Quantitative Binding of Antibiotics to Ribosomes from a Yeast Mutant Altered on the Peptidyl-Transferase Center

Eur. J. Biochem. 54,483-492 (1975)

Quantitative Binding of Antibiotics to Ribosomes from a Yeast Mutant Altered on the Peptidyl-Transferase Center Antonio JIMENEZ and David VAZQUEZ

Instituto de Biologia Celular, Madrid

(Received November 16, 1974/February 17, 1975)

Quantitative binding studies of [G-3H]anisomycin and [~cetyZ-~~C]trichodermin to sensitive and resistant 80-S ribosomes from yeasts are described in this work. A single mutation, most probably affecting the ribosome peptidyl transferase centre, appears to have pleiotropic effects on the ribosome leading to resistance to trichodermin and anisomycin and to an increased sensitivity to sparsomycin. Resistance to trichodermin is due to a reduced affinity of ribosomes from the mutant for the antibiotic. Ribosomes from the sensitive strain (Y166) bind [a~etyl-~~C]trichodermin with a dissociation constant of 0.99 pM while those from the resistant one (TR,) bind [~cetyZ-'~C]trichodermin with a dissociation constant of 15.4 pM. Similar results are obtained when the binding of [acetyZ-14C]- trichodermin to Y166 and TR, 60-S subunits is studied.

The mutant TR, is also resistant to anisomycin. Although trichodermin and anisomycin bind to the ribosome at mutually exclusive sites, the higher affinity binding of [G-3H]anisomycin that is responsible for the inhibition of the peptidyl transferase center is practically identical for Y166 and TR, ribosomes. Therefore, the mutation in the ribosome leading to resistance to trichodermin and anisomycin decreases the affinity for trichodermin but not for anisomycin.

Trichodermin, trichothecin and fusarenon X inhibit the binding of [G-3H]anisomycin to TR, ribosomes to a lower extent than to Y166 ribosomes, suggesting that the resistance of TR, ribosomes to the effects of trichothecin and fusarenon X is caused by a decrease in the affinity of the ribosomes for these drugs, as was seen with trichodermin. On the other hand, verrucarin A inhibits [G-3H]- anisomycin binding to Y166 and TR, ribosomes to a similar extent and therefore its affinity for the ribosome does not appear to be affected by the mutation leading to resistance.

Trichothecin, trichodermin and fusarenon X appear to have a common binding site on the 60-S ribosomal subunits, which overlaps or is closely linked to the binding sites of anisomycin and verrucarin A.

Abbreviations. Y166 and TR,, wild type and mutant resistant to trichodermin respectively; K& (80 S) and Ki,, (80 S), dissociation constants of trichodermin binding to Y166 and TR, ribosomes respectively; K:,, (60 S) and K& (60 S), dissociation constants of trichodermin binding to Y166 and TR, 60-S ribosomal subunits respectively; 4, (80s) and Kd, (80 S) dissociation constants of higher affinity anisomycin binding to Y166 and TR, ribosomes respectively; K&+ (80 S) and K;,z,a (80 S) dissociation constants of lower affinity binding of anisomycin to Y166 and TR, ribosomes respectively; K& (60 S) and K&, (60 S), dissociation constant of anisomycin binding to Y166 and TR1 60-S ribosomal subunits respectively; K:,l,a (60 S + 40 S) and Ki,,,= (60 S + 40 S), dissociation constants of higher affinity binding of anisomycin to Y166 and TR, reconstituted ribosomes respectively; K&a (60 S + 40 S) and K& (60 S + 40 S) dissociation constants of lower affinity binding of anisomycin to Y166 and TR, reconstituted ribosomes respectively.

