©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ricin A Chain Can Be Chemically Cross-linked to the Mammalian Ribosomal Proteins L9 and L10e (*)

Carol A. Vater (1)(§), Laura M. Bartle (1), John D. Leszyk (2), John M. Lambert (1), Victor S. Goldmacher (1)

From the (1) From ImmunoGen, Inc., Cambridge, Massachusetts 02139-4239 and the (2) Core Laboratory for Protein Chemistry, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Indirect immunofluorescence studies revealed that when fixed, permeabilized cultured human cells were incubated with ricin A chain, the toxin molecule localized in a staining pattern indicative of binding to the endoplasmic reticulum and to nucleoli. Chemical cross-linking experiments were performed to identify the cellular components that mediated the binding of ricin A chain. Conjugates were formed between I-labeled ricin A chain and two proteins present in preparations of total cell membranes and in samples of purified mammalian ribosomes. Specificity of the ricin A chain-ribosome interaction was demonstrated by inhibition of formation of the complexes by excess unlabeled ricin A chain, but not by excess unlabeled gelonin, another ribosome-inactivating protein. Complexes of ricin A chain cross-linked to the ribosomal proteins were purified and subjected to proteolytic digestion with trypsin. Amino acid sequencing of internal tryptic peptides enabled identification of the ricin A chain-binding proteins as L9 and L10e of the mammalian large ribosomal subunit.


INTRODUCTION

Ricin is a heterodimeric ribosome-inactivating protein, derived from the seeds of Ricinus communis (castor beans), that consists of a 30/32-kDa A chain linked by a disulfide bond to a 32-kDa B chain. The A chain exists in two major species, the 30-kDa A form which has a single complex oligosaccharide unit and the 32-kDa A form which has a high mannose type oligosaccharide in addition to the complex unit (1) . The B chain mediates binding to cell surface glycoproteins and thereby facilitates cellular uptake of the toxin (for review, see Ref. 2). The A chain, once in the cytoplasm and liberated from the B chain (3) , acts as an N-glycosidase that attacks the 60 S subunit of eukaryotic ribosomes, removing a specific adenine in 28 S rRNA (4) . While the precise mechanism underlying the catalytic activity of ricin A chain has been determined (5, 6, 7, 8, 9) , much less is known about how ricin A chain gains access to ribosomes and the roles, if any, that proteins play in the delivery and binding of ricin A chain to its ribosomal target.

Early work by Hedblom et al.(10) demonstrated that ricin A chain is capable of binding to 60 S subunits of rat liver ribosomes with a dissociation constant of 2.2 10M and a ratio of 1 mol of ricin A chain/mol of subunits. That ribosomal proteins play an important role in making rRNA highly susceptible to attack by ricin A chain was established by Endo and Tsurugi (6) , who showed that rat rRNA in the context of the ribosome is depurinated at adenine 4324 by ricin A chain with a K nearly 10-fold greater than that measured using naked 28 S rRNA. Naked 23 S rRNA of Escherichia coli is depurinated by ricin A chain at the homologous N-glycosidic bond, adenine 2660, at concentrations similar to those effective on mammalian naked rRNA, yet ricin is ineffective against intact E. coli ribosomes (4, 6, 11) .

Single-chain ribosome-inactivating proteins, such as gelonin, momordin I, and saporin-S6, and the A chains of double chain toxins, such as ricin and abrin c, have highly similar amino acid sequences at their putative active sites (12 and references therein; 13). These ribosome-inactivating proteins all have the same specificity for adenine 4324 of naked 28 S rRNA and exhibit comparable levels of inhibition in rabbit reticulocyte lysate translation systems. However, these same ribosome-inactivating proteins show very different levels of activity against ribosomes of other species, such as Homo sapiens (HeLa cells), E. coli, and various metazoan organisms (12 and references therein).

Taken together, these data suggest that the variations in sensitivity of ribosomes of different species to ribosome-inactivating proteins having identical rRNA substrate specificities reflect differences in ribosomal protein content or interactions. Ribosomal proteins may differentially bind various ribosome-inactivating proteins. Alternatively, ribosomal proteins may maintain the rRNA in a conformation susceptible to attack (4, 14) by some ribosome-inactivating proteins but not by others. Exactly which ribosomal proteins are involved and the nature of their contribution to the increased efficiency of catalysis by ribosome-inactivating proteins remains to be determined. This report is a first step toward elucidating the roles of ribosomal proteins in the inactivation of eukaryotic ribosomes by ricin A chain. We describe our success in identifying the first two ricin A chain-associating proteins to be isolated, mammalian ribosomal proteins L9 and L10e.


EXPERIMENTAL PROCEDURES

Cell Lines and Materials

The human small cell lung carcinoma line SW2 (15) was maintained as exponentially growing cultures in RPMI 1640 medium supplemented with 10% heat-inactivated iron-fortified bovine calf serum and 2 mML-glutamine. The human bladder carcinoma squamous cell line SCaBER (ATCC HTB 3) was maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum and 2 mML-glutamine.

Unless stated otherwise, reagents were purchased from Sigma. The protein cross-linking agents, disuccinimidyl suberate (DSS)() and 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and biotin-HPDP were purchased from Pierce. Bovine calf serum and fetal bovine serum were from JRH Biosciences (Lenexa, KS); media and other cell culture reagents were from BioWhittaker, Inc. (Walkersville, MD). Plasticware was purchased from Becton Dickinson and Co. Methanol was purchased from J. T. Baker Inc. Ricin A chain was obtained from Inland Laboratories (Austin, TX). Gelonin (Catalog No. G-2394) and the rabbit polyclonal anti-ricin antibody (Catalog No. R-1254) were purchased from Sigma.

Adsorption of Rabbit Anti-ricin Antibody

Prior to use in immunofluorescence experiments, the rabbit anti-ricin antibody was presorbed with fixed permeabilized SCaBER cells to remove any cell cross-reacting antibodies. SCaBER cells (10 cells) were removed from tissue culture plates by trypsinization, washed with 30 ml of PBS, and pelleted by centrifugation. The cell pellet was resuspended in 1 ml of -20 °C methanol for 5 min and then washed three times in PBS containing 5 mM NaN by resuspension and centrifugation. The buffer was aspirated, and the fixed cell pellet was resuspended in 50 µl of rabbit anti-ricin antibody at 17 mg/ml in PBS containing 5 mM NaN. Adsorption was allowed to proceed for 2 h at room temperature, after which time the cells were removed by centrifugation. The adsorbed antibody was transferred to a fresh tube and stored at 4 °C.

