tion, dissolved in 20 gl 60% formamide, 0.1 x TEP and analysed on 1.2% agarose, 50% formamide gels run at 20 mA for 2 h in 0.1 x TEP buffer (1 x TEP is 36 mM TRIS-HC1, pH 8.0, 30 mM NaH2PO4, 2 mM EDTA).

Mutagenesis of RTA-transformed yeast strains. Transfor- mant J19, which contains multiple, tandemly integrated copies of pJG2RA, was grown for 36 h at 30 ° C on a YPD plate. Approximately 10 s cells were resuspended in 10 ml 0.1 M sodium phosphate, pH 8.0, harvested and resuspended in 10 ml of the same buffer. Then 0.3 ml EMS (methane sulphonic acid, ethyl ester, Sigma Chemi- cal Co.) was added and the suspension incubated at 30 ° C with shaking for 1 h. The EMS was inactivated with 6% sodium thiosulphite and the cells washed three times with 0.1 M sodium phosphate, pH 8.0, followed by incubation in 10 ml YPD at 25 ° C with shaking for 1 h. Aliquots of mutagenized cells were grown at 25 ° C for 3 to 4 days on YPD, to determine cell survival rates, and on URA DO-galactose to select for mutants. Indi- vidual mutant colonies were maintained as patches on YPD plates at 4 ° C until analysed.

Isolation of ribosomal subunits and polysomes. Ribosomal fractions were isolated on sucrose density gradients by a modification of the method of Warner et al. (1985). Transformants containing one (J20) or two 019) copies of the RTA expression plasmid pJG2RA were grown to a cell density of approximately 107/ml in URA DO- ethanol, harvested, resupended in URA DO-galactose and grown for 5 h at 30 ° C to induce expression of RTA. Alternatively, mutant strains were grown in URA DO- galactose continuously. Cycloheximide (50 gg/ml) was added to the culture immediately before harvesting. The culture was then rapidly cooled in ice-water. Cells were harvested, washed twice in buffer A (50 mM TRIS-ace- tate, pH 7.0, 50 mM NH4C1, 12 mM MgC12, 50 gg/ml cycloheximide, 200 gg/ml heparin), resuspended in 1 ml buffer A and lysed with glass beads. The cell debris was removed by two rounds of centrifugation in a Sorvall RC-5B HS-4 rotor at 3000 g, for 5 min at 4 ° C and the cleared lysate layered onto 11 ml 7%-40% exponential sucrose density gradients. Gradients were made in 50 mM TRIS-acetate, pH 7.0, 50 mM NH4C1, 12 mM MgClz, 1 mM dithiothreitol. The gradients were centri- fuged in a Beckman SW41 rotor at 240000 g for I h 50 min at 4 ° C. 40S, 60S, 80S and polysomal fractions were collected through a UV analyser (Instrument Spe- cialities Co., model UA5) by upward displacement with 60% sucrose. Ribosomes were harvested by centrifuga- tion in a Beckman TL100 ultracentrifuge at 540000 g at 4 ° C for 30 rain. Samples to be treated in vitro with RTA were washed with and finally resuspended at ap- proximately 1 ttg/gl in 25raM TRIS-HC1, pH7.6, 25 mM KC1, 5 mM MgC1/. Samples for depurination assays were resuspended in sterile water and RNA ex- tracted in Kirby buffer by two phenol/chloroform ex- tractions followed by ethanol precipitation.

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Expression of mutant RTA in E. coli. Site-directed muta- genesis was used to change the AGC codon of Ser-203 to AAC (Ash) in pUC119-RTA (Schlossman et al. 1989) and the modified RTA BamHI fragment was then sub- cloned into the E. coli expression vector pJG200 (Schlossman et al. 1989). Recombinant, mutant RTA was produced as a tripartite fusion protein consisting of the RTA moiety joined by a collagen linker to /% galactosidase, and purified as previously described (Schlossman et al. 1989). After collagenase treatment the amount of RTA, which was positively identified by Western blotting, was determined by scanning the gels and comparing the signal with that obtained from known amounts of plant RTA (Inland Lab., Austin, Tex.). Enzymatic activity of mutant RTA was assessed by a protein synthesis inhibition assay using the rabbit reticulocyte lysate system (Schlossman et al. 1989). The concentration of mutant RTA necessary to inhibit incor- poration of [3H]leucine into protein by 50% (the IDso) was determined and compared with the values obtained using purified, native plant RTA and wild-type recom- binant RTA produced in E. coll.

