Eur. J. Biochem. 91, 441 -448 (1978)
Antibiotic Susceptibility of the Peptidyl Transferase Locus of Bovine Mitochondrial Ribosomes Nancy D. DENSLOW and Thomas W. O'BRIEN Department of Biochemistry and Molecular Biology, University of Florida (Received July 6 , 1978)
We have used a modified 'fragment reaction' to compare the susceptibility of isolated bovine mitochondrial ribosomes, bacterial (Escherichia coli) ribosomes and eukaryotic (bovine microsomal) ribosomes to several antibiotics. All of these ribosomes share certain structural features of their peptidyl transferase center, as indicated by their interaction with substrates, puromycin and gougerotin. Bovine mitochondrial ribosomes have other structural features in common with bacterial, but not cytoplasmic ribosomes, as revealed by their susceptibility to chloramphenicol and the streptogramin antibiotics. While mitochondrial ribosomes are susceptible to all inhibitors of bacterial ribosomes tested, their low susceptibility to the lincosamines and macrolides suggests that some component(s) of the binding sites for these antibiotics is altered. The 55-S ribosomes from mammalian mitochondria resemble bacterial and eukaryotic cytoplasmic ribosomes in their fundamental properties. They consist of two subunits containing RNA and proteins, and they function according to the same overall mechanism, using initiator tRNA, aminoacyl-tRNAs and soluble initiation and elongation factors to translate an mRNA molecule. In terms of their fine structure and physicochemical properties, however, they differ unexpectedly from both these kinds of ribosomes, as well as from other kinds of mitochondrial ribosomes 11-31. It might be expected that these differences would be reflected in some aspects of ribosome structure and function, such as antibiotic susceptibility. We undertook these experiments to explore this possibility and, also, to obtain information on the interaction of specific antibiotics with bovine mitochondrial ribosomes, as a model system for studies on mammalian mitochondrial ribosomes. This work focuses on the peptidyl transferase activity of the large subribosomal particle, using several antibiotics as molecular probes of the peptidyl transferase center of mammalian mitochondrial ribosomes. The results of these experiments provide a background for continuing studies on the structure and function of mammalian mitochondrial ribosomes, especially on the role of individual ribosomal components in defined functions. Abbreviations. A site, aminoacyl-tRNA binding site; P site, peptidyl-tRNA binding site.
MATERIALS AND METHODS Preparation of Mitochondrial and Cytoplasmic Ribosomes
Mitochondrial and cytoplasmic ribosomes were prepared from partially purified bovine liver mitochondria as described previously 14- 61. To obtain mitochondrial ribosomes free from cytoplasmic ribosomes, some preparations of mitochondria were treated with 0.5 % digitonin between the third and fourth washes [7,8]. Mitochondria were resuspended in buffer A, 10 mM Tris . HC1 pH 7.6,lO mM MgC12, 0.1 M KC1, 5 mM 2-mercaptoethanol and 0.1 mM EDTA and were lysed by the addition of Triton X-100 to 2%. The crude ribosomes were recovered by centrifugation and incubated for 5 min at 37 "C in buffer B, 20 mM MgClz, 100 mM KC1, 20 mM triethanolamine . HCl pH 7.5, 5 mM 2-mercaptoethanol, containing 1 mM puromycin to discharge nascent polypeptides 151. This solution was clarified by centrifugation and the ribosomes were separated by centrifugation in 10- 30 % sucrose density gradients in buffer B [6]. Fractions containing 55-S or 80-S monoribosomes and the mitochondrial small and large subribosomal particles were pooled separately and concentrated by centrifugation. The ribosomes were used immediately in peptidyl transferase assays. On occasion, mitochondrial 39-S subunits were further washed by resuspension in the high salt buffer C, 0.5 M KC1, 5 mM MgC12, 20 mM triethanolamine . HCl pH 7.5 and 5 mM 2-mercaptoethanol and freed from released
442
proteins by sucrose density gradient centrifugation in buffer C.
Preparation of E. coli Ribosomes E. coli ribosomes were prepared from E. coli K-12, strain 1200 Sup by the procedure of Nirenberg 191 with the following modifications : the cells were ruptured by sonication in a Bronwill Biosonik sonicator and no DNase was added. Crude ribosomes were washed by resuspension and centrifugation in the high salt buffer D, 10 mM MgClZ, 1 M NH4C1, 6 niM 2-mercaptoethanol and 10 mM Tris . HCl pH 7.5. Ribosomes were resuspended in buffer E, 10 mM Mg(OAc)z, 0.2 M KCl, 25 mM Tris pH 7.5, and stored frozen at - 70 "C.