Indirect Immunofluorescence Microscopy

The adherent cell line, SCaBER, was chosen for immunofluorescence experiments based on its good morphological characteristics, our previous experience in fixing and labeling these cells, and availability. Cells were plated at 2 10 cells per well onto sterile glass coverslips in 24-well tissue culture plates. Three days later, the medium was aspirated from the wells, and the cell monolayers were fixed and permeabilized by the addition of 1 ml of methanol at -20 °C for 5 min. The cells then were rinsed three times in PBS and blocked for 30 min with PBS containing 5% normal goat serum and 5 mM NaN. All subsequent reagent dilutions were made in PBS containing 5% normal goat serum and 5 mM NaN. Antibodies routinely were microfuged for 5 min at 14,000 rpm to remove any grossly aggregated material prior to use. A minimum reagent volume of 100 µl was added per well and incubated at room temperature with gentle rocking to ensure an even distribution of the sample over the monolayer. Ricin A chain at 10M was added to the fixed cell monolayers for 75 min; alternatively, PBS containing 5% normal goat serum and 5 mM NaN alone was added to control wells. Between incubations with reagents, three washes with 1 ml of PBS, for 5 min each with gentle rocking, were performed. Adsorbed rabbit anti-ricin antibody was diluted 1:130 and added for 1 h, followed by three washes. FITC-labeled goat anti-rabbit IgG (Catalog No. OB1420-FITC, Fisher) at 1:100 was used to probe for bound anti-ricin antibody; the incubation with the secondary antibody was for 1 h in the dark. After the final incubation, the coverslips were washed three times in PBS and mounted onto microscope slides using a polyvinyl-based mounting medium (16) . Fluorescence was viewed through a Nikon FXA microscope equipped with epifluorescence optics and the appropriate filter cube for the detection of fluorescein. Photographs were taken on Fujichrome film.

In some experiments, cells were co-stained with a monoclonal antibody specific for protein disulfide isomerase, a marker protein of the endoplasmic reticulum. The HP13 mouse monoclonal antibody (17) , a generous gift of Dr. C. Kaetzel of Case Western Reserve University, Cleveland, OH, was used at a final dilution of 1:20 and added to fixed, permeabilized cells simultaneously with ricin A chain. Bound HP13 antibody was detected using Texas Red-conjugated goat anti-mouse IgG (Catalog No. OB5000-TxRd, Fisher) at 1:100.

I-Labeling of Ricin A Chain and Gelonin

Ricin A chain and gelonin were labeled with [I]NaI by the IODO-GEN method (18) using IODO-GEN Iodination Reagent purchased from Pierce (Product No. 28600) and [I]NaI purchased from DuPont NEN (Catalog No. NEZ-033H). Twenty-five microliters of IODO-GEN Iodination Reagent at 50 µg/ml in chloroform was dried to form a film on 12- 75-mm glass test tubes. Ricin A chain (120 µg in 40 µl of PBS/glycerol, 1:1) or gelonin (100 µg in 100 µl of PBS) was added to the IODO-GEN-coated tubes together with 10 µl of 1 M sodium phosphate, pH 7.0, and 0.33 mCi of [I]NaI, and the mixtures were incubated for 30 min on ice. The reactions were stopped by transferring the protein solutions to fresh tubes that did not contain IODO-GEN. Non-radioactive NaI (10 µl of a 1 M solution in water) was added and the mixtures were incubated for 10 min at room temperature. Ricin A chain and gelonin then were separated from free [I]NaI on NAP-5 (Pharmacia Biotech Inc.) columns of Sephadex G-25 equilibrated in PBS. Pooled fractions of the I-labeled ribosome-inactivating proteins were diluted with an equal volume of glycerol and stored at -20 °C.

Preparation of SW2 Cell Homogenate, Membrane, and Cytosol Fractions

SW2 cells were pelleted by centrifugation, washed twice in 0.145 M NaCl, and stored at -80 °C. The pellet from 5 10 cells was resuspended in 4 ml of 25 mM Hepes-KOH buffer, pH 7.4, containing 0.25 M sucrose and 1 mM PMSF, and homogenized using 20 strokes of a tight-fitting borosilicate glass pestle homogenizer. The cell lysate was centrifuged for 10 min at 6,000 rpm in a Microfuge, and the supernatant was removed to a fresh tube. The pellet was resuspended in another 4 ml of 25 mM Hepes-KOH buffer, pH 7.4, containing 0.25 M sucrose and 1 mM PMSF, and the homogenization and centrifugation steps were repeated. The homogenate supernatants were pooled and, in some cases, subjected to two rounds of freezing in liquid nitrogen and quick-thawing at 37 °C. A portion of the homogenate was separated into membrane and cytosol fractions by centrifugation at 4 °C for 2 h at 160,000 g (42,000 rpm) in a Type 50.2 Ti rotor (Beckman Instruments). The resulting supernatant, taken free of any contaminating pellet material, was designated the SW2 cell cytosol. The pellet, which is referred to as the SW2 total membrane fraction, was resuspended by gentle homogenization in 800 µl of 25 mM Hepes-KOH buffer, pH 7.4, containing 0.25 M sucrose and 1 mM PMSF. The homogenate, membrane, and cytosol preparations were aliquoted, frozen in liquid nitrogen, and stored at -80 °C.

Isolation of Ribosomes from Rabbit Reticulocytes, Rat Liver, and E. coli

Rabbit reticulocyte blood was purchased from Pel-Freeze Biologicals, and ribosomes were purified from the rabbit reticulocytes according to standard procedures (19, 20, 21) . Ribosomes were isolated from fresh rat livers (purchased from Taconic, Germantown, NY) and from the E. coli strain DHFF` according to protocols described by Spedding (22) , with the exception that the reducing agent was omitted. The final pelleting step for the ribosomes was made through a sucrose cushion prepared in 20 mM Hepes-KOH, pH 7.4, a buffer compatible with the cross-linking agents used, rather than Tris-HCl. The ribosomal pellets were rinsed gently with 25 mM Hepes-KOH, pH 7.4, containing 0.1 M KCl, and resuspended in the same buffer at 53 A units/ml, 580 A units/ml, and 410 A units/ml for the rabbit reticulocyte, rat liver, and E. coli preparations, respectively. The concentrations of the ribosome preparations were estimated by adsorbance at 260 nm, where 1 A unit is equal to 18 pmol of 80 S ribosomes or to 23 pmol of 70 S ribosomes (22) .

I-Labeled Ricin A Chain Cross-linking Protocol

Cell fractionation samples were tested for the ability to be cross-linked to ricin A chain. SW2 cell membrane fractions, SW2 cytosol fractions, or purified rabbit, rat, or E. coli ribosome preparations were diluted into 25 mM Hepes-KOH buffer, pH 7.4, containing 0.25 M sucrose, and mixed with 1 µl of I-labeled ricin A chain (approximately 0.25 µg) in a total volume of 20 µl. The mixtures were incubated for 1 h at room temperature to allow binding to occur. Then, one of two different amine-reactive cross-linking agents, DSS or DFDNB, was added to the reaction mixtures at a final concentration of 0.3 mg/ml from freshly prepared stock solutions in dimethyl sulfoxide (3.3 mg/ml). Cross-linking was allowed to proceed for 2 h at room temperature, after which time the reaction was quenched with the addition of glycine, pH 7.4, to 0.1 M and further incubation for 30 min at room temperature. Portions of the reaction mixtures (10 to 20 µl) were subjected to SDS-PAGE and autoradiography.