Bacterial resistance to antibiotics at the ribosomal level has been widely studied [I]. In most cases it has been found that mutant ribosomes have a reduced affinity for the drug to which they are resistant. Such mutations have been described for ribosomes resistant to streptomycin [2], erythromycin [3 - 51, chlor- amphenicol [4] and spectinomycin [6 ] . On the other hand an Escherichiu coZi erythromycin-resistant mutant has been described recently in which resistance to erythromycin occurs without decreasing the affinity of the ribosome for the antibiotic [5]. This erythromycin-resistant mutant has an altered protein of the 50-S subunit (protein L22) but the erythromycin-

Eur. J. Biochem. 54 (1975)

484 Antibiotic Binding to Yeast-Mutant Ribosomes

resistant ribosomes appear to bind the antibiotic as efficiently as those from the wild-type strain [5].

A number of mutants with altered ribosomes resistant to antibiotics have been isolated from eukaryotic cells [7- 101 but antibiotic binding studies were not reported. There are data, however, on quantitative binding studies of [G-3H]anisomycin and [acety/-14C]trichodermin binding to eukaryotic ribosomes [ll,12]. Since we have recently isolated a yeast mutant resistant to anisomycin and to the sesquiterpene antibiotics of the trichodermin group [13], we decided to carry out comparative quantitative binding studies of [G-3H]anisomycin and [a~etyl-'~C]- trichodermin to ribosomes of the wild sensitive strain and to the resistant mutant. The results obtained show that resistance to trichodermin in our mutant is due to a lower binding affinity of the ribosomes for the antibiotics, whereas resistance of the same mutant to anisomycin occurs without any significant change in the ribosomal affinity for the drug.

MATERIALS AND METHODS

Yeast Strains

Wild type Y166 and the spontaneous mutant TR, resistant to sesquiterpene antibiotics and to anisomycin were used and have been described elsewhere ~ 3 1 .

Subcellular Fractions and Assays

Wild-type and mutant polysomes, ribosomes and their subunits were prepared as previously described [ 131. Poly(U)-directed polyphenylalanine synthesis and endogenous mRNA-programmed polypeptide synthesis were studied as reported [13,14]. Peptidyl- puromycin formation was carried out basically following methods described elsewhere [15] but the ionic conditions were 50 mM Tris-HC1 buffer pH 7.4, 12.5 mM MgClz and 80 mM KC1 to avoid the non- enzymic translocation which takes place in eukaryotic polysomes at high K + concentration [16,17]. Binding of radioactive labelled antibiotics to yeast ribosomes was studied following the method of sedimentation in the ultracentrifuge [18], in 60- 100 p1 reaction mixtures containing 50 mM Tris-HC1 buffer pH 7.4, 12.5 mM MgCl,, 80 mM KC1, and 0.1 mM dithio- threitol. Sedimentation was carried out at 0 "C at 140000 x g in the Spinco 40 rotor for 6 h in the case of 60-S subunits and 2.5 h in the cases of 80-S ribosomes and reconstituted (60-S + 40-S) ribosomes. Data obtained from quantitative assays are presented as Scatchard plots [19]. The lines were drawn by the least-squares method with the aid of a computerized

program. The lines for the low-affinity type of [G-3H]- anisomycin binding were obtained by selecting the best coefficient of correlation. [a~ety/-'~C]Tricho- dermin and [G-3H]anisomycin were prepared following methods reported previously [12,20]. The sources of non-radioactive antibiotics used in this work were as indicated [12,21].

RESULTS Quantitative Binding of [G-3 HIAnisomycin and [a~etyl-'~C/Trichodermin to Y166 and TR, Ribosomes

It has been shown previously that the binding of [G-3H]anisomycin to yeast ribosomes depends on the ionic environment while the binding of [a~ety/-'~C]- trichodermin is hardly affected by changes of the ionic conditions [ll, 121. The ionic conditions used in our cell-free systems for polypeptide synthesis were identical to those used in the antibiotic binding assays in order to permit comparison of the results obtained with both systems. Fig. 1 presents the results of quantitative studies of [a~etyl-~~C]trichodermin to Y166