Peptidyl Transferase Assay Ac13H]Leu-tRNA was prepared [lo, 111 from E. coli B tRNA. The peptidyl transferase activity was measured at 25 "C by a modified 'fragment reaction' 112,131. The reaction mixture contained: 33 mM Tris . HCl pH 7.5,267 mM KC1, 13.3 mM Mg(OAc)z, 0.66 mM puromycin, 15 - 25 pmol of ribosomes, 9.3 nM AcljHILeu-tRNA (6000- 10000 counts/min) and 33% (v/v) ethanol in a volume of 0.15 ml 141.
Antibiotic Susceptibility of Bovine Mitochondrial Ribosomes Table 1. Peptidyl transferase activity of ribosomes Ribosomes (15-25 pmol) were incubated for 10 min with puromycin and A c [ ~ H ] L ~ u - ~ R N described A ~ s inMaterials and Methods. Activity of 28-S mitochondria1 subribosomal particles is the average value of two experiments; other values are averages of 6-21 experiments Ribosome
counts. min-' . pmol-' Mitochondrial 55-S Mitochondrial 39-S Mitochondrial 28-S E . coli 70-S E. coli 50-S Cytoplasmic 80-S
3
0
RESULTS Bovine mitochondrial 55-S ribosomes and 39-S subribosomal particles show high activities in the transpeptidation reaction. In fact, mitochondrial ribosomes are about as active (80 - 90 %) as E. coli ribosomes in the peptidyl transferase assay (Table 1). This is in marked contrast to the low activity of cytoplasmic ribosomes in this system (Table l), as was noted earlier 141. The kinetics of the peptidyl transferase reaction of 55-S ribosomes are compared to those of E. coli 70-S ribosomes in the Lineweaver-Burk plots in Fig. 1. Under these reaction conditions, we calculate
143 123 1 159 I48 7
I
Materials Puromycin dihydrochloride was obtained from Nutritional Biochemical Corp. and chloramphenicol was purchased from Sigma Chemical Co. LincomycinHCl and celesticetin were obtained from Dr George B. Whitfield, Upjohn & Co. PA1 14A and carbomycin were donated by Dr Nathan Belcher, Pfizer Co., tylosin tartrate was a gift from Dr Robert Hosley, Lilly Research Labs, and vernamycin A was obtained from Ms Barbara Stearns, Squibb Co. E. coli B de-aminoacylated tRNA was purchased from General Biochemicals and 14.5-3H2]leucine, 55 Ci/mmol, from Schwarz-Mann.
A~[~H]Leu-puromycin formed
m
400 ~ / [ ~ u r o m y c i n ](FM-~)
J
600
Fig. 1. Kinetic analysis of the peptidyl transferase activity of mitochondrial and E, coli monoribosomes using the Lineweaver-Burk plot. Ribosomes were assayed at a concentration of 0.12 pM, and the concentration of N-AC-[~H]L~U-~RNA was 2.6 nM. The points are averages of duplicate values. ( 0 ) Mitochondrial 55-S ribosomes and (A)E. coli 70-S ribosomes
an apparent Km, puromycin of 170 pM, for mitochondrial ribosomes, and 63 pM for E. coli ribosomes. These values fall within the range of those reported for bacterial ribosomes under a variety of conditions 114- 171. The antibiotic gougerotin acts on both bacterial and eukaryotic cytoplasmic ribosomes 1181. Not surprisingly, we find that gougerotin is also inhibitory to the peptidyl transferase activity of 55-S mitochondrial ribosomes (Table 2). Mitochondrial and cytoplasmic ribosomes have essentially the same susceptibility to this antibiotic, requiring somewhat higher concentrations than E. coli ribosomes to achieve comparable levels of inhibition. However, the differences in the response of the three ribosome types is not pronounced, and it is clear that the binding site for gougerotin is conserved in these diverse ribosome types.