SDS-PAGE and Autoradiography

SDS-PAGE was performed under reducing conditions using 9% or 10% polyacrylamide gels (analytical gels, 12 cm 14 cm 0.75 mm; preparative gels, 12 cm 14 cm 1.5 mm or 3 mm) according to the method of Laemmli (23) . Samples were prepared in 62.5 mM Tris-HCl buffer, pH 6.8, containing 2% (w/v) SDS, 10% (v/v) glycerol, 0.001% bromphenol blue (w/v), and 5% (v/v) -mercaptoethanol and heated for 5 min in a boiling water bath. When mini-gels (8 cm 8 cm 1 mm) were used, the acrylamide concentration was 10% or 12%, and the gels were purchased from Novex (San Diego, CA). Estimates of relative molecular mass were made using Rainbow Markers (Amersham). Gels containing samples of I-labeled ricin A chain were dried and exposed to Fuji x-ray film at -80 °C for 6 to 16 h using a Cronex Lightning Plus intensifying screen (DuPont NEN).

Reduction and Biotinylation of Ricin A Chain

Ricin A chain at 3 mg/ml in PBS/glycerol (1:1) was reduced by reaction with 30 mM dithiothreitol for 50 min at 30 °C. Excess dithiothreitol was removed by passage of the mixture through a PD-10 column (Pharmacia Biotech Inc.) using PBS containing 1 mM EDTA as eluent. The reduced ricin A chain was reacted with biotin-HPDP (N-(6-[biotinamido]hexyl)-3`-(2`pyridyldithio)propionamide) according to the manufacturer's instructions in order to link biotin to the thiol of cysteine 259 near the C terminus of ricin A chain (24) .

Purification of the Cross-linked Ricin A Chain-Ribosomal Protein Species

The complex of ricin A chain cross-linked to the protein subsequently identified as ribosomal protein L9 was purified as follows. Rat liver ribosomes (9 mg in 350 µl of 25 mM Hepes-KOH, pH 7.4, containing 0.1 M KCl) and biotinylated ricin A chain (280 µg in 500 µl of PBS) were mixed together in 8 ml of 25 mM Hepes-KOH, pH 7.4, containing 50 mM KCl, 0.125 M sucrose, and 1 mM PMSF, and incubated for 1 h at room temperature. The final concentrations of ribosomes and ricin A chain during the binding step were approximately 4 10M and 1 10M, respectively. Control samples, in which either the ribosomes or the biotinylated ricin A chain were omitted, were performed in parallel and resulted in the absence of any protein comigrating with the cross-linked species at the end of the experiment (data not shown). DSS was added to 0.3 mg/ml from a 10 mg/ml stock solution freshly prepared in dimethyl sulfoxide. Cross-linking was allowed to proceed for 2 h at room temperature, after which time the reaction was quenched with the addition of 0.1 M glycine, pH 7.4, and incubation for an additional 30 min. The reaction mixture was layered over a 5-ml cushion of 50 mM Tris-HCl, pH 7.8, containing 0.5 M sucrose, 12.5 mM MgCl, and 80 mM KCl, and centrifuged for 2 h at 160,000 g (42,000 rpm) in a Beckman 50.2 Ti rotor.

The ribosomal pellet was resuspended by homogenization in 1 ml of 25 mM Tris-HCl, pH 7.5, containing 0.076 M NaCl, 5% (w/v) SDS, 1 mM PMSF, 2 mM EDTA, 1 µM leupeptin, 1 µM pepstatin, and 100 µg/ml RNase A Type II-A, using 12 strokes of a tight-fitting borosilicate glass pestle homogenizer. The resuspended sample was transferred to a screw-cap Microfuge tube, heated for 5 min in a boiling water bath to enhance solubilization, and subsequently microfuged for 10 min at 14,000 rpm. The supernatant was removed from the residual insoluble material in the pellet, transferred to a 50-ml screw cap tube, and diluted to 48 ml with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl, 1 mM PMSF, 2 mM EDTA, 1 µM leupeptin, and 1 µM pepstatin. Avidin-agarose (ImmunoPure Immobilized Avidin, Product No. 20225, Pierce) was treated with bovine serum albumin (0.1 mg/ml for 30 min) to block any nonspecific binding sites. 1 ml of a 50% slurry of the bovine serum albumin-blocked avidin-agarose was added to the solubilized, diluted ribosomal material, and the mixture was rotated at 4 °C overnight. The avidin-agarose beads were pelleted by centrifugation, and the supernatant was discarded. The beads were washed 5 times by resuspension in 10 ml of 10 mM Tris-HCl, pH 8, containing 0.1% SDS, 0.1% Triton X-100, and 2 mM EDTA, followed by centrifugation and aspiration of the supernatant. The beads (approximately 500 µl) then were transferred, by multiple washings, into a screw-cap Microfuge tube and collected by centrifugation. The supernatant was aspirated.

The avidin-agarose beads were resuspended in 1 ml of reducing (2)SDS-PAGE sample buffer and heated for 5 min in a boiling water bath. The beads were removed by centrifugation and the solubilized proteins that were released into the supernatant were electrophoresed on 10% polyacrylamide preparative gels. A second extraction of the avidin-agarose beads using 300 µl of reducing (1)SDS-PAGE sample buffer was performed to yield additional material. Biotinylated ricin A chain (10 µg/well) was run on several other lanes of the same gels. Proteins on the gels were electroblotted onto Immobilon-P (Millipore Corp.) for 90 min at 400 mA in 10 mM CAPS, pH 11, containing 10% methanol, at 4 °C. The blots were rinsed with water, then with 100% methanol, and stained for several minutes with Amido Black (0.1% Amido Black, 1% acetic acid, 40% methanol) to visualize the protein bands. Following destaining in several changes of water, bands of the 55-kDa cross-linked species were excised and pooled. In all, bands excised from 8 lanes, in which 150-µl samples had been loaded per well, were sufficient to generate enough material for internal peptide sequencing. We estimate that this corresponded to approximately 8 µg of total protein. Control bands of non-cross-linked biotinylated ricin A chain also were excised and used to generate an HPLC profile of the trypsin fragments derived from ricin A chain alone.

