Binding of Cycloheximide to Ribosomes from Wild-Type and

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1980, p /80/ /05$02.00/0 Vol. 18, No. 6 Binding of Cycloheximide to Ribosomes from Wild-Type and Mutant Strains of Saccharomyces cerevisiae WALTER STOCKLEIN* AND WOLFGANG PIEPERSBERG Lehrstuhl fur Mikrobiologie der Universitat Munchen, Munich, West Germany Cycloheximide bound to cytoplasmic (80S) ribosomes of the yeast Saccharomyces cerevisiae with an association constant (Ka) of 2.0 (+ 0.5) x 107 M-1. The number of binding sites found per ribosome was between 0.4 and 0.6; it was reduced by high-salt treatment of ribosomes. 60S particles prepared in the presence of high salt had a lower affinity (Ka: 5.5 [± 0.5] x 106 M-1) than did 80S ribosomes, but a greater proportion of particles (0.8) were able to bind. No specific binding to 40S subunits was observed. The addition of supematant fractions (S100, high-salt wash fraction) increased the number of binding sites found per 80S ribosome up to 0.8, leaving the association constant unchanged. In contrast, the affinity of 60S subunits was enhanced to a Ka value of 3.5 x 10-7 M-1 by the addition of supernatant fractions, whereas the number of binding sites stayed constant. A model to explain these facts is proposed. 80S ribosomes, as well as 60S subunits of strain cy32, which is highly resistant to cycloheximide and altered in ribosomal protein L29 (18), showed a drastically reduced affinity for the drug (Ka values of 2.0 x 106 M-1). Cycloheximide is one of the most extensively studied antibiotics inhibiting translation exclusively on cytoplasmic (80S) ribosomes of eucaryotes (reviewed in 10, 20). Recent investigations indicate that all energy-dependent stages in the protein-synthesizing process are affected by this compound, though initiation seems to be the most sensitive step (5, 9). A large number of mutants resistant to cycloheximide are described from organisms belonging to completely different taxonomic groups of eucaryotes, including yeasts (3, 4, 7), other fungi (1, 6, 13), Tetrahymena (19), and Chinese hamster and human cell lines (11, 12). In nearly all cases investigated, the large (60S) ribosomal subunit was altered in resistant mutants. The only exception known is a mutant of Tetrahymena in which the 40S subunit seems to be affected (19). These results, together with the observation that, in yeasts, a factor necessary for cycloheximide susceptibility is easily washed off the ribosome by high-salt treatment (17), raise the question as to which component of the translational apparatus the drug binds and whether more than one binding site exists. By the use of tritium-labeled antibiotic (kindly provided by R. S. P. Hsi, The Upjohn Co.), we demonstrate here that in Saccharomyces cerevisiae ribosomal binding of cycloheximide (i) is a feature of the large (60S) ribosomal subunit, (ii) is changed in ribosomes from a cycloheximide-resistant mutant with an altered 863 ribosomal protein L29 (18), and (iii) is affected by high-salt treatment of ribosomes. MATERIALS AND METHODS Strains and growth conditions. The wild-type strain of S. cerevisiae used in this study was A364A (from L. H. Hartwell). Cy32 is a spontaneous cycloheximide-resistant isolate from A364A and is described elsewhere (18). Yeast strains were grown at 30 C in YPAD medium (15) supplemented with threonine (20,jg/ml), adenine (20,ug/ml), and uracil (20,tg/ml). Preparation of ribosomal and supernatant fractions. S100 fraction, as well as 80S, 60S, and 40S ribosomal particles were prepared as described in a preceding publication (18). Ribosome-free supematant (S100) and ribosomes (80S) separated from crude extracts in buffer 1 [100 mm tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (ph 7.4), 80 mm KCl, 12.5 mm magnesium acetate, 1 mm dithiothreitol] were designated low salt; ribosomes separated in buffer 2 (20 mm Tris-hydrochloride (ph 7.4), 0.1 M magnesium acetate, 0.5 M NH4Cl, 5 mm mercaptoethanol) and ribosomal subunits (separated by addition of 0.5 M KCl to buffer 1) were called high salt fractions. A high-salt ribosomal wash fraction was obtained by extracting low-salt ribosomes with buffer 2 for 10 mi at 30 C and by sedimenting them again by ultracentrifugation. All fractions were dialyzed against buffer la (buffer 1 with the concentration of Tris lowered to 20 mm) and were either used immediately or stored frozen at -700C. The patterns of proteins present in the ribosomal, as well as the supernatant, fractions were analyzed by two-dimensional gel electrophoresis as described earlier (18). Downloaded from on May 4, 2018 by guest