N. D. Denslow and T. W. O’Brien
443 150
1
I
I
I
10-5
0-4
10-3
10-2
I
D
I U ~
10-7
10-5
10-4
10-3
10-7
1 ~ 6
Antibiotic concentration ( M )
Fig. 2. Effect of several antibiotics on the peptidyl transferase activity of mitochondrial 55-S (e), mitochondrial 39-S ( 0 ) E. coli 70-S (A), E. coli 50-S [A) and microsomal 80-S (m) ribosomes. Antibiotics are: (A) chloramphenicol, (B) PA114A, (C) vernamycin A, (D) lincomycin, (E) celesticetin, (F) carbomycin and (G) tylosin. The values are averages of 2-4 experiments Table 2. Fffect of gougerotin on the peptidyl transjerase activity of bovine mitochondrial (5.54) and microsomal (SO-S) ribosomes and E. coli [ 7 0 3 ) ribosomes Assays performed as described in legend of Table 1 ; values are averages of two experiments Gougerotin concn
Ribosome -
E. coli 703 mM
% control
0.01 0.1 1.0
90 60 40
mitochondrial cytoplasmic 55-s 80-S __ -
-
96 60
113 60
Some antibiotic inhibitors of protein synthesis do discriminate between ribosomes of the prokaryotic and eukaryotic classes. In this work we have concentrated on the susceptibility of 55-S ribosomes to a series of inhibitors of prokaryotic specificity that selectively inhibit the peptidyl transferase activity of bacterial ribosomes. Bovine mitochondrial ribosomes
appear to be nearly as sensitive as E. coli ribosomes to antibiotics like chloramphenicol and the streptogramins (Fig. 2A-C and Table 3). In this respect, they resemble typical prokaryotic ribosomes. It appears that the binding site for these antibiotics is highly conserved on the large subunit of bovine mitochondrial ribosomes. The streptogramins bind with high affinity to mitochondrial ribosomes as well as bacterial ribosomes. The 50 % inhibitory levels of the streptogramins that we determined for isolated bovine mitochondria1 ribosomes (Table 3), are close to the K d (0.3 pM) calculated for rat liver mitochondrial ribosomes, using vernamycin A to inhibit protein synthesis within intact mitochondria. These results are in strong contrast to the findings with cytoplasmic ribosomes from the same cells (Fig. 2A-C). Compared to E. coli ribosomes, bovine mitochondrial ribosomes show a diminished susceptibility to the lincosamines, lincomycin and celesticetin (Fig. 2 D, E), and the macrolides, carbomycin and tylosin (Fig. 2F, G). Relatively high concentrations (1 10 mM) of these antibiotics are required to reach 50 % inhibitory levels for mitochondrial ribosomes (Table 3).
Antibiotic Susceptibility of Bovine Mitochondrial Ribosomes
444 Table 3. Antibiotic susceptibility of E. coli and bovine mitochondrial ribosomes Assay conditions are described in legend of Table 1. Values represent concentrations of antibiotics required to inhibit peptidyl transferase activity by 50 % Antibiotic
E. coli ribosomes
Mitochondria1 ribosomes
703
55-s
393
0.6 0.9 100 1000 2000 700 10000
0.3 0.7 80 200
PM Vernamycin A PA1 14A Chloramphenicol Lincomycin Celesticetin Carbomycin Tylosin
0.1 0.2 40 10 200 1.0 10
A
~-
-
500 -
- 6 . P 4
2
While mitochondrial ribosomes have about the same susceptibility to these lincosamines and macrolides, E. coli ribosomes show a differential response. For example, both celesticetin and lincomycin appear to interact poorly with mitochondrial ribosomes, but lincomycin is a more effective inhibitor than celesticetin for E. coli ribosomes in this system (Fig. 3 D, E). This effect is even more apparent in the case of the macrolides, where the response of bacterial and mitochondrial ribosomes differs by three orders of magnitude (Fig. 2F, G). The resistance of isolated mitochondrial ribosomes to the lincosamines and macrolides may be an intrinsic property of these ribosomes. Alternatively, it may be possible to account for this observation on another basis. For example, the binding site for these antibiotics may be partially obscured or otherwise affected by exogenous proteins that are bound or adsorbed to the ribosome 1201. To test this possibility, assays were also performed using mitochondrial ribosomes that had been washed in a high salt buffer (buffer C) to remove loosely adsorbed proteins. Since the derived subunits obtained by this washing procedure have the same sensitivity to these antibiotics as do the native 39-S subunits of mitochondrial ribosomes (Fig. 2), we conclude that the diminished susceptibility to these inhibitors is an intrinsic property of bovine mitochondrial ribosomes. It can be noted that the mitochondrial ribosomal subunits are somewhat more susceptible (than 55-S ribosomes) to certain antibiotics, especially lincomycin (Fig. 2D). These modest effects are not apparent with bacterial ribosomes. They may reflect partial occlusion of the antibiotic binding site by the small subunit of mitochondrial ribosomes or, perhaps, different conformational states of the large subunits, whether as a free subunit or in a monoribosome couple.