The complex of ricin A chain cross-linked to the protein later identified as ribosomal protein L10e was isolated using a purification scheme similar to that described above for the ricin A chain-L9 complex, with the following modifications. A preparation of total SW2 cell membranes, derived from 1.4 10 cells and resuspended in 43 ml, was used as the source of crude protein to be cross-linked to ricin A chain. DFDNB, rather than DSS, was used as the cross-linking agent and was added to a final concentration of 0.6 mg/ml from a stock solution of 20 mg/ml in dimethyl sulfoxide. Following quenching and ultracentrifugation of the sample, the pellet of biotinylated ricin A chain-bound ribosomes and membranes was resuspended in 8 ml of solubilization buffer and incubated for 30 min at 37 °C, rather than for 5 min at 100 °C. Insoluble material was removed by centrifugation. The solubilized sample was diluted to 0.1% (w/v) SDS and purified using avidin-agarose, SDS-PAGE, and electroblotting, as described above for ribosomal protein L9.

Trypsin Digestion, HPLC Separation of Peptides, and Amino Acid Sequencing

In situ trypsin digestions, HPLC separation of the peptides, and microsequence analyses were performed at the W. M. Keck Foundation Protein Chemistry Facility of the Worcester Foundation for Experimental Biology in Shrewsbury, MA. Electroblotted, excised bands of ricin A chain and the ricin A chain-ribosomal protein cross-linked species were digested in situ with trypsin, as described by Fernandez et al.(25) . The tryptic fragments were separated using a Brownlee Aquapore C8 microbore column (1 250 mM) on a Hewlett Packard 1090M HPLC system equipped with a UV diode array detector. With 0.1% trifluoroacetic acid as solvent A and 0.08% trifluoroacetic acid in acetonitrile/water (70:30 v/v) as solvent B, a linear gradient from 0 to 55% in solvent B was developed over 90 min at a flow rate of 50 µl/min. Peptide peaks were collected manually in 1.5-ml Microfuge tubes. Comparison of the HPLC peptide profiles derived from the ricin A chain-ribosomal protein cross-linked species with that derived from ricin A chain alone enabled the selection of peptides unique to the putative ribosomal proteins. The most prominent non-ricin A chain peptide from each complex was subjected to automated Edman degradation on an Applied Biosystems 477A Sequencer equipped with a Model 120A in-line phenylthiohydantoin analyzer or a Model 492 Procise Sequencer.


RESULTS

Localization of Ricin A Chain Binding by Indirect Immunofluorescence

Indirect immunofluorescence microscopy was used as an initial approach toward determining what specific ricin A chain binding sites existed, if any, in permeabilized human cells. As shown in Fig. 1a, the indirect immunofluorescence staining pattern obtained with ricin A chain was indicative of ricin A chain binding to the endoplasmic reticulum, where many ribosomes are localized, and to nucleoli, where ribosomal particles are assembled. Only a low level of background staining was observed in a control sample, which was not treated with ricin A chain but received both the primary and secondary antibodies (Fig. 1b). Double labeling experiments (data not shown), performed as described under ``Experimental Procedures,'' confirmed the association of ricin A chain with the endoplasmic reticulum, by demonstrating that ricin A chain co-localized with protein disulfide isomerase, a marker enzyme of that organelle.


Figure 1: Localization of ricin A chain binding by indirect immunofluorescence microscopy. Fixed, permeabilized SCaBER cells were incubated for 1 h with 10M ricin A chain (a) or buffer alone (b). The cell monolayers then were washed, and bound ricin A chain was detected by processing with a polyclonal rabbit anti-ricin antibody followed by a FITC-labeled goat anti-rabbit IgG secondary antibody.



I-Labeled Ricin A Chain Cross-linking to Proteins from Fractionated Cells

Chemical cross-linking experiments were performed to identify cellular components to which ricin A chain can bind. Because these studies required bulk cell culture, the nonadherent human small cell lung carcinoma line SW2 was chosen, rather than the adherent SCaBER cell line which was used for immunofluorescence. Cells were homogenized and fractionated into cytosol and membrane fractions, and these preparations were tested for the presence of putative ricin A chain-binding proteins by incubating them for 1 h with I-labeled ricin A chain. Either DSS or DFDNB then was added and the cross-linking reactions were allowed to proceed for 2 h at room temperature. The reaction mixtures were analyzed by SDS-PAGE and autoradiography; results using DSS and DFDNB are shown in Fig. 2, a and b, respectively.


Figure 2: Cross-linking of I-labeled ricin A chain to cell fractions. I-labeled ricin A chain at a final concentration of 4 10M was cross-linked to proteins in various cell fractions using either DSS (a) or DFDNB (b). Lane 1, buffer alone; lane 2, SW2 cell homogenate from 4.8 10 cells; lane 3, SW2 cell cytosol from 4.8 10 cells; lane 4, SW2 cell membranes from 4.8 10 cells; lane 5, 10 pmol of rat liver ribosomes; lane 6, 10 pmol of E. coli ribosomes. Following quenching of the cross-linking reactions, the mixtures were analyzed by SDS-PAGE and autoradiography. Only half of the rat liver ribosome sample cross-linked with DSS was loaded (a, lane 5) to avoid overexposure by the prominent 55-kDa band generated in this reaction mixture. The arrows point to the major ricin A chain-cross-linked species generated with each cross-linking agent. The arrowheads indicate minor cross-linked species. The major doublet running at 30-32 kDa is monomeric I-labeled ricin A chain. Positions of the molecular weight standard markers are indicated as M 10.



In control samples in which I-labeled ricin A chain was incubated with buffer alone (Fig. 2, a and b, lane 1), the only radiolabeled species observed were the prominent I-labeled doublet of monomeric ricin A chain at 30 kDa/32 kDa, a fainter diffuse band representing cross-linked I-labeled ricin A chain-ricin A chain dimers running between 60 kDa and 64 kDa and heavily cross-linked large aggregates of I-labeled ricin A chain that failed to enter the separating gel. We have noted that ricin A chain tends to aggregate more in the absence of other protein, leading to higher levels of cross-linked ricin A chain dimers and multimers in buffer control samples than in samples containing various cell fractions.

In reaction mixtures containing proteins from the homogenate of SW2 cells in addition to I-labeled ricin A chain and DSS (Fig. 2a, lane 2), a new band was observed running as a tight doublet at approximately 55 kDa (indicated by the large arrow). Since monomeric ricin A chain itself runs as a doublet, the 55-kDa doublet likely represents a single SW2 cell protein cross-linked to both of the differentially glycosylated forms of ricin A chain. The 55-kDa species was enriched in samples containing the SW2 total cell membrane fraction as the source of binding proteins (Fig. 2a, lane 4). Under optimal binding and cross-linking conditions, the amount of I-labeled ricin A chain present in this cross-linked product represented 25% of that present in the monomer bands of I-labeled ricin A chain, as determined by excision from the gel and counting of the respective bands in a counter. The 55-kDa species was not generated in the SW2 cell cytosol reaction mixture (Fig. 2a, lane 3).