and TR, ribosomes. In agreement with a previous study [12], a single type of [a~etyl-'~C]trichodermin binding to yeast ribosomes was observed corresponding to one binding site of the antibiotic per ribosome. The dissociation constant for trichodermin binding to Y166 ribosomes, Kit (80 S) = 0.99 pM, is about half the value previously reported for ribosomes from a different yeast strain [12]. In the case of TR, resistant ribosomes another single type of binding appeared but with a much lower value of affinity for trichodermin, KAt (80 S) = 15.4 pM. This value is approximate, however, since the high ribosome and antibiotic concentrations required in these experi- ments could result in non-specific binding with a concomitant decrease in the value of the dissociation constant. Quantitative binding studies of [a~etyl-'~C]- trichodermin to 60-S subunits from Y166 and TR, ribosomes gave similar results to those obtained for the interaction with 80-S ribosomes (Fig. 2). The dissociation constant of [a~ety/-'~C]trichodermin for Y166 60-S subunit K;i, (60 S) = 0.97 pM (n = I), was 10 times lower than the corresponding one for TR, 60-S subunits, Kit (60 S) = 10 pM (n = 1). The above results show that resistance to trichodermin is caused by a lower affinity of the antibiotic for the mutant ribosomes.

The Scatchard plots corresponding to the binding of [G-3H]anisomycin to Y166 and TR, ribosomes (Fig. 3A and B) show the two types of interaction previously observed with yeast ribosomes [l l] . The higher affinity binding, (80 S) = 0.68 pM

Eur. .I. Biochem. 54 (1975)

48 5

1 . 1

A. Jimenez and D. Vazquez

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Fig. 1. Scatchard plots of [a~etyl-'~C]trichodermin binding to Y166 and TR, ribosomes. Data were obtained by the sedimentation method in the ultracentrifuge described under Materials and Methods. Final incubation volume was 100 pl and 3O-pl samples were taken prior to and after centrifugation for determination of radioactivity. Y166 ribosome concentration was 3.45 pM for [~cetyl-'~C]trichodermin concentrations ranging from 0.8 pM to 10 pM. TR1 ribosome concentration was 6.25 pM for [ ~ c e t y l - ' ~ C ] - trichodermin concentrations ranging from 2 pM to 35 pM. Y166

ribosomes ( O d ) . TRl ribosomes (0-0)

(n = 0.4) and Ki,l,a (80 S) = 0.80 pM (n = 0.39), was practically the same for both types of ribosomes. The lower affinity binding, K:,2,a (80 S) = 2.9 pM (n = 0.89) and K&a (80 S) = 3.8 pM (n = 0.8), was significantly different for sensitive and resistant ribosomes, but the low-affinity binding has been shown to be an artefact which results from the high-salt washing procedure and hence the values of the dissociation constant could differ from one batch of ribosomes to another [22]. However, only ribosomes that bind anisomycin with the higher affinity are active in peptide bond formation, the step of protein synthesis specifically inhibited by the antibiotic [ll]. Since the higher affinity binding of anisomycin is responsible for the inhibition of the peptidyl transferase center of ribosomes sensitive to the antibiotic and since the value for this type of binding to Y166 and TR1 ribosomes was similar in the resistant and

I

Fig. 2. Scatchard plots of [a~etyl-'~C]trichodermin binding to Y,,, and TR, 603 subunits. Data were obtained as described under Materials and Methods. Y166 60-S subunit concentrations were 2 and 3.5 pM for [a~etyl-'~C]trichoderrnin concentrations ranging from 0.8 to 4 pM and from 4 to 7 pM respectively. TR1 60-S subunit concentration was 6 pM for [a~etyl-'~C]trichoderrnin concentrations ranging from 0.8 to 10 pM. Y166 6 0 3 subunits (M). TR, 60-S subunits (0-0)

in the sensitive strains, it appears that the mutant ribosomes have been altered in such a manner that the peptidyl transferase is little affected by the anisomycin bound.