Results

Expression of RTA in yeast

Yeast strain JRY188 was transformed with a plasmid (pJG2RA, Fig. 1) that contains a galactose-regulated copy of RTA, linearized within either the leu2-d or URA3 DNA sequences. Southern analysis of URA3 ÷ transformants showed that J19 contains tandemly inte- grated copies of RTA expression plasmid pJG2RA (Fig. 2, lane 3). The copy number of the integrated plas-

• mid was not determined precisely, but the data in Fig. 2 suggest that two copies were integrated in tandem. In- duction of RTA expression in J19, by addition of galac- tose to the medium, resulted in immediate arrest of

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Fig. 2. Southern analysis of yeast transformants. Total yeast DNA, cut with EcoRI was separated on a 0.8% agarose gel and trans- ferred to a nylon membrane (Schleicher and Schuell). The filter was hybridized to a 32P-labelled RTA DNA fragment and autora- diographed overnight at - 80 ° C. Lane 1, RTA expression plasmid pJG2RA; 2, host strain JRY188; 3, parental transformant J19; 4, mutant 19.55

growth presumably due to the potent toxicity of RTA to yeast ribosomes (Fig. 3C). Transformant J19 pro- duced a protein of the expected size for cytoplasmically expressed RTA, which was immunoreactive with anti- RTA antibodies (Fig. 4, lane 2). The protein produced was slightly larger than recombinant RTA produced in E. coli (Fig. 4, lane C) since it contains an additional 24 amino acid residues at the N-terminus. Two smaller RTA-related bands, which were presumably degradation products, were also detected on the Western blot. One band, larger than RTA, was detected in all samples, including the non-induced transformants and the host strain, which does not contain RTA, and was presumed to be a yeast protein that reacts non-specifically with the polyclonal anti-RTA antiserum.

rRNA from induced J19 cells was shown to be suscep- tible to aniline cleavage (Fig. 5), suggesting that active RTA that can modify yeast ribosomes in vivo is pro- duced by this transformant. This RNA analysis revealed that 26S rRNA was partially modified 1 h after addition of galactose to the medium (Fig. 5, lane 6); after 4 h a high proportion (> 70%) of the 26S rRNA had been modified by the expressed RTA, as shown by the de- creased amount of intact 26S rRNA in Fig. 5, lane 12.

Isolation of RTA mutants

A transformant containing two copies of the galactose- regulated RTA expression plasmid (J19) was used as the parental strain for mutagenesis in an attempt to de- crease the probability of isolating mutations within the RTA DNA or associated promoter sequences and to facilitate isolation of mutations within yeast genes that would confer resistance to RTA. Mutagenized strains that have apparently acquired resistance to RTA could have alterations within yeast genes, giving rise to RTA- resistant yeast strains, or within the promoter sequences or RTA-encoding DNA itself thus preventing expression of the toxin or giving rise to mutant toxin. Many hundreds of mutants that were able to survive induction of RTA expression were obtained. To eliminate those mutations that inactivate the GAL-CYC1 promoter, to- tal RNA from 11 mutants was analysed on Northern blots probed with RTA DNA. All of the mutants tran- scribed full-length RTA RNA (data not shown). Further analysis, by treatment with aniline, of RNA isolated after induction of RTA expression, showed that five out of nine mutant strains had partially modified 26S rRNA (data not shown).

Those mutants that did not possess modified rRNA could have mutations in yeast genes that block modifica- tion. However, all of these mutants had ribosomes that were susceptible in vitro to depurination by native RTA, suggesting that they produce non-functional RTA.