2 864 STOCKLEIN AND PIEPERSBERG Equilibrium dialysis. Equilibrium dialysis was carried out as described elsewhere (2) with all reactants dissolved in buffer la. Tritium-labeled cycloheximide was used in a concentration range between 0.22,uM and 1.79,uM for the determination of association constants (K.), and at 0.36 MM in those experiments where only one concentration of the drug was employed. Ribosomes and ribosomal subunits were adjusted to a concentration of about 0.4 MuM each, assuming 14 units' absorbancy at 260 nm to correspond to 1 mg of ribosomes and the molecular weights to be 3.6 x 106, 2.57 x 106, and 1.01 x 106 for 80S, 60S, and 40S ribosomal particles, respectively (18). Final concentrations of 8 and 0.31 to 0.42 mg of protein per ml of S100 and high-salt wash fractions, respectively, were added to ribosomes when indicated. Chemicals. [3H]cycloheximide was a generous gift from The Upjohn Co. (Kalamazoo, Mich.) and had a specific activity of 1.2 Ci/mmol. Emetine was from Serva (Heidelberg); trichodermin, verrucarin, anisomycin, and hygromycin B were gifts from Leo Pharmaceuticals (Ballerup), Sandoz (Basel), D. Vazquez (Madrid), and Eli Lilly & Co. (Indianapolis, Ind.), respectively. 4.0o A 80 s 3.0\ RESULTS Binding of cycloheximide to 80S ribosomal particles. In equilibrium dialysis experiments with tritium-labeled cycloheximide and a standard preparation (low salt) of 80S ribosomes from wild-type yeast strain A364A, a Ka value of 2.0 (± 0.5) x 107 M- (average from four independent experiments) was obtained. The number of binding sites found was below one in each case and varied considerably between dif- o2.0- ANTIMICROB. AGENTS CHEMOTHER. ferent ribosomal preparations. The Scatchard plot of a typical experiment is shown in Fig. 1A. Within the range of drug concentrations used (up to fourfold molar excess over ribosomes), no indication for additional binding sites was found, and the number of drug molecules bound per ribosome plotted versus drug concentration followed a saturation curve with a single plateau (data not shown). The affinity of low-salt 80S ribosomes from strain cy32 to cycloheximide was considerably decreased (Fig. 1A). The K. value obtained was 2.0 x 106 M-1. This result is consistent with the findings that the minimal inhibitory concentration for cycloheximide and the drug concentration necessary for 50% inhibition of in vitro protein synthesis are 100-fold higher than those for susceptible yeast strains (18). The Scatchard plot (not shown) for mutant 80S ribosomes extrapolated to 0.2 to 0.3 molecules of cycloheximide bound per particle. When 0.5 M NH4Cl was used to wash ribosomes, the fraction of wildtype 80S ribosomes able to bind the drug decreased considerably, but their affinity was unchanged (Fig. 1A). The same effect was demonstrated for mutant 80S ribosomes. Binding of cycloheximide to ribosomal subunits. Next, we studied whether the antibiotic binds to only one of the ribosomal subunits, or to both, or only to intact 80S particles. For this purpose 60S and 40S subunits were separated and purified by two successive gradient centrifugations in the presence of 0.5 M KCl as I 00 I3NC 1.0 ' FIG. 1. (A) Scatchard plots for cycloheximide binding to 80S ribosomes from strain A364A prepared in presence of high salt without (0) and with S100 added (0), and from strain cy32 (low salt) with S10() from wild type added (A). S100 protein concentration was 8 mg/ml. (B) Scatchard plots for cycloheximide binding to 60S subunits from strains A364A (0) and cy32 (A).