lo
tl
6
nl "0
0.6 0.2 0.4 Inhibitor concentration (mM)
1.0
Fig. 3. Analysis of the peptidyl transferase activity of ribosomes in Henderson [26] plots. The data in Fig.2 are plotted using the equation u o / v i = m[I], 1 where: 00 is the velocity of the reaction in the absence of inhibitor, u i is the velocity in the presence of inhibitor, m, the slope, = Z ( N i / & ) / D , an expression containing terms that relate to the mechanism of the reaction and to the interaction of the inhibitor with the enzyme, Ki,and [I], is the concentration of the inhibitor. Bovine mitochondrial 55-S (0)and 3 9 3 (0),E. coli 7 0 3 (A) and 5 0 3 (A), and bovine cytoplasmic 80-S (I ribosomes ) were tested with (A) chloramphenicol, (B) lincomycin and (C) carbomycin
+
Because of the diminished susceptibility of mitochondrial ribosomes to these lincosamines and macrolides, it is possible to identify concentrations of these antibiotics which are clearly inhibitory to bacterial ribosomes, but which are without effect on mitochondrial or cytoplasmic ribosomes. However, since mitochondrial ribosomes are susceptible to these antibiotics, higher concentrations will discriminate the three types of ribosomes from each other. For example, the peptidyl transferase activity of E. coli ribosomes is reduced to 50% or less by a 10 pM concentration of lincomycin or carbomycin, levels which are not
445
N. D. Denslow and T. W. O'Brien
Table 4. lnhibiror constants (Ki) and apparent dissociation constants (Ki) f o r the interaction of chloraniphenicol, lincomycin nnd carbomycin with mitochondrial and E. coli ribosomes 55-S Ribosomes calculated from
Antibiotic
70-S Ribosomes calculated from
-~
-
~~
Scatchard plot
Henderson plot ~
Ki ~
~~
~~
Scatchard plot
Ki ~~
Henderson plot
~-
.~
Ki
Ki
~
~
Ki
Ki
~~
-~
~
~
PM ~~
100 390 -
Chloramphenicol Lincomycm Carbomycm ~
~
20.4" 80" - 390
21 ~
500
~
~
~
29 5.1 -
2 5" 0.49"- 5.7b
29
-
1.9'
-
~
~
+
For competitive inhibition: K ; = KA/[I ([S]/K,,,)],where Ki is the inhibitor constant, KA is the apparent dissociation constant, obtained frpm the slope of the Scatchard plot, [S] is the puromycin concentration and K,,, is the Michaelis constant for puromycin. For non-competitive inhibition: Ki = KA. For non-competitive inhibition: Ki = l / m in the Henderson plot, Fig. 3. a
inhibitory to mitochondrial or cytoplasmic ribosomes (Fig. 2D, F). On the other hand, discriminatory levels of these antibiotics (1 mM, for example) which inhibit mitochondrial ribosomes by 50 %, are without effect on cytoplasmic ribosomes, while completely suppressing the activity of E. coli ribosomes (Fig. 2D, Fj. This level of lincomycin susceptibility observed for isolated bovine mitochondrial ribosomes appears to be characteristic for mitochondrial ribosomes of mammals [4,21,22] and insects (231. In fact, the KA (250 pM) calculated for lincomycin from studies on the inhibition of protein synthesis in intact rat liver mitochondria 1221 is in the range of that found for isolated bovine mitochondrial ribosomes (Table 4). On the other hand, the susceptibility observed for mammalian mitochondrial ribosomes to carbomycin depends on the system used. Mitochondria1 ribosomes isolated from bovine liver (Fig.2), rat liver 1241 and even from Neurospora [24], appear to have the same susceptibility to carbomycin. These results reflect intrinsic properties of the isolated mitochondrial ribosomes and not secondary effects, as are observed in studies with intact mitochondria [22] which appear able to concentrate carbomycin 1241. It should be noted that ribosomes isolated from yeast mitochondria are an exception to this pattern, in that they appear as susceptible to lincomycin and carbomycin as bacterial ribosomes 1251.
Linear Transformation of the Inhibition Data To better compare the response of the different kinds of ribosomes to representative antibiotics from each group, the inhibition data is analyzed in Fig. 3 using a linear transformation (Henderson plot) of the rate equation for inhibited reactions 1261. One effect of this transformation is to amplify the apparent differences between bacterial and mitochondrial ribosomes, especially when their susceptibilities differ by
more than a factor of 10. As a consequence, the mitochondrial ribosomes tend to resemble cytoplasmic ribosomes moreso than prokaryotic ribosomes in their response to lincomycin (Fig. 3 Bj and especially to carbomycin (Fig. 3C). A major advantage of this transformation is that the slope of the linear plot obtained is proportional to the inhibitor constant, Ki. Since the relationship between Ki and the slope of such plots depends upon the mechanism of inhibition, inhibitor constants can be calculated for those antibiotics whose mechanism of action is known. For competitive inhibitors of puromycin in the peptidyl transferase assay,
K i = - (1 .