Mutant 19.55 contains a single copy of RTA with one amino acid substitution

One mutant (19.55) that had partially modified 26S rRNA when grown in galactose medium was selected

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Fig. 3A-F. Yeast growth curves. Cells were grown in 2% ethanol medium, harvested and resuspended in 2% galactose medium at the times indicated by the arrows. A Host strain JRY188; B, trans- formant J20 (single copy of RTA); C transformant J19 (two copies of RTA); D mutant 19.55 (single copy of mutant RTA); E mutant

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for detailed analysis. To investigate the possibility that the R T A D N A in mutant 19.55 had been rearranged, total genomic D N A was subjected to Southern analysis. Figure 2 shows that 19.55 contains a single, integrated copy of pJG2RA, whereas its parental strain, J19, con- tains two copies. More detailed restriction mapping of p J G 2 R A in 19.55 showed that this single copy appears to be similar to the original plasmid used to t ransform yeast (data not shown). To determine whether this re- maining copy of R T A in 19.55 contains any alterations it was cloned into pUC19, as described in Materials and methods. D N A sequencing of the resultant R T A clone and par t of the p romoter contained within this clone revealed a single base change (G 608 to A), which results

in an amino acid alteration of serine 203 to asparagine in R T A protein.

Growth properties of yeast strains containing mutant RTA

To determine whether the observed mutan t phenotype of 19.55 was due to the single base change in R T A and not due to a yeast gene mutat ion, mutan t R T A D N A from 19.55 was recloned into the yeast expression vector pJG2, generating plasmid pJG2.55RA. This was then linearized within the leu2-d DNA, transformed into the host strain JRY188 and URA3 ÷ t ransformants selected. Southern analysis identified t ransformants that contain

86

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Fig. 4. Western immunoblot analysis of yeast transformants. Trans- formants were grown to 5 x 106 cells per mt in URA DO-ethanol, harvested and induced to express RTA by growth in URA DO- galactose at 30 ° C for 9 h. As negative controls, protein samples were prepared from the host strain JRY188 induced with galactose and from RTA-containing strains grown in glucose medium. ~Ibtal cell extracts were prepared from the following strains: lane 1, host strain JRY188; 2, transformant J19 (two copies of RTA); 3, mu- tant 55.4 [multiple recloned copies (> 2) of mutant RTA recovered from 19.55]; 4, 7, transformant J20 (single copy of RTA); 5, 8, mutant 19.55 (single copy of mutant RTA); 6, 9, mutant 55.30 (single recloned copy of mutant RTA recovered from 19.55); C, 300 ng recombinant RTA. The arrowhead indicates RTA, contain- ing an additional 24 amino acid residues at the N-terminus, pro- duced by yeast transformants. GAL, galactose-indueed samples; GLU, glucose-repressed samples

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Fig. 5. Time course of rRNA modification by RTA, A transfor- mant 019) containing two copies of the RTA expression plasmid pJG2RA was grown to approximately 107 cells per ml in URA DO-ethanol and then induced to express RTA by resuspension in URA DO-galactose. Samples were taken at the intervals indicat- ed below, the RNA extracted and treated with aniline (+) or ana- lysed directly ( - ) . * indicates RTA-modified 26S rRNA from which the 369-base 3' fragment (>) has been removed by hydroly- sis with aniline. Lane 1, control RTA-treated ribosomes; 2, 3, 0 rain; 4, 5, 30 min;, 6, 7, i h; 8, 9, 2 h; 10, 11, 3 h; 12, 13, 4 h after induction

copies of pJG2.55RA integrated into the leu2 locus (data not shown). Transformant 55.30 contains a single copy of the RTA expression plasmid. The precise number of tandemly integrated copies of pJG2.55RA in transfor- mant 55.4 was not determined, but was judged to be greater than two based on the intensities of the hybridiz-

ing bands (data not shown). The growth properties of transformants 55.30 and 55.4 were compared with strains containing one (J20) or two 0 t 9 ) integrated cop- ies of the original RTA expression plasmid (pJG2RA) and with mutant strain 19.55. Figure 3 B shows that the strain containing just a single copy of wild-type RTA (J20) was unable to grow in galactose-containing medi- um. In contrast, mutant 19.55, which contains a single copy of the mutant RTA, and the transformants con- taining recloned RTA rescued from 19.55, grow at ap- proximately wild-type rates in galactose (Fig. 3 D, F, E). It is possible that growth in galactose of the mutant RTA-containing strains for longer than 40 h would re- sult in accumulation of sufficient mutant RTA to impair growth, however we do not believe this to be the case since it is possible to grow the mutants continuously in galactose. The ability of the original mutant, 19.55, to survive induction of RTA expression thus appears to be caused by the alteration of serine 203 to asparagine RTA in the single remaining copy of RTA.