3 VOL. 18, 1980 a dissociating condition. The result is shown in Fig. 1B. The large (60S) ribosomal subparticle of wild-type strain A364A was able to bind the drug specifically, though with an association constant (K.: 5.5 x 106 M-') about fourfold lower than complete (80S) ribosomes. This correlates well with the finding that in those cycloheximide-resistant yeast strains analyzed so far, resistance is a property of the large ribosomal subunits (3, 7, 14, 18). Surprisingly, the number of drug molecules bound per particle (0.8; Fig. 1) was higher than with 80S ribosomes and did not vary for two independent subunit preparations. 60S subunits from the cycloheximide-resistant strain cy32 also bound the antibiotic (Fig. 1B). In contrast to the result with wild-type subunits, their association constant (Ka: 2.0 x 106 M-l) was in the same range as that of the respective 80S ribosomes. Neither wild-type nor mutant cy32 40S subunits exhibited any specific binding of cycloheximide within the concentration range employed. Effect of supernatant factors. The observation that 80S ribosomes bind cycloheximide with high affinity, but with low and variable efficiency, prompted the investigation of whether a factor is necessary for drug binding which can be washed off from the ribosome. To this end, cycloheximide binding to low- and high-salt ribosomes was studied by equilibrium TABLE 1. CYCLOHEXIMIDE BINDING TO RIBOSOMES 865 dialysis in the presence and absence of supernatant fractions. It was found that S100 or the high-salt wash ribosomal fraction from strain A364A, as well as from mutant cy32, significantly enhanced the binding capacity of wild-type 80S ribosomes. The results are summarized in Table 1. The highest ratio of binding sites obtained was 0.8 per ribosome (data not shown). In each case the corresponding association constants remained unchanged. S100 and high-salt wash factors did not bind the drug themselves. Similar results were obtained with wild-type S100 and 80S ribosomal preparations from mutant cy32 (Fig. 1A; Table 1). Also, supernatant fractions of mutant cy32 enhanced the capacity of wild-type, as well as mutant, 80S ribosomes for binding cycloheximide (Table 1). This indicates that the stimulating supernatant factor is not identical with the altered protein L29 in strain cy32 (18), because in that case one would expect only wild-type S100 to increase the affinity of mutant ribosomes. A totally different effect was seen upon addition of S100 to 60S ribosomal subunits from the wild type (Fig. 2). The association constant shifted to that of 80S particles and even higher, depending on the amount of supernatant fraction added. This indicates that the supernatant factor is needed in stoichiometric amounts and that its effect is not via any enzymatic action. In Effect ofpreparation conditions and addition of supernatant fraction on binding efficiency of 80S ribosomes Espt Source of ribosomes Supematant fraction addedb Molar ratio antibiotic Expt (Prepn condition') (source) bound/ribosomesc I A364A (1s) _d A364A (is) S100 (A364A) A364A (ls) hsw (A364A) A364A (hs) A364A (hs) S100 (A364A) A364A (hs) hsw (A364A) II A364A (hs) A364A (hs) S100 (cy32) A364A (hs) hsw (cy32) III cy32 (is) cy32 (is) S100 (A364A) cy32 (hs) cy32 (hs) S100 (A364A) cy32 (hs) hsw (A364A) cy32 (hs) S100 (cy32) cy32 (hs) hsw (cy32) a s, Low-salt preparation; hs, high-salt preparation (see text). b S100 and hsw (high-salt wash) are the ribosome-free supernatants of the low- and high-salt ribosome preparation, respectively. c Molar ratio antibiotic/ribosomes input was 0.58 for A364A and 1.20 for cy32 ribosomes. d _, None.