The results of binding studies of [G-3H]anisomycin to 60-S subunits from the sensitive and resistant strains are presented as Scatchard plots in Fig. 4A and 4B, respectively. A single type of interaction in both preparations of 60-S subunits was obtained. Anisomycin interacted with TRI 60-S subunits, (60 S) = 1.9 pM (n = 0.69), with an even higher affinity than with Y166 60-S subunits, K:,a (60s) = 3.0 pM (n = 0.98); the number of TR1 60-S subunits active in binding anisomycin was 69% while this value was 98 % in the case of Y166 60-S subunits. This difference might be due to an alteration of the mutant 60-S subunits, which appears to concord with the activity of reconstituted ribosomes in synthesis of polyphenylalanine.

Fig. 5A and 5B show Scatchard plots for the binding of [G-3H]anisomycin to 80-S ribosomes reconstituted from 60-S and 40-S subunits of Y166 ribosomes and TR1 ribosomes, respectively. The higher affinity binding, K:,l,a (60 S + 40 S) = 0.94pM (n = 0.56) and Ki,l,a (60 S + 40 S ) = 0.91 pM (n = 0.43), and the lower affinity binding, K:,2,a (60 S + 40 S) = 3.15 pM and K:,2,a (60 S + 40 S) = 5.75 pM (approximate values), are practically identical for both types of reconstituted ribosomes and are very similar to the results obtained with undissociated 80-S ribosomes.


486 Antibiotic Binding to Yeast-Mutant Ribosomes 0.6

0.5

0.4

- - 9 - I

c - 5 0.3 a ._ c 4

IP

- ..

0.2

0.1

C 0.2 0.4 0.6 0.8 1 .o f

Fig. 3. Scatchard plots of [G-3HJanisomycin binding to Y166 and TR1 ribosomes. Data were obtained from assays performed as described under Materials and Methods. Incubation volume was 100 pl and 30-p1 samples were taken before and after sedimentation

0.4.

- v Fig. 4. Scatchard plots for [G-'H]anisomycin binding to Y,,, and TR, 6 0 3 subunits. Data were obtained following the sedimentation method described in Materials and Methods. The subunit concentration was 2 pM for [G-3H]anisomycin concentrations ranging from 0.2 to 0.6 pM, 3.5 pM for [G-3H]anisomycin concentrations

B

3

0.2 0.4 0.6 0.8 1 .O - v

of ribosomes. The ribosome concentration was 3 pM for [G3H]- anisomycin concentrations ranging from 0.2 to 2.5 pM and 5.5 pM for a [G-3H]anisomycin range of 2.5 to 20 pM. (A) Ribosomes from Y,,, strain; (B) ribosomes from TR, strain

- V

ranging from 0.6 to 4 pM, and 6 pM for [G-3H]anisomycin concentrations ranging from 4 to 10 pM. The incubation volumes were 100,IOand 60 pl and 30,20 and 10-p1 samples were taken respectively for determination of radioactivity. (A) 60-S subunits from Y166 ribosomes; (B) 604 subunits from TR, ribosomes


A. Jimenez and D. Vazquez 487

A

0.2 0.4 0.6 0.8 1.0 - V

Fig. 5 . Scatchard plots of data for [G-3H]anisomycin binding to Y16, and TR1 reconstituted ribosomes. Data were obtained by the ultracentrifugation method described under Materials and Methods. Equimolar amounts of homologous 60-S and 404 subunits were preincubated at 30 "C for 10 min to reconstitute the 80-S ribosomes. The final incubation volumes and the samples taken prior to and after centrifugation were as described in the legend to Fig. 4.

From the results presented above and those previously reported we can conclude that (a) the mutation leading to resistance to anisomycin is expressed in the 60-S subunit [13], and (b) the resistance is due to a conformational and/or structural alteration in mutant ribosomes which does not alter the higher affinity binding of anisomycin to the ribosomes but which actually increases the affinity of the antibiotic for the 60-S subunits from the resistant ribosomes. Upon reconstitution of 80-S ribosomes from their subunits, the ribosomal heterogeneity is restored and the higher affinity type of [G-3H]anisomycin binding becomes identical in both types of ribosomes.