When the shorter cross-linking reagent, DFDNB, was used, a second I-labeled ricin A chain cross-linked product was observed running at approximately 70 kDa, indicated by the large arrow in Fig. 2b. As with the 55-kDa species, the 70-kDa product was prominent in reaction mixtures containing SW2 cell membranes (Fig. 2b, lane 4), but not in those containing SW2 cell cytosol (Fig. 2b, lane 3).

Neither the indirect immunofluorescence experiments using whole cells nor the cross-linking studies using cell membrane fractions could resolve whether the ricin A chain-binding proteins were membrane proteins of the endoplasmic reticulum or components of the ribosomes that stud the endoplasmic reticulum and sediment with the membrane fraction. To address this question, we isolated ribosomes from rat liver and from E. coli and tested the purified ribosome preparations in cross-linking experiments with I-labeled ricin A chain. The 55-kDa and 70-kDa I-labeled ricin A chain-cross-linked species were enriched in samples containing rat liver ribosomes (Fig. 2, a and b, lane 5), suggesting that these ricin A chain-binding proteins were ribosomal or ribosome-associated proteins. The E. coli ribosome preparation did not support the generation of either of these ricin A chain-cross-linked species, although several high molecular weight products were observed in the sample of E. coli ribosomes cross-linked with DFDNB (Fig. 2b, lane 6).

In addition to the major 55-kDa and 70-kDa cross-linked species indicated by the large arrows in Fig. 2 (a and b), several less prominent bands can be seen on these autoradiographs; their positions are indicated by the small arrowheads in Fig. 2, a and b. These proteins, some of which can be generated more efficiently using other cross-linking agents, are being investigated currently and will be the subject of a future report.

Formation of the I-Labeled Ricin A Chain Cross-linked Species Is Inhibited by Excess Unlabeled Ricin A Chain but Not by Excess Unlabeled Gelonin

Specificity of the ricin A chain-ribosome interaction was demonstrated by competition experiments using either ricin A chain or gelonin, another ribosome-inactivating protein having the identical mechanism of ribosome inactivation (26) in a broad spectrum of species including rat and rabbit (12, 26) . A 50-fold molar excess of unlabeled ricin A chain or a 70-fold molar excess of gelonin was added to the reaction mixtures during the time in which I-labeled ricin A chain was preincubated with the ribosomes, before the addition of the cross-linking reagent. Whereas unlabeled ricin A chain produced nearly complete inhibition of formation of the I-labeled ricin A chain-cross-linked products (Fig. 3, a and b, lane 3), unlabeled gelonin was unable to prevent I-labeled ricin A chain cross-linking to either of the ribosomal proteins (Fig. 3, a and b, lane 5). The samples run in lane 4 of Fig. 3, a and b, represent an additional buffer control since the stock solution of ricin A chain was in PBS containing 50% glycerol. Note that in the experiment shown in Fig. 3a we used ribosomes purified from rabbit reticulocytes as the substrate for generation of the 55-kDa cross-linked species; similar results were obtained with rat liver ribosomes. Additional evidence that gelonin exhibits binding characteristics different from those of ricin A chain, even though they inactivate ribosomes by acting on the rRNA in a similar manner, was provided by experiments in which gelonin was labeled with [I]NaI and substituted for I-labeled ricin A chain in the standard cross-linking protocol using rat ribosomes and DSS. No I-labeled gelonin-cross-linked products were observed (data not shown).


Figure 3: Cross-linking of I-labeled ricin A chain to ribosomes: competition by ricin A chain but not by gelonin. I-labeled ricin A chain was cross-linked to ribosomes in reaction mixtures containing buffer or an excess of an unlabeled competing ribosome-inactivating protein. In a, ribosomes were purified from rabbit reticulocytes and cross-linked to I-labeled ricin A chain using DSS. In b, ribosomes were isolated from rat liver and cross-linked to I-labeled ricin A chain using DFDNB. The reaction mixtures contained the following components. Lane 1, I-labeled ricin A chain; lane 2, ribosomes + I-labeled ricin A chain; lane 3, ribosomes + a 50-fold molar excess of unlabeled ricin A chain + I-labeled ricin A chain; lane 4, ribosomes + glycerol-containing buffer + I-labeled ricin A chain; lane 5, ribosomes + a 70-fold molar excess of unlabeled gelonin + I-labeled ricin A chain. Arrows mark the positions of the 55-kDa and 70-kDa cross-linked species in a and b, respectively. The prominent doublet running at 30-32 kDa is monomeric I-labeled ricin A chain. The positions of the molecular weight standard markers are indicated as M 10.



Purification of the Ricin A Chain-Ribosomal Protein Complexes

We performed large-scale cross-linking experiments to generate sufficient amounts of the ricin A chain-ribosomal protein complexes for purification and internal peptide sequence analyses. SW2 cell membranes were used as the source of ribosomal proteins to produce the DFDNB-cross-linked 70-kDa complex, and rat liver ribosomes were used to produce the DSS-cross-linked 55-kDa complex. As described in detail under ``Experimental Procedures,'' the use of biotinylated ricin A chain enabled us to purify the complexes on avidin-agarose. The ricin A chain-ribosomal protein complexes were recovered from the resin by heating the avidin-agarose samples in SDS-containing buffer. The solubilized proteins then were subjected to SDS-PAGE and electroblotted onto Immobilon-P. A blot of the purified 55-kDa complex stained with Amido Black in shown in Fig. 4. Biotinylated ricin A chain alone was run in lanes 1 and 4, and the purified complex was run in lanes 2 and 3. The highly purified 55-kDa complex ran as a doublet, as does ricin A chain, and was easily resolved from contaminating traces of non-cross-linked ricin A chain and monomeric avidin, which ran at the dye front and which presumably leached off the agarose beads during boiling in SDS. Similar results were obtained for the 70-kDa complex, purified using the same strategy.


Figure 4: Western blot of the purified 55-kDa ricin A chain-ribosomal protein cross-linked complex. Biotinylated ricin A chain was cross-linked to ribosomes, and the ricin A chain-ribosomal protein complex was purified on avidin-agarose. The complex was eluted from the avidin-agarose beads by boiling in SDS-containing buffer and purified by SDS-PAGE and Western blotting onto a PVDF membrane. The membrane was stained with Amido Black; positions of the molecular weight standard markers are indicated as M 10. Biotinylated ricin A chain alone was run in lanes 1 and 4, 10 µg and 5 µg, respectively. The ricin A chain-ribosomal protein complex was run in lanes 2 and 3. The arrow points to the ricin A chain-cross-linked doublet species.