4 866 STOCKLEIN AND PIEPERSBERG 3-0~ FIG. 2. Effect ofthe addition ofwild-type high-salt wash and S100 on cycloheximide binding by wildtype 60S subunits. 60S alone (0); with high-salt wash (0.12 mg ofprotein per ml) (0); with high-salt wash (0.6 mg of protein per ml) (A); with S100 (8 mg of protein per ml) (A). contrast to the results obtained with 80S ribosomes, the Scatchard plots for 60S subunits extrapolated to the same number of binding sites (0.8), irrespective of whether supernatant was present or not. From this we conclude that the high-affinity binding sites of 80S particles are completely retained in 60S subunits and that their affinity is totally restored upon the addition of supematant. The possibility that this effect is brought about by the presence of subunits in the S100 (not precipitated during ultracentrifugation) was excluded by separating the proteins from this fraction on two-dimensional gels. No ribosomal proteins could be detected. In contrast, the low-affinity binding site on 60S subunits of strain cy32 was unaffected by t-he addition of wild-type S100 fraction (data not shown), which could mean either that the supematant factor no longer binds to the mutant 60S subparticles or that it is no longer able to enhance its affinity due to the presence of the altered protein L29. Effect of other antibiotics on cycloheximide binding. The influence of other antibiotics on cytoplasmic ribosomes of eucaryotes was studied with trichodermin, verrucarin, anisomycin, emetine, and hygromycin B. None of these, when used in fivefold molar excess over cycloheximide, was able to compete with cycloheximide binding (Table 2). DISCUSSION Although most cycloheximide-resistant mutants of eucaryotic organisms have been shown to possess an altered 60S ribosomal subunit, nothing has been known about the binding affinity of the subunits, the number of ribosomal sites for cycloheximide, or the specificity of binding. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Effect of other antibiotics on cycloheximide binding by 80S ribosomes from strain A364A Antibiotic' cpm Bound (%) -b.. 2,448 (100) Hygromycin B... 2,451 (100) Emetine ,494 (102) Anisomycin... 2,509 (102) Trichodermnin... 2,498 (102) Verrucarin... 2,435 (99) Cycloheximide (unlabeled) (26) All antibiotics indicated were added in fivefold molar excess over [3H]cycloheximide. The concentration of [3H]cycloheximide was 0.36 gm, and each value is the mean from two determinations. b, None. The results presented here show that ribosomes from S. cerevisiae have one high-affinity binding site for this drug on the 60S subunit. This binding site is specific for cycloheximide and shows no overlap with those for other 80S inhibitors. It was also found that the 80S ribosomes lose a significant proportion of this binding capacity during preparation and that this deficiency could be partially restored by the addition of (a) soluble protein factor(s) either from the S100 or the ribosomal wash fraction. The affinity of binding, however, was the same, irrespective of whether 80S ribosomes had been treated with high salt or not. In the case of 60S subunits, addition of supernatant factors did not affect the binding capacity but rather increased the affinity for cycloheximide. The following conclusions can be drawn from these results: (i) 40S ribosomal subunits abolish the accessibility for the antibiotic in the 80S complex in the absence of a certain factor, i.e., they exhibit a "masking" effect; (ii) a factor only loosely bound to ribosomes and easily washed off during preparation is necessary for the high affrinity of 60S subunits for cycloheximide, as well as for the availability of the binding site in 80S ribosomes. This factor antagonizes the masking effect of 40S particles. Thus, 80S ribosomes either bind the drug or do not, whereas 60S subunits bind it with either high or low affinity, in the presence or absence of the factor, respectively. In view of this interpretation, it is also evident why certain mutations in the 40S subunits of Tetrahymena ribosomes (19), which lead to cycloheximide resistance, may occur in addition to those located in the 60S particles. We predict that this type of 40S alteration either enhances the masking effect of the small ribosomal subunit on binding of the antibiotic to 60S subunits or prevents interaction between the 80S ribosome and the factor necessary for high affinity.