.The Effect of Several Antibiotics on Protein Synthesis

The results presented above suggested that the mutation leading to trichodermin resistance has pleio-

0.5

tropic effects on the peptidyl transferase center of the 60-S subunit. Therefore we extended our studies to a number of antibiotics which are known to act on eukaryotic cells at the ribosome level. Blasticidin S, gougerotin and sparsomycin block peptide bond formation by ribosomes from both eukaryotic and prokaryotic organisms [23]. On the other hand cycloheximide inhibits the translocation step [23]. Therefore we have studied the effects of these antibiotics on poly(U)-directed polyphenylalanine synthesis by Y166 and TR1 ribosomes. The results obtained show that blasticidin S, gougerotin and cycloheximide similarly inhibit polyphenylalanine synthesis by Y166 and TR1 ribosomes whereas sparsomycin is a stronger inhibitor of polyphenylalanine synthesis in ribosomes from TR, than from Y166 (Table 1). This striking effect of sparsomycin was confirmed in an endogenous mRNA-programmed polysomal system and in the poly(U)-directed cell-free system using a wider range



Table 1. Effect of several antibiotics on poly(U)-directed ['"C]- phenylalanine incorporation Reaction mixtures contained 50 mM Tris-HC1 buffer pH 7.4, 12.5 mM MgCI,, 80 mM KCl, 4 m M creatine phosphate, 2 pg creatine phosphokinase, 125 pg yeast tRNA, 1 mM ATP, 0.05 mM GTP, crude supernatant fraction containing EF-1 and EF-2 (about 2 mg/ml protein), 0.8 A,,, unit of high-salt washed ribosomes and 15 pg of poly(U) in 50 pl of solution. Incubation was carried out at 30 "C for 30 min, stopped by precipitation with 1 ml 10% trichloroacetic acid and the samples processed. 390 and 392 pmol of ['4C]phenylalanine were incorporated in the controls of Y,,, and TR, ribosomes, respectively

Antibiotic Concn [14C]Phenylalanine incorporation by ribosomes from

y166

PM % control

C ycloheximide 5 72 50 66

Blasticidin S 10 45 100 25

Sparsomycin 10 42 100 30

Gougerotin 100 72 1000 48

Anisomycin 2 36 20 11

61 64

44 25

25 13

72 48

75 22

of antibiotic concentrations (Fig. 6). Furthermore, we have shown that the alteration leading to a higher sensitivity to sparsomycin is located in the 60-S subunit of TR, ribosomes as might be expected (Table 2).

These observations indicate that the mutant ribosomes have an altered peptidyl transferase center that responds differentially to several antibiotics. Our finding showing that blasticidin S and gougerotin behave in a similar manner in both systems is com- patible with the proposal that both antibiotics act on the same area of the peptidyl transferase center of the 60-S subunit [23].

Effect of Antibiotics on Peptidyl-[3H]puromycin Formation

The results presented above suggest that TR, ribosomes have a conformational modification located at the peptidyl transferase center. This would imply that the modified response to several antibiotics in TR, ribosomes is expressed at the level of peptide bond formation. Therefore we studied the effect of inhibitors of peptide bond formation on peptidyl- [3H]puromycin synthesis. The effects of the inhibitors were basically similar to those obtained with either

g 20 ._ E a

0 1 0 . ~ 10-6 10-5 10-4

[~parsomycin] ( M j

Fig. 6. The effect of sparsomycin on endogenous mRNA-programmed polypeptide synthesis and on poly( U)-directed polyphenylalanine synthesis. In the case of poly(U)-directed ['"Clphenylalanine incorporation by Y166 (-0) and TR, (0-0) ribosomes, reaction mixtures were as indicated in Table 1, but the incubation was carried out for 10 min at 30 "C. In the absence of any antibiotic 180 and 191 pmol ['4C]phenylalanine were incorporated in the controls of Y166 and TR, ribosomes respectively. Similar reaction mixtures were prepared for endogenous mRNA-directed [14C]- phenylalanine incorporation but poly(U) and ribosomes were replaced by 0.6 A,,, unit of polysomes. Incubation was carried out for 7 min at 30 "C. In the absence of any antibiotic 8.8 and 7.0 pmol ['4C]phenyIalanine were incorporated in the controls of Y,,, and TR, polysomes respectively. ['4C]Phenylalanine incorporation by Y,,, (*--+) and TR, (0----0) polysomes