Internal Peptide Sequence Analyses Identify the Ribosomal Proteins as L9 and L10e

Bands of the purified ricin A chain-ribosomal protein complexes and of ricin A chain alone were excised from the blots and subjected to in situ digestion with trypsin. The peptides generated from each sample were separated by HPLC, and the resulting chromatographs, shown in Fig. 5, were compared in order to identify peptides of non-ricin A chain origin. The peptide digest derived from the 55-kDa ricin A chain-ribosomal protein complex, shown in the middle tracing (Fig. 5b), and the peptide digest derived from the 70-kDa complex, shown in the bottom tracing (Fig. 5c), both contained peaks that were not present in the peptide digest derived from the sample of ricin A chain alone, shown in the upper tracing (Fig. 5a). The unique peptides marked with arrows in Fig. 5, b and c, were chosen for amino acid sequencing.


Figure 5: HPLC separation of tryptic peptides derived from ricin A chain and the ricin A chain-ribosomal protein complexes. Bands of ricin A chain and the ricin A chain-ribosomal protein complexes were excised from Western blots and subjected to in situ proteolytic digestion with trypsin. The peptides were separated by HPLC and the resulting chromatograms are shown. a, ricin A chain alone; b, the 55-kDa ricin A chain-ribosomal protein complex; c, the 70-kDa ricin A chain-ribosomal protein complex. Tracings b and c, therefore, reflect peptides derived from both ricin A chain and the cross-linked ribosomal protein. The peptides eluting in the peaks designated with arrows in b and c were chosen for amino acid sequencing.



Residues 2 through 14 of the peptide derived from the 55-kDa complex were unambiguously identified as FNHINVELSLLGK. The 13-amino acid sequence of the peptide was used to perform a BLAST data base search and found to be a perfect match to residues 38 to 50 of rat ribosomal protein L9, as deduced from the nucleotide sequence obtained by Suzuki et al.(27) . Rat ribosomal protein L9 contains 192 amino acids and has an molecular weight of 21,879 (27) , making it an appropriately sized protein to form a 55-kDa complex with ricin A chain. The sequence of 20 residues of the peptide derived from the 70-kDa complex was determined to be AFLADPSAFVAAAPVAAATT. A BLAST data base search revealed a perfect match to residues 267 to 286 of human ribosomal protein L10e, as deduced from the cDNA sequence obtained by Rich and Steitz for the human ribosomal phosphoprotein originally termed P0 (28, 29) . Human ribosomal protein L10e contains 317 amino acids and has a molecular weight of 34,273 (28) .


DISCUSSION

We have established that ricin A chain associates with eukaryotic ribosomes in the proximity of two protein components, forming covalent complexes of 55 kDa or 70 kDa when cross-linked to ribosomes using the homobifunctional amine-reactive cross-linking agents DSS or DFDNB, respectively. Through purification of the complexes and internal peptide sequence analyses, we have identified the polypeptides as L9 and L10e of the large ribosomal subunit. These identifications are consistent with data generated by indirect immunofluorescence, localizing ricin A chain binding in permeabilized cells to the nucleoli and the endoplasmic reticulum, sites of eukaryotic ribosome biogenesis (30) and protein synthesis, respectively. The ability of ricin A chain to form a similar 55-kDa protein complex with ribosomes prepared from three different mammalian species (human, rabbit, and rat), but not with ribosomes from E. coli, suggests that the L9 protein is highly conserved among eukaryotes, but not between eukaryotes and prokaryotes. This conclusion is supported by reports of eukaryotic homologs of rat L9 cloned from H. sapiens(31) and S. cerevisiae(32) , having amino acid sequence identity over a 192-residue overlap to rat ribosomal L9 of 99% and 49%, respectively. Significantly less homology, 26%, was found between rat ribosomal L9 and its E. coli homolog L6 (27, 33) .

The 70-kDa ricin A chain complex was generated readily using ribosomes purified from either cultured human cells or rat liver, less efficiently when rabbit reticulocyte ribosomes were used, and not at all when E. coli ribosomes were used as the ricin A chain-binding substrate. Deduced amino acid sequence identities between human L10e and equivalent proteins from other species have been reported to be 95% for Rattus norvegicus(34) , 90% for Rattus rattus (GenPept Updates) and Mus musculus (35), 84% for Gallus gallus (GenBank Updates), 55% for Saccharomyces cerevisiae(36, 37) , 25% for Halobacterium cutirubrum(29) , and 15% for E. coli(29) .

Although the precise roles of L9 and L10e in the process of eukaryotic protein synthesis are unknown, clues to the functions of these proteins may be deduced by analogy to their prokaryotic homologs, L6 and L10 (29, 33, 38) . Recent studies showing that L10 homologs in yeast and rat are required for the assembly of a multiprotein complex onto eukaryotic ribosomes that is similar to the (L7/L12)L10 complex of E. coli ribosomes indicate that analogies between the eukaryotic and bacterial ribosomal systems are likely to be valid (39, 40) . In E. coli, both L6 and L10 have been ascertained by chemical cross-linking and immunoelectron microscopy studies to be located in close proximity to each other on the large ribosomal subunit, in the translocation domain positioned at the interface between the large and small subunits (41-44). Furthermore, both L6 and L10 are believed to be functionally involved in the GTP hydrolysis step mediated by elongation factor G during translocation. Both L10 and L6 have been cross-linked to L7/12 dimers (44, 45) , and both proteins have been shown to stimulate the elongation factor G-dependent GTP hydrolysis associated with L7/L12 (46, 47, 48) . L6 has been identified as one of several proteins to which elongation factor G can be cross-linked upon binding to the ribosome (42, 49) . In eukaryotic ribosomes, L9 has been cross-linked to elongation factor EF-2 (50) .

Our finding that ricin A chain can be cross-linked to two ribosomal protein components of the translocation domain is likely to be of physiological relevance, because EF-2-catalyzed GTP hydrolysis and translocation are the steps of protein synthesis inhibited by ricin (51). Indeed, EF-2 has been shown to protect ribosomes from inactivation by ricin A chain (52, 53) and to prevent binding of ricin A chain to ribosomes in the presence of a nonhydrolyzable GTP analog (54). Likewise, ricin can inhibit the binding of EF-2 to the ribosome (55, 56). These results suggest that EF-2 and ricin A chain compete for identical or closely located binding sites, such that prior binding of one protein precludes access of the other to the ribosome.