m
Km
IS]
+Km
)
'
where m is the slope, K , is the Michaelis constant for puromycin and [S] is the puromycin concentration [26]. Since chloramphenicol is a competitive inhibitor of the puromycin reaction for bacterial ribosomes 1271, we can evaluate the chloramphenicol inhibitor constant Ki from the above relationship. This value, calculated for E. coli ribosomes in our system as 2.9 pM, compares well with the published values (in the range of 2.2 pM) for the chloramphenicol dissociation constant at 0°C [17,28]. It is reasonable to assume that chIoramphenico1 acts as a competitive inhibitor for mitochondrial ribosomes as well. In this case, the competitive inhibition constant calculated for mitochondrial 55-S ribosomes is 27 pM. This value, about 10-fold higher than that for E. coli ribosomes in this system, reflects the difference noted earlier in the inhibitor-response profiles in Fig. 2A. Carbomycin and lincomycin inhibit the binding of substrates to both the aminoacyl-tRNA binding (A) site and the peptidyi-tRNA binding (P) site [29,30] of E. coli ribosomes, complicating the calculation of inhibitor constants from Henderson plots 1261. However, the relative affinity of mitochondrial and bac-
446
Antibiotic Susceptibility of Bovine Mitochondria1 Ribosomes I
I
7
I
I
1
A
1.6 -
6
i
E
. 2.
4
2
0
40
t
t -0
B
Q2
06
0.4
0.8
1.0
I
Fig. 4. Analysis of the inhibition of the peptidyl transferase activity of mitochondrial and E. coli monoribosomes by lincomycin, using a (n/KJ. The symbols modified Scatchard plor: i/(I/ = - (i/KJ used are: i, the fraction of inhibition of the peptidyl transferase reaction effected by a concentration of inhibitor [I]; n, the number of binding sites per ribosome affecting the measured activity and K & the apparent dissociation constant for the inhibitor. Ki is equal to Ki [(K,,, [S])/K,,,] for competitive type inhibitors and to K i for non-competitive type inhibitors. The intercept on the x-axis is a direct measure of n, the number of binding sites. ( 0 ) 55-S rnitochondrial ribosomes, and (A)7 0 6 E. coli ribosomes
+
+
terial ribosomes for these antibiotics can be ascertained directly from the slopes of the Henderson plots (Fig. 3B, C). Thus it appears that E. coli ribosomes have a 50-fold higher affinity for lincomycin, and a 250-fold higher affinity for carbomycin, than do bovine mitochondrial ribosomes. Assuming that carbomycin acts mainly as a noncompetitive inhibitor in this reaction [27], we calculate inhibitor constants for carbomycin of 1.9 pM for E. coli ribosomes, and 500 pM for mitochondrial ribosomes. The lincomycin inhibition data (Fig. 2D) can be plotted in a Scatchard format (Fig.4) to provide an estimate of the apparent dissociation constant K& as well as the number of ribosomal binding sites at which lincomycin inhibits the peptidyl transferase reaction. The affinity of lincomycin for mitochondrial ribosomes is about 65-fold lower than for E.coli ribosomes (Table 4). Nevertheless, we find 0.7 lincomycin binding site/mitochondrial ribosome, a value close to that found for E. coli ribosomes in this system, 0.8 site/ ribosome (Fig. 4). Analysis of the chloramphenicol inhibition data (Fig.2A) in a Scatchard plot shows that mitochondrial ribosomes, like bacterial ribosomes, contain one
Fig. 5. Analysis of the inhibition of the peptidyl transferase activity of mitochondria1 and E. coli monoribosomes by chloramphenicol, using a modified Scatchard plot, as in Fig. 4
binding site at which chlorarnphenicol inhibits peptidyl transferase activity (Fig. 5). Apparent dissociation constants for chloramphenicol calculated from these Scatchard plots show that E. coli ribosomes have a slightly higher affinity for chloramphenicol than mitochondrial ribosomes (Table 4). The inhibitor constants calculated from these apparent dissociation constants agree well with those calculated for mitochondrial and bacterial ribosomes from the Henderson plots (Table 4) and also with the literature values available for E. coli [31].
DISCUSSION We have used the peptidyl transferase assay to examine the susceptibility of bovine mitochondrial ribosomes to several antibiotics. With the exception of gougerotin, which inhibits all types of ribosomes, the antibiotics used in this study are selective inhibitors of bacterial ribosomes. We find that all of these antibiotics are inhibitory to isolated bovine mitochondrial ribosomes and, by this criterion alone, it would appear that these mitochondrial ribosomes are of the prokaryotic type. Ribosomes from E. coli and bovine mitochondria are equally susceptible to chloramphenicol and the streptogramins, PA-1 14A and vernamycin A (Table 3). However, bovine mitochondrial ribosomes are much less sensitive than E. coli ribosomes to the lincosamine and macrolide antibiotics tested, requiring 100- 1000-fold higher levels of these antibiotics to achieve comparable inhibition.