poly(U)-directed endogenous mRNA-directed polypeptide synthesis [13] (Tables 1 and 2). Cross resistance to anisomycin and sesquiterpene antibiotics and an enhancement of sensitivity to sparsomycin was observed in TR, ribosomes (Table 3). However, there is no clear cut difference between polysomes from the TR, mutant and the Y,66 strain in their sensitivity to anisomycin when peptidyl-puromycin formation was studied (Table 3). This is in agreement with results presented above showing the non-identity of the trichodermin and anisomycin binding sites at the ribosomal level. It is interesting to note that TR, polysomes were more active than those from the Y,66 strain, clearly suggesting an alteration on the peptidyl transferase center of the mutant ribosomes. A similar finding has been found for another trichodermin-resistant mutant [ 101 and also in an Escherichia coli mutant resistant to erythromycin [5].

Effect of Antibiotics on [G-3H]Anisomycin Binding to Ribosomes

The binding of [acetyl-'4C]trichodermin and [G-3H]anisomycin to yeast ribosomes is unevenly affected by a number of sesquiterpene antibiotics [ 11,121. Since anisomycin binds to ribosomes from both Y166 and TR, strains, the effects of sesquiterpene antibiotics in the binding reaction was studied to


A. Jimenez and D. Vazquez 489

Table 2. Effects of sparsomycin and anisomycin on reconstituted ribosomes Equimolar amounts of 60-S and 40-S subunits were combined to obtain 1.2 A,,, units of the reconstituted ribosomes per 50 pl. Reaction mixtures were otherwise as described in the legend to Table 1. Incubation at 30 "C was continued for 10 min. Values in brackets represent per- centage of incorporation compared to the controls without antibiotic

Source of ribosomal subunits [14C]Phenylalanine incorporated

60-S 40-S control + sparsomycin + anisomycin (20 pM)

y166 y166 141.62 (100) 118.30 (84) 60.33 (43) 31.95 (23) TRl TR1 99.68 (100) 62.32 (63) 25.73 (23) 63.02 (63) TRl y166 110.18 (100) 69.68 (63) 27.05 (25) 69.82 (63) y166 TRl 130.57 (100) 95.57 (73) 58.33 (45) 29.83 (23)

Table 3. Effects of several antibiotics on peptidyl-[3H]puromycin formation 50 p1 of the reaction mixture contained 50 mM Tris-HC1 buffer pH 7.4,12.5 mM MgC12, 80 mM KCl, 5 mM GTP, 2.4 A2,, units of polysomes, and 2 pl of crude supernatant fraction. After a 7-min preincubation, [3H]puromycin was added to a final concentration of 1 pM, and reaction was allowed to proceed for 1 min before adding 1 ml of 10 % trichloroacetic acid to stop the reaction. 1.79 and 2.40 pmol of peptidyl-[3H]puromycin were synthesized in samples of Y,,, and TR, polysomes respectively

~ ~~ _______ ~~

Polysomes Peptidyl-[3H]puromycin formation with antibiotic

trichodermin verrucarin A trichothecin fusarenon X anisomycin sparsomycin

20pM 200pM 20pM 200pM 20pM 200pM 20pM 200pM 0.7pM 2 p M 0.4pM 2 p M ~

% control

y166 16 4 103 80 20 6 45 8 64 44 65 18 TRl 76 44 99 80 90 55 76 40 75 59 41 6

characterize more precisely their site of interaction with the ribosome. Trichodermin, trichothecin, and fusarenon X inhibited the binding of [G-3H]anisomycin to TR1 ribosomes to a much lower extent than the binding to Y166 ribosomes (Fig. 7A, B and C). This result suggests that resistance to trichothecin and fusarenon X in TR, cells is due to a lower affinity of these antibiotics for the mutant ribosomes as it has been shown above for trichodermin. Furthermore, the results presented above suggest that trichodermin, trichothecin and fusarenon X bind at the same ribosomal site. On the other hand verrucarin A inhibits the binding of the radioactive antibiotic to ribosomes from the Y166 and TR1 strains to a similar extent (Fig. 7D). It appears, therefore, that the affinity of the ribosomes for verrucarin A is not affected by the mutation leading to resistance to trichodermin.