60S ribosomal subunits are preferentially modified by Asn203 RTA

The surprising observation that mutant strains with par- tially modified ribosomes are viable prompted us to in- vestigate how these strains avoid translational arrest, since it has been shown that the presence of modified 60S subunits in polysomes inhibits translation (Fodstad and Olsnes 1977). To address this question transfor- mants containing wild-type or mutant copies of RTA were induced to express RTA and ribosomes from them were fractionated on sucrose density gradients. The su- crose density gradients were designed to optimize separa- tion of 40S and 60S ribosomal subunits and as a result the polysome fraction was compressed towards the bot- tom of the gradient (Fig. 6A, B). rRNA was extracted from 40S and 60S subunits, 80S ribosomes and poly- somes, rRNA from the latter fraction contained a repre- sentative sample from the whole polysome mix at the bot tom of the gradient, rRNA from the various fractions was treated with aniline and separated on denaturing agarose gels. As expected, all 26S rRNA-containing frac- tions from strains containing wild-type RTA were modi- fied by the expressed RTA (e.g. Fig. 6 C, lanes 3, 5, 7). In contrast, a higher proport ion of 26S rRNA in the free 60S subunits was modified by mutant RTA com- pared with that in the 80S or polysomal fractions (Fig. 6D, compare lane 3, 60S, with lane 7, polysome fraction). This difference was observed both when Asn203 RTA was expressed in the original mutant yeast strain (19.55, data not shown) and when the recloned mutant RTA was expressed in transformant 55.30 (Fig. 6D), demonstrating that it is a property of the mu- tant RTA and not of the yeast strain itself.

As further evidence that this difference is due to a modified RTA, ribosomes or polysomes were isolated from the parental and mutant strains after growth in glucose to prevent RTA expression and treated in vitro with purified RTA. RNA modification was then assessed

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Fig. 6A-D. RTA modification of ribosomal subunits. Transformant J20, which contains one copy of RTA, and mutant strain 55.30, which contains a single, recloned copy of mu- tant RTA, were grown in URA DO-ethanol to approximately 107 cells per ml and induced to express RTA by resuspension and growth in URA DO-galactose for 5 h. A, B UV absorp- tion prolifes of ribosomal subunits separated on sucrose density gradients, from transfor- mant J20 (A) and mutant 55.30 (B). 40, 60, 80, and poly indicate peaks of 40S and 60S sub- units, 80S ribosomes and polysomes respective- ly. Arrows show the positions of half-mer poly- ribosomes. C, D Denaturing agarose gels of RNA extracted from ribosomal fractions shown in A and B. + , aniline-treated RNA; - , untreated RNA; I~, position of 369-base fragment released from RTA-modified 26S rRNA by aniline. C Transformant J20. Lanes t, 2, 40S; 3, 4, 60S; 5, 6, 80S; 7, 8, po- lysomes; 9, 10, total RNA from lysate prior to sucrose density gradient centrifugation; 11, 12, control RTA-treated ribosomes. D Mutant 55.30. Lanes 1, 2, 40S; 3, 4, 60S; 5, 6, 80S; 7, 8, polysomes; 9, t0, control RTA-treated ribo- somes

by aniline t rea tment as described in Materials and meth- ods. N o difference in sensitivity between mu tan t and parental r ibosomes or po lysomes was observed even at R T A concent ra t ions tha t only partially modif ied the 26S r R N A (data no t shown). The apparen t increase in resis- tance to r ecombinan t R T A in m u t a n t 19.55 therefore

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Fig. 7. Protein synthesis inhibition assay. Brome Mosaic virus mRNA was translated in a rabbit reticulocyte lysate system in the presence of increasing concentrations of native (open squares), recombinant wild-type (closed circles) or mutant RTA (open cir- cles). Protein synthesis is expressed as a percentage of the 3[H]leu- cine incorporated in the absence of RTA

seems to be a funct ion o f the R T A produced, a l though it is possible tha t u p o n purif icat ion o f 19.55 r ibosomes the associated factor(s) that alters the degree o f modifi- cat ion is lost.