N.D. Denslow and T. W. O’Brien
It is noteworthy that bovine mitochondrial ribosomes are sensitive to all of the antibacterial antibiotics used, especially in view of the fact that cytoplasmic ribosomes from the same cells are refractory to these inhibitors. This observation shows that binding sites for these antibiotics are conserved, albeit to different degrees, in the mitochondrial ribosomes, but not in the cytoplasmic ribosomes. This observation is of special interest, if ribosomal proteins comprise antibiotic binding sites 132,331, since the proteins which confer this ‘prokaryotic’ aspect to mitochondrial ribosomes are themselves, like cytoplasmic ribosomal proteins, encoded in the nuclear genome and synthesized on cytoplasmic ribosomes. Despite their common biosynthetic origin the mitochondrial ribosomal proteins are nevertheless very different from those of cytoplasmic ribosomes, on the basis of their 2-dimensional electrophoretic properties [34,35] (and T. W. O’Brien, unpublished observation). More significantly, the mitochondrial ribosomal proteins also differ extensively from those of bacterial ribosomes [34] (T. W. O’Brien, unpublished observation), suggesting that only selected sequences are conserved in the mitochondrial ribosomal proteins. From studies on the interaction of antibiotics with ribosomes, it appears that several functional groups on the ribosome may contribute to the actual binding site for a given antibiotic 1361. In the case of E. coli ribosomes, at least three separate groups on the ribosome appear to be involved in binding chloramphenicol 136,371. Operationally, these functional groups are located within the A site of the peptidyl transferase center, where chloramphenicol blocks the entry of the 3’-terminal fragment of aminoacyl-tRNA [30]. However, the exact nature of the actual binding site for chloramphenicol is unknown. The various groups comprising this binding site may be contributed by one 1381 or more proteins 1391 and perhaps even by a region of the ribosomal RNA. The fact that mitochondrial and bacterial ribosomes bind chloramphenicol with comparable affinities (Table 4), suggests that the chloramphenicol-binding site on mitochondrial ribosomes contains equivalent functional groups, in the same spatial orientation, as those on bacterial ribosomes. A similar situation appears to exist for the binding sites of PA-114A and vernamycin A, since both kinds of ribosomes are sensitive to very low concentrations (0.1 - 1 pM) of these antibiotics. In E. coli, the ribosomal binding site of the streptogramins overlaps that of chloramphenicol ; the streptogramins can inhibit the binding of the 3’ terminus of aminoacyl-tRNA, as well as that of chloramphenicol [40,41]. Thus, the same functional groups that interact with chloramphenicol in the A site, conserved in the mitochondrial ribosome, may also be involved in binding the streptogramins. In E. coli ribosomes, the binding
441
domain for streptogramins extends into the P site of the peptidyl transferase center [40], where additional groups may be involved in binding these antibiotics. These extra interactions may account for the higher affinity of bacterial ribosomes for the streptogramins, compared to chloramphenicol [42] (Fig. 2). Since mitochondrial ribosomes have an equally high affinity for the streptogramins (Fig. Z), the additional groups involved are probably conserved in mitochondrial ribosomes. We interpret the diminished susceptibility of mitochondrial ribosomes (relative to E. coli ribosomes) to the lincosamines (Fig. 2D, E) and the macrolides (Fig. 2F, G), as resulting from the absence or improper positioning, on mitochondrial ribosomes, of one or more of the equivalent functional groups that are involved in binding these antibiotics to bacterial ribosomes. The binding domain for these antibiotics extends into both the A and the P site regions of the peptidyl transferase center of bacterial ribosomes [29,30]. The binding sites for these antibiotics also overlap that for chloramphenicol [41] suggesting the involvement of common functional groups at the A site. Since these functional groups appear to be conserved at the A site of mitochondrial ribosomes (above), the diminished susceptibility of mitochondrial ribosomes observed for the lincosamines and macrolides suggest that the additional groups required for stabilizing the. binding of these antibiotics to the P site are missing, or improperly oriented, on mitochondrial ribosomes. Thus, the disposition of functional groups within the A site of mitochondrial ribosomes appears to be more conserved, relative to bacterial ribosomes, than that of analogous groups in the P site. It is expected that essential structural features of the peptidyl transferase locus would be shared by prokaryotic and eukaryotic ribosomes alike, since both kinds of ribosomes interact with the common substrates, aminoacyl-tRNA and peptidyl-tRNA. This intrinsic property of the peptidyl transferase center is apparently responsible for the binding of antibiotics like puromycin and the 4-aminohexose pyrimidine nucleosides, including gougerotin [18]. In this sense, these antibiotics probably bind to some of the same functional groups in the A site [18] that are required for the binding and manipulation of the natural substrates by either kind of ribosome. We find that bovine mitochondrial ribosomes are just as susceptible to gougerotin as are bacterial and eukaryotic cytoplasmic ribosomes (Table 2). This result suggests that the components of the gougerotin binding site that are conserved in these three structurally dissimilar ribosomes, are absolutely essential for this central mechanism of protein synthesis. In addition to this universal ribosomal domain, there exists another set of functional groups at the
448
N. D. Denslow and T. W. O’Brien: Antibiotic Susceptibility of Bovine Mitochondria] Ribosomes
ribosomal A site, common to the prokaryotic type of ribosomes. These groups, responsible for the susceptibility of mitochondrial ribosomes to the antibacterial antibiotics (above), delineate a structural domain which is fundamentally different in ribosomes of the prokaryotic and eukaryotic types. In this respect, it is most interesting that mitochondrial ribosomes have not lost their susceptibility to chloramphenicol, as have eukaryotic ribosomes, since mitochondrial ribosomes not only comprise a most heterogeneous group [I], but they also appear to be evolving more rapidly than other ribosomes [43- 461. This work was supported by grant GM-15438 from the United States Public Health Service, National Institutes of General Medical Sciences. We are grateful for the technical assistance of Warren Clark and Mark Critoph.