DISCUSSION

Resistance to antibiotics at the ribosome level can be explained in at least three different ways: (a) a

reduced binding affinity of the antibiotic for the resistant ribosome ; (b) a conformational alteration in the ribosome that does not change the antibiotic binding affinity but uncouples its action, and (c) a combination of both (a) and (b).

Trichodermin resistance in the yeast strain TR, is at least partly due to a reduced binding affinity for the mutant ribosomes. We have shown in the present work that the affinity for [~cetyl-'~C]trichodermin by resistant ribosomes, Ki,t (80 S) = 15.4 pM, is about 15 times lower than the affinity shown by sensitive ribosomes, K,& (80 S) = 0.99 pM. This result agrees with the observation of a smaller inhibitory effect of trichodermin on [G-3H]anisomycin binding to TR1 than to Y166 ribosomes; the concentration of trichodermin required to give 50% inhibition of [G-3H]- anisomycin binding to mutant ribosomes is 20 times higher than in the case of wild-type ribosomes (Fig. 7A). In addition, the affinity of [ucetyl-l4C]- trichodermin to Y166 60-S subunits is approximately 10 times higher than its affinity for TR1 60-S subunits.

Our quantitative binding studies of [G-3H]anisomycin to sensitive and resistant ribosomes clearly



100

80

60

40 - - e c c

20

c V ._

F o .? 100 F m

(3 - 8 0 ul U .- ._ m

60

40

20

0

I I I I I I I

10-6 1 0 . ~ 1 0 . ~ 1 0 . ~ 1 o - ~ [Antibiotic] (M)

Fig. 7. Inhibition of (G-3H]anisomycin binding to Y166 and TR, ribosomes by trichodermin, trichothecin, fusarenon X and verrucarin A . Data were obtained from sedimentation assays. [G-3H]Aniso- mycin and ribosome concentrations were 1 and 3 pM respectively. The final incubation volume was 100 pl. 30-p1 aliquots were taken in all cases for estimation of radioactivity. The percentages of

show that the higher affinity binding is practically identical for the two kinds of ribosomes. Moreover the isolated 60-S subunits from mutant ribosomes have a higher affinity for anisomycin, Ki,a (60 S) = 1.9 pM, than the subunits from sensitive ribosomes, K& (60 S) = 3.0 pM. This result could not been explained on the basis of an alteration of the mutant 60-S subunits due to the procedure of preparation since upon reconstitution of the 80-S ribosomes from their subunits the two types of binding affinities are observed to take place with similar values as those in normal 80-S ribosomes. A previous report has presented evidence strongly suggesting that this high affinity binding is responsible for the inhibitory action of anisomycin on peptide bond formation [ll]. The mutant ribosomes are resistant to the inhibition of anisomycin although they have a higher affinity binding similar to that of ribosomes from the sensitive

[G-3H]anisomycin bound to Y166 and TR, ribosomes were 41 and 44 % respectively. Antibiotics added were : (A) trichodermin; (B) trichothecin; (C) fusarenon X and (D) verrucarin A. In all cases binding of [G-3H]anisomycin to Y166 ( 0 4 ) and TR, (0-0) ribosomes was studied

strain. Therefore, the resistance cannot be explained on the basis of a reduced binding affinity of the antibiotic for the resistant ribosomes. It can be explained, however, on the basis of a conformational and/or structural change at the peptidyl transferase center of the 60-S subunits which allows the binding of the antibiotic to take place but lowers the degree of inhibition of peptide bond formation. A similar situation appears to occur in erythromycin-resistant mutants in which protein L22 is modified [5].