Effect o f altering Ser 203 to Asn on inhibition of protein synthesis in reticulocyte lysate

Mutan t R T A expressed in E. coli was tested for enzymat- ic activity by assessing its ability to inhibit t ranslat ion o f Brome Mosaic virus r R N A in a rabbit reticulocyte lysate prote in synthesis react ion as described previously (Schlossman et al. 1989) (Fig. 7). Plant R T A inhibited protein synthesis in this system with an IDs0 o f approxi- mately 2 x 10 -11 M. In the same assay recombinan t R T A produced in E. coli was slightly less active, as pre- viously observed (Schlossman et al. 1989), with an IDso of 6 x 10 - z 1 M. Asn 203 mu tan t R T A was approximate- ly 100-fold less effective at inhibiting protein synthesis, having an IDso o f 7 x 10 -9 M.

Discussion

These experiments and those o f Frankel et al. (1989) show that expression o f R T A in yeast is lethal and induc- t ion o f R T A expression in yeast t ransformants contain- ing a single, integrated copy o f wild-type R T A causes immediate cessation o f g rowth (Fig. 3 B). Figure 4 shows

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that a low level of RTA-immunoreactive material could be detected in transformants containing single or multi- ple integrated copies of the RTA expression plasmid after induction of RTA expression by growth in galac- tose. Frankel et al. (1989) were unable to detect RTA- immunoreactive material expressed from a low copy number plasmid, although a band of the expected size was seen on Western blots of extracts from induced cells carrying a high copy number RTA expression plasmid. Our ability to detect RTA-immunoreactive material from low copy number vectors in these experiments is most likely due to the different constructs used; the RTA expressed from pJG2RA has an additional 24 amino acids at its N-terminus compared with native RTA, which may render it slightly less toxic and allow detect- able quantities to accumulate. Two smaller RTA-related proteins were also detected on the Western blot: interest- ingly the larger of these was the same size as recombinant RTA produced in E. coli that has the correct RTA N- terminus. It is possible that RTA produced in yeast may be fortuitously processed to yield mature RTA since it has been observed that proricin can be processed to gen- erate RTA- and RTB-sized polypeptides by a crude yeast extract (S. Harley and J.M. Lord, unpublished data). In protein bodies an identical protease may be responsi- ble for both removal of the linker and maturation of the RTA N-terminus (Lord and Robinson 1986).

Using a depurination assay for RTA modification of 26S rRNA we have shown that the RTA that is pro- duced in transformant J19 is active and that depurinated 26S rRNA can be detected I h after induction of RTA expression (Fig. 5). This modification of 26S rRNA pre- sumably causes inhibition of protein synthesis and leads to the arrest of cell growth observed when transformants containing wild-type RTA are grown in galactose (Fig. 3).

Mutant RTA-transformed strains that are capable of growing during galactose induction of the RTA gene have been isolated. All the mutants tested contain RTA RNA suggesting that GALl-10 promoter-RTA muta- tions have not been obtained. A proportion of these mutant strains no longer have RTA-modified rRNA. Treatment of ribosomes from such strains with purified RTA in vitro demonstrated that none possessed RTA- resistant ribosomes; their ability to survive induction of RTA expression is thus most likely due to production of inactive RTA.