REFERENCES 1. O’Brien, T. W. (1977) in International Cell Biology (Brinkley, B. R. & Porter, K. R., eds) pp. 245-255, Rockefeller University Press, New York. 2. OBrien, T. W. & Matthews, D. E. (1976) in Handbook qf Genetics (King, R. C., ed.) vol. 5, pp. 535-580, Plenum Publishing Co., New York. 3. O’Brien, T. W. (1976) in Protein Synthesis (McConkey, E., ed.) vol. 2, pp. 249-307, Marcel Dekker, New York. 4. Denslow, N. D. & O’Brien, T. W. (1974) Biochem. Biophys. Res. Commun. 57, 9-16. 5. Hamilton, M. G. & O’Brien, T. W. (1974) Biochemistry, 13, 5400 - 5403. 6. O’Brien, T. W. (1971) J . Bid. Chenz. 246, 3409-3417. 7. Schnaitman, C., Erwin, V. G. & Greenawalt, J. W. (1967) J . Cell Biol. 32, 719-735. 8. Loewenstein, J., Scholte, H. R. & Wit-Peeters, E. M. (1970) Biochim. Biophys. Acta, 223, 432-436. 9. Nirenberg, M. W. (1963) Methods Enzymol. 6, 17-23. 10. Nishizuka, Y., Lipman, F. & Lucas-Lenard, J. (1968) Methods Enzymol. 12B, 708 - 721. 11. Haenni, A. L. & Chapeville, F. (1966) Biochim. Biophys. Acta, 114, 135-148. 12. de Vries, H., Agsteribbe, E. & Kroon, A. M. (1971) Biochim. Biophys. Acta, 246, 11 1 - 122. 13. Miskin, R., Zamir, A. & Elson, D. (1970) J. Mol. Biol. 54, 355- 378. 14. Pestka, S. (1972) Proc. Natl Acad. Sci. U.S.A. 65, 624-628. 15. Fahncstock, S., Neumann, H., Shashoua, V. & Rich, A. (1970) Biochemistry, 5, 2477 - 2483. 16. Fico, R. & Coutsogeorgopoulos, C. (1972) Biochem. Biophys. Res. Commun. 47,645-651. 17. Fernandez-Muiioz, R. & Vazquez, D. (1973) Mol. Biol. Rep. I , 75-79.
18. Barbacid, M. & Vazquez, D . (1974) Eur. J . Biochem. 44, 445-453. 19. Dixon, H., Kellerman, G. M., Mitchell, C. H., Towers, N. H. & Linnane, A. W. (1971) Biochem. Biophys. Res. Commun. 43, 780 - 786. 20. Spirin, A. S. & Asatryan, L. S. (1976) FEBSLett. 70, 101- 104. 21. Kroon, A. M. & de Vries, H. (1971) in Autonomy and Biogenesis of Mitochondria and Chloroplasts (Boardman, N. K., Linnane, A. W. & Smillie, R. M., eds) pp. 318 - 327, American Elsevier, New York. 22. Towers, N. R., Kellerman, G . M. & Linnane, A. W. (1973) Arch. Biochem. Biophys. 155, 159- 166. 23. Williams, K. L. & Birt, L. M. (1976) FEBS Lett. 66, 120- 123. 24. de Vries, H., Arendzen, A. J. & Kroon, A. M. (1973) Biochim. Biophys. Acta, 331, 264-275. 25. Grivell, L. A., Netter, P., Borst, P. & Slonimski, P. P. (1973) Biochim. Biophys. Acta, 312, 358 - 367. 26. Henderson, P. J. F. (1972) Biochern. J. 127, 321-333. 27. Pestka, S. (1970) Arch. Biochem. Biophys. 136, 80-88. 28. Pestka,S. (1975) in Antibiotics (Corcoran, J. W. & Hahn,F.E., eds) vol. 3, pp. 370- 395, Springer-Verlag, New York. 29. Celma, M. L., Monro, R. E. & Vazquez, D. (1970) FEBS Lett. 6, 273 -277. 30. Celma, M. L., Monro, R. E. & Vazquez, D. (1971) FEBS Lett. 13, 247-251. 31. Fernandez-Muiioz, R., Monro, R. E., Torres-Pinedo, R. & Vazquez, D. (1971) Eur. J. Biochem. 23, 185-193. 32. Pellegrini, M. & Cantor, C. R. (1977) in Molecular Mechanisms of’ Protein Synthesis (Weissbach, H. & Pestka, S., eds) pp. 468 - 555, Academic Press, New Y ork. 33. Vazquez, D., Barbacid, M. & Fernandez-Mufioz, R. (1975) in Topics in Infectious Diseases (Drews, J. & Hahn, F. E., eds) vol. 1, pp. 193-216, Springer-Verlag, New York. 34. Van den Bogert, C. & de Vries, H. (1976) Biochim. Biophys. Acta, 442, 227-238. 