5 VOL. 18, 1980 The effect of that factor on the binding affinity of 60S subunits for cycloheximide should be dependent on the presence of stoichoimetric amounts. This could be clearly demonstrated. Further, the increase of the association constant of 60S subunits parallels the increase of the fraction of drug-binding 80S ribosomes upon addition of increasing amounts of ribosomal wash preparation, favoring the assumption that the same factor is responsible for both effects. 80S ribosomes from a cycloheximide-resistant strain, cy32, show a 10-fold lower affinity for the antibiotic than do wild-type ribosomes; this is in good agreement with the previously described resistance phenotype (18). Mutant 80S ribosomes show the same response upon addition of ribosomal wash fractions from wild-type and from cy32 cells; mutant 60S subunits, however, are completely unaffected. Two explanations are possible: (i) the alteration of L29 directly or indirectly influences both the affinity for the drug and for the factor in 60S subunits, whereas the masking effect remains unchanged; (ii) the factor binds with similar affinity to mutant and to wild-type 60S particles, but has no influence on the drug-binding affinity. The postulated factor could be identical to factor "P" described by Somasundaran and Skogerson (17), which is necessary for sensitivity of in vitro protein synthesis to cycloheximide and which also could be washed off ribosomes in the presence of high salt (0.5 M NH4Cl) (16). Its identity with ribosomal protein L29 is ruled out by the fact that addition of wild-type supernatant increases the number of binding sites on 80S ribosomes of strain cy32 without enhancing the affinity of binding. ACKNOWLEDGMENT We are very much indebted to R. S. P. Hsi for his generous gift of radioactive cycloheximide and to the colleagues supplying us with samples of the antibiotics used. We thank A. Bock for reading the manuscript and for comments. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to W.P. LITERATURE CITED 1. Begueret, J., M. Perrot, and M. Crouzet Ribosomal proteins in the fungus Podospora anserina: evidence for an electrophoretically altered 60S protein in a cycloheximide resistant mutant. Mol. Gen. Genet. 156: Bock, A., A. Petzet, and W. Piepersberg Ribosomal ambiguity (ram) mutations facilitate dihydrostreptomycin binding to ribosomes. FEBS Lett. 104: Coddington, A., and R. Fluri Characterization of the ribosomal proteins from Schizosaccharomyces CYCLOHEXIMIDE BINDING TO RIBOSOMES 867 pombe by two-dimensional polyacrylamide gel electrophoresis. Mol. Gen. Genet. 158: Cooper, D., D. Banthorpe, and D. Wilkie Modified ribosomes conferring resistance to cycloheximide in mutants of Saccharomyces cerevisiae. J. Mol. Biol. 26: Cooper, T. G., and J. Bossinger Selective inhibition of protein synthesis initiation in Saccharomyces cerevisiae by low concentrations of cycloheximide. J. Biol. Chem. 251: Haugli, F. B., W. F. Dove, and A. Jimenez Genetics and biochemistry of cycloheximide resistance in Physarum polycephalum. Mol. Gen. Genet. 118: Jimenez, A., B. Littlewood, and J. Davies Inhibition of protein synthesis in yeast p In E. Munoz, F. Garcia-Ferrandiz, and D. Vazquez (ed.), Molecular mechanisms of antibiotic action on protein synthesis and membranes. Elsevier/North Holland Publishing Co., New York. 8. Mazelis, A. G., and M. L. Petermann Physicalchemical properties of stable yeast ribosomes and ribosomal subunits. Biochim. Biophys. Acta 312: Oleinick, N. L Initiation and elongation of protein synthesis in growing cells: differential inhibition by cycloheximide and emetine. Arch. Biochem. Biophys. 