The other class of mutants possesses modified rRNA when grown in galactose. This was a surprising observa- tion since the mutant strains grow with a generation time similar to wild type (Fig. 3) and it is widely accepted that a single molecule of RTA is sufficient to cause cell death (Eiklid et al. 1980). The phenotype of these mu- tants could be explained in several ways: they may pro- duce RTA with altered activity such that the yeast cells can tolerate its expression; they may produce a lower level of RTA, such that ribosomes can be made faster than they become inactivated; alternatively these mu- tants may contain alterations in yeast ribosomal protein genes giving rise to a population of RTA-resistant ribo- somes. This latter possibility was considered since many

ribosomal protein genes in yeast are duplicated (Warner 1989) and a situation could therefore be envisaged in which the mutant strain possesses two populations of ribosomes, one of which is wild type and sensitive to RTA and thus give rise to the modified rRNA and the other of which contains mutant ribosomal proteins which enable the cells to grow in the presence of RTA.

To investigate these possibilities one mutant (19.55) was chosen for detailed analysis. The RTA in this strain was shown by DNA sequencing to have a single nucleo- tide change (G608 to A) which causes an amino acid alteration of Ser203 to Asn (data not shown). Figure 6D shows that 26S rRNA in free 60S ribosomal subunits appears to be preferentially modified by this mutant RTA. An alternative explanation for the increased level of modification in the free 60S subunits observed in Fig. 6D could be that modified 60S subunits have be- come dissociated from polysomes and are unable to rein- itiate protein synthesis, resulting in an accumulation of modified subunits within the 60S subunit pool. We be- lieve the former explanation is more likely to be correct for two reasons. Firstly it has been shown that once a 60S ribosomal subunit has been modified within a polysome it remains in place and prevents further trans- lation on that polysome (Fodstad and Olsnes 1977). Sec- ondly, the patterns of modification caused by expression of wild-type and mutant RTA and detected after isola- tion of the various rRNA fractions are different; if all fractions were equally available to both toxins in vivo, one would expect to see the same patterns of modifica- tion in Fig. 6 C and 6 D. Clearly this is not the case.

It might be expected that a modified free 60S subunit would be impaired in its ability to form part of a poly- some. The work of Osborn and Hartley (1990) has dem- onstrated that RTA-modified 60S subunits are slow to initiate in protein synthesis. The presence of half-mers on polysomes (e.g. Fig. 6A, B) lends support to this observation since it has previously been shown that a deficiency of 60S ribosomal subunits causes the appear- ance of half-reefs (Rotenberg etal. 1988). Half-mers have not been detected on polysomes from yeast cells grown in the absence of RTA (data not shown). These observations, in conjunction with the preferential modi- fication of free 60S subunits by the mutant RTA in 19.55, may explain how the cells can tolerate induction of RTA expression, i.e. modified 60S subunits may be preferentially excluded from entering polysomes.

To provide further evidence that the Ser 203 to Asn RTA alteration is responsible for the ability of 19.55 yeast cells to tolerate expression, the sensitivities to na- tive RTA of ribosomes or polysomes from parental and mutant strains were assessed in vitro. No differences were observed (data not shown), suggesting that the ri- bosomes from both sources are identical and that the mutant RTA is sufficiently less toxic than native RTA to permit growth of expressing yeast cells. It is possible, however, that in vitro results such as these may be mis- leading since protective factors associated with 19.55 ri- bosomes, which alter the degree of RTA modification, may be lost during the isolation procedure. We believe that this explanation is unlikely to account for the ob-

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served differences since expression of mutant RTA rec- loned into the original expression vector and trans- formed into the wild-type strain gives the same growth phenotype (compare Fig. 3D and F) and ribosomal modification pattern in vivo (Fig. 6B, D; identical re- sults for 19.55 not shown) as does expression of RTA in 19.55.