We believe this to be the first report identifying ribosomal proteins in the immediate vicinity of the site of ricin A chain association with the ribosome. Gelonin, a protein having precisely the same mechanism of action as ricin A chain (26) , was incapable of inhibiting the association of ricin A chain with L9 or L10e in our cross-linking experiments. One explanation for this result may be that gelonin associates with the ribosome at the same binding site as ricin A chain but with much lower affinity. Alternatively, gelonin may bind to the ribosome with a different orientation than ricin A chain. The observed differential sensitivity of protein translation systems from various sources to inactivation by ricin A chain and by single chain ribosome-inactivating proteins such as gelonin (12) may reflect differences in the way these distinct ribosome-inactivating proteins associate with ribosomal proteins of various species. Indeed, even though ricin A chain and gelonin catalyze the same reaction, there is only 35.5% amino acid sequence identity between them (13) . Interestingly, saporin-6, a ribosome-inactivating protein extracted from the seeds of Saponaria officinalis and having the identical site of rRNA depurination as ricin A chain, has been shown by chemical cross-linking experiments to form a complex of approximately 60 kDa with a component of the 60 S subunit of yeast ribosomes (57) . The identity of the yeast ribosomal saporin-binding protein is as yet unreported, but it is intriguing to speculate that it will turn out to be YL9, the yeast homolog of mammalian ribosomal protein L9 (32) .

What is the role of ribosomal proteins L9 and L10e in ricin A chain activity? Endo and Tsurugi (6) demonstrated that the context provided to eukaryotic rRNA by ribosomal proteins is required for efficient catalysis of rRNA depurination by ricin A chain. Although ricin A chain has similar K values measured with rat ribosomes, naked 28 S RNA, and a synthetic 19-mer oligonucleotide, the k measured for ricin A chain using intact ribosomes as substrate is 10-fold greater than that using naked 28 S RNA or the synthetic oligonucleotide (6, 8) . This major difference in turnover number has prompted investigators to propose a role for eukaryotic ribosomal proteins in inducing or maintaining the proper rRNA secondary structure required to make the specific adenine residue highly susceptible to attack by ricin A chain (14) . However, although the ribosomes of E. coli contain GAGA loop structures similar to that present in the ricin A chain-susceptible site of eukaryotic rRNA, and although naked 23 S RNA is depurinated by ricin A chain with a k comparable to that of naked 28 S RNA, intact E. coli ribosomes are not inactivated by ricin A chain (6, 11) . On the basis of our observation that ricin A chain can be cross-linked to ribosomal proteins L9 and L10e, we propose that ricin A chain becomes an efficient catalyst of eukaryotic ribosome depurination only upon binding to these ribosomal proteins, and that ricin A chain is inactive against E. coli ribosomes due to its inability to associate with the more divergent L6 and L10 proteins. Our proposal is supported by the observation that ricin A chain cannot be cross-linked to these E. coli ribosomal proteins under conditions used successfully to cross-link ricin A chain to the homologous proteins from human, rabbit, and rat ribosomes.

Regardless of the importance of the association of ricin A chain with L9 or L10e for ricin A chain function, our data should be of interest to those attempting to build a macromolecular model of the mammalian ribosome. Although much progress has been made in solving the structure of the E. coli organelle, much less is known about the protein-protein and protein-rRNA contacts and functional interactions in eukaryotic ribosomes. The observation that ricin A chain binds to the ribosome in close proximity to ribosomal proteins L9 and L10e now can be combined with pre-existing knowledge that ricin A chain depurinates the 28 S rRNA specifically at adenine 4324 to provide new insight toward assembling a structural model of the 60 S ribosomal subunit.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 617-661-9312; Fax: 617-661-9334.

The abbreviations used are: DSS, disuccinimidyl suberate; DFDNB, 1,5-difluoro-2,4-dinitrobenzene; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline, 10 mM potassium phosphate, pH 7.2, containing 0.145 M NaCl; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; HPLC, high performance liquid chromatography; CAPS, 3-(cyclohexylamino)propanesulfonic acid.


ACKNOWLEDGEMENTS

We wish to express our gratitude to Dr. Nancy Kedersha and Gail McKenzie for assistance with fluorescence microscopy and to Dr. C. Kaetzel of Case Western Reserve University, Cleveland, OH, for the generous gift of the HP13 anti-protein disulfide isomerase antibody. We thank Drs. Walter A. Blättler and Rajeeva Singh for advice, Dr. Walter A. Blättler for critical reading of the manuscript, and Rita Steeves and Carlos Acevedo for assistance in the preparation of biotinylated ricin A chain.