Our results on polypeptide synthesis have shown that the effects of sparsomycin is greater in ribosomes and polysomes from TR, than from Y166 (Fig. 6). This differential action of sparsomycin on the two types of ribosomes operates at the level of peptidyl transferase as shown by the results of the effect of the antibiotic on peptidyl-[3H]puromycin formation (Table 3). Sparsomycin, gougerotin and blasticidin S


A. Jimenez and D. Vazquez 49 1

appear to act on the peptidyl transferase centre in binding sites which appear to be common to both bacterial and eukaryotic ribosomes and are indepen- dent from the binding sites of antibiotics such as anisomycin and trichodermin which selectively block the peptidyl transferase centre of eukaryotic ribosomes (for review see [23]). Therefore our finding that similar levels of sensitivity to gougerotin and blasticidin S were observed in ribosomes from the wild Y166

strain and the trichodermin-resistant and anisomycin- resistant TR, strain is not unexpected. However, it is interesting to note that the mutation in TR, leading to an increase in the resistance to trichodermin and anisomycin induces an increase in sensitivity to sparsomycin. Thus, pleiotropy of mutation in the peptidyl transferase center of eukaryotic ribosomes is demonstrated in the present study by the differential effects of sparsomycin, trichodermin and anisomycin on Y166 and TR, ribosomes. Mutations in one part of the bacterial ribosome exerting pleiotropic effects on other ribosomal components also have been describ ed by others [24].

Trichodermin and trichothecin most probably bind to the same ribosomal site on the peptidyl transferase center. This is suggested by their common blocking effects on peptide bond formation [21,25] and polypeptide chain termination [25,26], the decrease in their binding affinity to resistant ribosomes from the TR, mutant (Fig. 1 and 7), and their competi- tive inhibition for binding to sensitive yeast ribosomes [12]. For the same reasons, fusarenonx appears to block the peptidyl transferase center at the same site as trichodermin and trichothecin [21,22]. Besides blocking peptide bond formation, however, furare- non X appears to inhibit the initiation of the polypeptide chain since it causes polysome breakdown [27,28].

Verrucarin A blocks peptide bond formation in the fragment reaction assay [21] but not in peptidyl- puromycin formation by polysomes [17,29] (Table 3). Furthermore, verrucarin A only partially inhibits binding of anisomycin and trichodermin to yeast ribosomes [11, 121 and the affinity of TR, ribosomes for verrucarin A does not appear to be affected by the mutation leading to trichodermin resistance (Fig. 7 D). All these results suggest that verrucarin A does not act on the ribosome precisely at the same site as trichodermin and trichothecin. Indeed verrucarin A has been shown to cause polysome breakdown and hence has been proposed as an inhibitor of initiation of the polypeptide chain [30].

Finally, it is important to note that interaction of an antibiotic with the peptidyl transferase center does not necessarily imply only inhibition of peptide bond formation. Since peptidyl transferase is implicat-

ed in the peptidyl-tRNA hydrolysis in the termination phase, all the peptidyl transferase inhibitors which has been tested in model assays block termination in bacterial and mammalian systems with the same specificity shown in peptide bond formation (for review see [15]). Furthermore, a number of antibiotics interacting with the peptidyl transferase center also appear to act as inhibitors of initiation because they interfere with the attachment of the 3’-terminal end of peptidyl-tRNA and Met-tRNA. Antibiotics of the lincomycin and streptogramin A groups and macrolides having the - mycaminose - mycarose moiety (carbomycin, spiramycins, leucomycins, nidda- mycin and tylosin) behave in this manner in bacterial systems (for review see [23]) and verrucarin A is probably the counterpart antibiotic in eukaryotic systems [17,29]. Antibiotics of these groups do not appear to interact with polysomes since they do not inhibit peptidyl-puromycin formation in polysomal systems. However fusarenon X appears to behave as verrucarin A in causing breakdown of polysomes, suggesting its inhibition on initiation. But unlike verrucarin A, it also inhibits peptide bond formation in polysomal systems (Table 3).

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Quantitative Binding of Antibiotics to Ribosomes from a Yeast Mutant Altered on the Peptidyl-Transferase Center