The three-dimensional structure of ricin determined by X-ray crystallography shows RTA to have a promi- nent cleft which has been proposed to be the active site (Montfort et al. 1987). Amino acid residues within this region that may be involved in catalytic activity have been identified by homology with two other plant toxins, trichosanthin and barley protein synthesis inhibitor, and with E. coli RNase H and Rous Sarcoma virus reverse transcriptase (Robertus 1988), all proteins that share the common function of RNA binding. Four conserved resi- dues, Glu 177, Ash 209, Trp 211 and Arg 180 (number- ing as in RTA), are found in the proposed active site. Analysis of RTA mutations produced in yeast (Frankel et al. 1989) lends support to the idea that this region is the enzymatic active site: mutations were obtained that changed Glu 177 to Asp or Lys, Trp 211 to Arg, Gly 212 to Trp or Glu, Ser 215 to Pro and Ile 252 to Arg, and that lowered the catalytic activity of RTA both when expressed in yeast and when produced in E. coli. All of these amino acids have side chains in the proposed active site cleft. The mutation described in this paper alters an amino acid residue (Set 203) that does not form part of the proposed active site cleft (J. Robertus, per- sonal communication) and which, to date, has not been implicated in the catalytic activity of RTA. This residue is conserved in the related plant lectin R. communis ag- glutinin and in the ribosome-inactivating protein tricho- santhin. There is a conservative change to Ala in the plant toxin abrin and the related shiga-like toxin from E. coli. Asn does not occur at this position in any plant or bacterial ribosome-inactivating protein known to date.

Using the conventional in vitro assay for RTA activi- ty, recombinant Asn 203 RTA from mutant 19.55, pro- duced in E. coli, was shown to be approximately 100-fold less toxic to a rabbit reticulocyte lysate protein synthesis system, compared with native plant RTA or with recom- binant wild-type RTA expressed in E. coli, despite the fact that the mutated residue (Set 203) does not lie in this proposed active site cleft. Based on the crystal struc- ture of the ricin holoenzyme it appears that once the subunits separate, Set 203 is partially exposed on the surface of RTA. Ser 203 is the third residue on a kinked c~-helix that makes up part of the proposed active site cleft. It is possible that mutation of Ser 203 might alter the activity of RTA by distorting the a-helix and/or mov- ing other residues involved in catalysis (T. Quinn, per- sonal communication). There are several other possibili- ties as to why a mutation distant from the active site could affect catalytic activity. It is possible that the alter- ation of Ser 203 to Asn may affect the solubility or turn- over of RTA such that less toxin is available to interact with the ribosomes. Figure 4 shows that yeast strains induced to express Asn 203 RTA produce at least as

much RTA-immunoreactive material as strains express- ing wild-type RTA, thus suggesting that accelerated turnover of the mutant protein is not a significant factor affecting its activity. The protein samples used for West- ern analysis were supernatants from a crude yeast cell lysate isolated after removal of insoluble matter by cen- trifugation at 12 000 g. Under these conditions aggregat- ed proteins would most likely be pelleted, as is the case when ricin B chain is expressed cytoplasmically in Sac- charomyces cerevisiae (Richardson et al. 1988). Insolu- bility of mutant RTA protein therefore probably does not contribute to its decreased activity. It is possible that a mutation located away from the active site may affect catalytic activity indirectly by impairing binding of RTA to the 60S ribosomal subunit. No residues have as yet been implicated in ribosome binding.

The isolation of mutations in yeast genes that confer resistance to the N-glycosidase activity of RTA has proved to be extremely difficult. Frankel et al. (1989) reported a double selection procedure in which RTA was expressed in strains cotransformed with plasmids carrying URA3 or LEU2 linked to RTA. None of the mutant strains they analysed had mutations in yeast genes and they concluded that the frequency of mutation to RTA resistance must be less than 1 in 1011. The rea- sons for this low frequency may be that alteration of the necessary protein to confer resistance is deleterious to the cell or it may be necessary to alter more than one protein simultaneously. Recently, CHO cell lines have been isolated that have ribosomes that are approxi- mately 35-fold more resistant to RTA than their wild- type equivalents (Sallustio and Stanley 1990). This sug- gests that it is possible to alter ribosomes in such a way as to increase their resistance to RTA while maintaining their functional capabilities. A novel screening proce- dure, using a genetic approach, is currently being investi- gated in an attempt to identify the rare RTA r mutations that may arise in yeast genes.

Acknowledgements. We thank Jon Robertus and Torn Quinn for help in correlating the Ser 203 mutation with X-ray crystallograph- ic data. This work was supported by SERC grants GR/E/65296 and GR/F/37825 and a Wellcome travel grant, KBS/TG/LEC/wj.

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C o m m u n i c a t e d by C. Hol lenberg