182: Pestka, S Inhibitors of protein synthesis. p In H. Weissbach and S. Pestka (ed.), Molecular mechanisms of protein biosynthesis. Publisher Inc., New York. 11. Poche, H., J. Junghahn, E. Geissler, and H. Bielka Cycloheximide resistance in Chinese hamster cells III. Characterization of cell-free protein synthesis by polysomes. Mol. Gen. Genet. 138: Poche, H., S. Zakrzewski, and K. H. Nierhaus Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes. Mol. Gen. Genet. 175: Pongratz, M., and W. KHingmiller Role of ribosomes in cycloheximide resistance of Neurospora mutants. Mol. Gen. Genet. 124: Rao, S. S., and A. P. Groilman Cycloheximide resistance in yeast. A property of the 60S ribosomal subunit. Biochem. Biophys. Res. Commun. 29: Sherman, F., G. R. Fink, and C. W. Lawrence Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 16. Skogerson, L., and E. Wakatama A ribosomedependent GTPase from yeast distinct from elongation factor 2. Proc. Natl. Acad. Sci. U.S.A. 73: Somasundaran, U., and L. Skogerson A cycloheximide sensitivity factor from yeast required for N- acetylphenylalanyl-puromycin formation. Biochemistry 15: Stdcklein, W., and W. Piepersberg Altered ribosomal protein L29 in a cycloheximide-resistant strain of Saccharomyces cerevisiae. Curr. Genetics 1: Sutton, C. A., M. Ares, and R. L. Hallberg Cycloheximide resistance can be mediated through either ribosomal subunit. Proc. Natl. Acad. Sci. U.S.A. 75: Vazquez, D Inhibitors of protein biosynthesis. In A. Kleinzeller, G. F. Springer, and H. G. Wittmann (ed.), Molecular biology, biochemistry, and biophysics, vol. 30. Springer Verlag, Berlin.

6 ERRATA Binding of Cycloheximide to Ribosomes from Wild-Type and Mutant Strains of Saccharomyces cerevisiae WALTER STOCKLEIN AND WOLFGANG PIEPERSBERG Lehrstuhl fir Mikrobiologie der Universitait Munchen, Munich, West Germany Vol. 18, no. 6, p. 863, Abstract, line 10: "3.5 x 10i M"' should read "3.5 x 107 M-1.'' P. 864, column 1, line 13: Reference "(18)" should be "(8)." Bactericidal Activity of Cerumen TUU-JYI CHAI AND TOBY C. CHAI Laboratory of Biochemistry and Metabolism, National Institute ofarthritis, Metabolism and Digestive Diseases, Bethesda, Maryland Vol. 18, no. 4, p. 640, column 1: Lines 3 and 4, which read "test cultures was dependent upon the concentrations of cerumen used. Perhaps a concentration," should follow line 34. Susceptibility of Eikenella corrodens to Newer Beta-Lactam Antibiotics ELLIE J. C. GOLDSTEIN, MYLES E. GOMBERT, AND EMMANUEL 0. AGYARE Infectious Diseases Division, SUNY-Downstate Medical Center, Brooklyn, New York Vol. 18, no. 5, p. 832, column 2, line 25: Add "It is known that bacampicillin is converted to an active agent, ampicillin, in the presence of blood. Our use of Mueller-Hinton agar supplemented with 5% sheep blood may have caused this conversion in our test system." P. 833, Table 1, column 3, line 6: Value range for moxalactam, presently " ," should read " " Tentative Interpretive Criteria for the Diffusion Susceptibility Test Using 30-,ug Netilmicin Disks RONALD N. JONES, ARTHUR L. BARRY, AND CLYDE THORNSBERRY Department ofpathology, Kaiser Foundation Laboratory (Oregon Region), Clackamas, Oregon 97015; Clinical Microbiology Laboratory, University of California Davis Medical Center, Sacramento, California 95817; and Antimicrobics Investigations Section, Center for Disease Control, Atlanta, Georgia Vol. 18, no. 3, p. 489, Table 2, footnote b: "(P < 0.5, > 0.02)" should read ("P < 0.05, > 0.02)." 208