REFERENCES
  1. Foxwell, B. M. J., Donovan, T. A., Thorpe, P. E., and Wilson, G. (1985) Biochim. Biophys. Acta 840, 193-202 [Medline] [Order article via Infotrieve]
  2. Johnson, V. G., and Youle, R. J.(1991) in Intracellular Trafficking of Proteins (Steer, C. J., and Hanover, J. A., eds) pp. 183-225, Cambridge University Press, Cambridge, UK
  3. Olsnes, S., Refsnes, K., and Pihl, A.(1974) Nature 249, 627-631 [Medline] [Order article via Infotrieve]
  4. Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K.(1987) J. Biol. Chem. 262, 5908-5912 [Abstract/Free Full Text]
  5. Endo, Y., and Tsurugi, K.(1987) J. Biol. Chem. 262, 8128-8130 [Abstract/Free Full Text]
  6. Endo, Y., and Tsurugi, K.(1988) J. Biol. Chem. 263, 8735-8739 [Abstract/Free Full Text]
  7. Ready, M. P., Kim, Y., and Robertus, J. D.(1991) Proteins: Struct. Funct. Genet. 10, 270-278 [Medline] [Order article via Infotrieve]
  8. Gluck, A., Endo, Y., and Wool, I. G.(1992) J. Mol. Biol. 226, 411-424 [Medline] [Order article via Infotrieve]
  9. Monzingo, A. F., and Robertus, J. D.(1992) J. Mol. Biol. 227, 1136-1145 [Medline] [Order article via Infotrieve]
  10. Hedblom, M. L., Cawley, D. B., Boguslawski, S., and Houston, L. L. (1978) J. Supramol. Struct. 9, 253-268 [Medline] [Order article via Infotrieve]
  11. Olsnes, S., Heiberg, R., and Pihl, A.(1973) Mol. Biol. Rep. 1, 15-20
  12. Barbieri, L., Battelli, M. G., and Stirpe, F.(1993) Biochim. Biophys. Acta 1154, 237-282 [Medline] [Order article via Infotrieve]
  13. Nolan, P. A., Garrison, D. A., and Better, M.(1993) Gene (Amst.) 134, 223-227 [Medline] [Order article via Infotrieve]
  14. Endo, Y., Gluck, A., and Wool, I. G.(1991) J. Mol. Biol. 221, 193-207 [CrossRef][Medline] [Order article via Infotrieve]
  15. Smith, A., Waibel, R., Westera, G., Martin, A., Zimmerman, A. T., and Stahel, R. A.(1989) Br. J. Cancer 59, 174-178 [Medline] [Order article via Infotrieve]
  16. Fukui, Y., Yumura, S., and Yumur, T. K.(1987) Methods Cell Biol. 28, 347-356 [Medline] [Order article via Infotrieve]
  17. Kaetzel, C. S., Rao, C. K., and Lamm, M. E.(1987) Biochem. J. 241, 39-47 [Medline] [Order article via Infotrieve]
  18. Fraker, P. J., and Speck, J. C.(1978) Biochem. Biophys. Res. Commun. 80, 849-857 [Medline] [Order article via Infotrieve]
  19. Hardesty, B., McKeehan, W., and Culp, W(1971) Methods Enzymol. 20, 316-330
  20. Martin, T. E., Rolleston, F. S., Low, R. B., and Wool, I. G.(1969) J. Mol. Biol. 43, 135-149 [Medline] [Order article via Infotrieve]
  21. Martin, T. E., Wool, I. G., and Castles, J. J.(1971) Methods Enzymol. 20, 417-429 [CrossRef]
  22. Spedding, G.(1990) Ribosomes and Protein Synthesis: A Practical Approach, Oxford University Press, New York, NY
  23. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  24. Lamb, F. I., Roberts, L. M., and Lord, J. M.(1985) Eur. J. Biochem. 148, 265-270 [Abstract]
  25. Fernandez, J., Andrews, L., and Mische, S. M.(1994) Anal. Biochem. 218, 112-117 [CrossRef][Medline] [Order article via Infotrieve]
  26. Endo, Y., Tsurugi, K., and Lambert, J. M.(1988) Biochem. Biophys. Res. Commun. 150, 1032-1036 [Medline] [Order article via Infotrieve]
  27. Suzuki, K., Olvera, J., and Wool, I. G.(1990) Gene (Amst.) 93, 297-300 [Medline] [Order article via Infotrieve]
  28. Rich, B. E., and Steitz, J. A.(1987) Mol. Cell. Biol. 7, 4065-4074 [Medline] [Order article via Infotrieve]
  29. Shimmin, L. C., Ramirez, C., Matheson, A. T., and Dennis, P. P.(1989) J. Mol. Evol. 29, 448-462 [Medline] [Order article via Infotrieve]
  30. Larson, D. E., Zahradka, P., and Sells, B. H.(1991) Biochem. Cell Biol. 69, 5-22 [Medline] [Order article via Infotrieve]
  31. Hori, N, Murakawa, K., Matoba, R., Fukushima, A., Okubo, K., and Matsubara, K.(1993) Nucleic Acids Res. 21, 4395 [Medline] [Order article via Infotrieve]
  32. Jones, D. G. L., Reusser, U., and Braus, G. H.(1991) Nucleic Acids Res. 19, 5785 [Medline] [Order article via Infotrieve]
  33. Wittmann-Liebold, B.(1984) Adv. Protein Chem. 36, 56-78
  34. Wool, I. G., Chan, Y.-L., Glueck, A., and Suzuki, K.(1991) Biochimie (Paris) 73, 861-870 [CrossRef][Medline] [Order article via Infotrieve]
  35. Krowczynska, A. M., Coutts, M., Makrides, S., and Brawerman, G.(1989) Nucleic Acids Res. 17, 6408 [Medline] [Order article via Infotrieve]
  36. Newton, C. H., Shimmin, L. C., Yee, J., and Dennis, P. P.(1990) J. Bacteriol. 72, 579-588
  37. Mitsui, K., and Tsurugi, K.(1988) Nucleic Acids Res. 16, 3573 [Medline] [Order article via Infotrieve]
  38. Golden, B. L., Ramakrishnan, V., and White, S. W.(1993) EMBO J. 12, 4901-4908 [Abstract]
  39. Santos, C., and Ballesta, J. P. G.(1994) J. Biol. Chem. 269, 15689-15696 [Abstract/Free Full Text]
  40. Uchiumi, T., and Kominami, R.(1992) J. Biol. Chem. 267, 19179-19185 [Abstract/Free Full Text]
  41. Lambert, J. M., and Traut, R. R.(1981) J. Mol. Biol. 149, 451-476 [Medline] [Order article via Infotrieve]
  42. Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G.(1986) in Structure, Function, and Genetics of Ribosomes (Hardesty, B., and Kramer, G., eds) pp. 286-308, Springer-Verlag New York Inc., New York
  43. Stoffler, G., and Stoffler-Meilicke, M.(1986) in Structure, Function, and Genetics of Ribosomes (Hardesty, B., and Kramer, G., eds) pp. 28-46, Springer-Verlag New York Inc., New York
  44. Walleczek, J., Schuler, D., Stoffler-Meilicke, M., Brimacombe, R., and Stoffler, G.(1988) EMBO J. 7, 3571-3576 [Abstract]
  45. Traut, R. R., Lambert, J. M., and Kenny, J. W.(1983) J. Biol. Chem. 258, 14592-14598 [Abstract/Free Full Text]
  46. Schrier, P. I., Maassen, J. A., and Moller, W.(1973) Biochem. Biophys. Res. Commun. 53, 90-98 [Medline] [Order article via Infotrieve]
  47. Kischa, K., Moller, W., and Stoffler, G.(1971) Nat. New Biol. 233, 62-63 [Medline] [Order article via Infotrieve]
  48. Hamel, E., Koka, M., and Nakamoto, T.(1972) J. Biol. Chem. 247, 805-814 [Abstract/Free Full Text]
  49. Skold, S.-E.(1982) Eur. J. Biochem. 127, 225-229 [Abstract]
  50. Uchiumi, T., Kikuchi, M, Terao, K., Iwasaki, K., and Ogata, K.(1986) Eur. J. Biochem. 156, 37-48 [Abstract]
  51. Montanaro, L. Sperti, S., and Stirpe, F.(1973) Biochem. J. 136, 677-683 [Medline] [Order article via Infotrieve]
  52. Fernandez-Puentes, C., Benson, S., Olsnes, S., and Pihl, A.(1976) Eur. J. Biochem. 64, 437-443 [Abstract]
  53. Holmberg. L., and Nygard, O.(1994) Biochemistry 33, 15159-15167 [Medline] [Order article via Infotrieve]
  54. Cawley, D. B., Hedblom, M. L., and Houston, L. L.(1979) Biochemistry 18, 2648-2654 [Medline] [Order article via Infotrieve]
  55. Montanaro, L., Sperti, S., Mattioli, A., Testoni, G., and Stirpe, F. (1975) Biochem. J. 146, 127-131 [Medline] [Order article via Infotrieve]
  56. Nolan, R. D., Grasmuk, H., and Drews, J.(1976) Eur. J. Biochem. 64, 69-75 [Abstract]
  57. Ippoliti, R., Lendaro, E., Bellelli, A., and Brunori, M.(1992) FEBS Lett. 298, 145-148 [CrossRef][Medline] [Order article via Infotrieve]

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