35. Leister, D. E. & Dawid, I. B. (1974) J . Biol. Chem. 245, 510851 18. 36. Hahn, F. E. & Gund, P. (1975) in Topics in Infectious Diseases (Drews, J. & Hahn, F. E., eds) vol. 1, pp. 245 - 266, SpringerVerlag, New York. 37. Cheney, B. V. (1974) J . Med. Chem. 17, 590-599. 38. Pongs, O., Bald, R. & Erdmann, V. A. (1973) Proc. Natl Acad. Sci. U.S.A. 70, 2229-2233. 39. Sonenberg, N., Wilchek, M. & Zamir, A. (1973) Proc. Natl Acad. Sci. U.S.A. 70, 1423-1426. 40. Ennis, H. L. & Duffy, K . E. (1972) Biochim. Biophys. Acra, 281,93- 102. 41. Vazquez, D. (1966) Biochim. Biophys. Acta, 114, 277-288. 42. Contreras, A. & Vazquez, D. (1977) Eur. J . Biochem. 74, 539 - 547. 43. Matthews, D. E., Hessler, R. A. & O’Brien, T. W. (1 978) FEBS Lett. 86, 76-80. 44. Groot, G. S. P., Flavell, R. A. & Sanders, J. P. M. (1975) Biochim. Biophys. Acta, 378, 186- 194. 45. Jakovcic, S., Casey, J. & Rabinowitz, M. (1975) Biochemistry, 14, 2043 - 2050. 46. Leister, D. E. & Dawid, I. B. (1975)J. Mol. Biol. Y6, 119-123.
N. D. Denslow and T. W. OBrien, Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box J-245, J. Hillis Miller Health Center, Gainesville, Florida, U.S.A. 32610
Antibiotic Susceptibility of the Peptidyl Transferase Locus of Bovine Mitochondrial Ribosomes Nancy D. DENSLOW and Thomas W. O'BRIEN Department of Biochemistry and Molecular Biology, University of Florida (Received July 6 , 1978)
We have used a modified 'fragment reaction' to compare the susceptibility of isolated bovine mitochondrial ribosomes, bacterial (Escherichia coli) ribosomes and eukaryotic (bovine microsomal) ribosomes to several antibiotics. All of these ribosomes share certain structural features of their peptidyl transferase center, as indicated by their interaction with substrates, puromycin and gougerotin. Bovine mitochondrial ribosomes have other structural features in common with bacterial, but not cytoplasmic ribosomes, as revealed by their susceptibility to chloramphenicol and the streptogramin antibiotics. While mitochondrial ribosomes are susceptible to all inhibitors of bacterial ribosomes tested, their low susceptibility to the lincosamines and macrolides suggests that some component(s) of the binding sites for these antibiotics is altered. The 55-S ribosomes from mammalian mitochondria resemble bacterial and eukaryotic cytoplasmic ribosomes in their fundamental properties. They consist of two subunits containing RNA and proteins, and they function according to the same overall mechanism, using initiator tRNA, aminoacyl-tRNAs and soluble initiation and elongation factors to translate an mRNA molecule. In terms of their fine structure and physicochemical properties, however, they differ unexpectedly from both these kinds of ribosomes, as well as from other kinds of mitochondrial ribosomes 11-31. It might be expected that these differences would be reflected in some aspects of ribosome structure and function, such as antibiotic susceptibility. We undertook these experiments to explore this possibility and, also, to obtain information on the interaction of specific antibiotics with bovine mitochondrial ribosomes, as a model system for studies on mammalian mitochondrial ribosomes. This work focuses on the peptidyl transferase activity of the large subribosomal particle, using several antibiotics as molecular probes of the peptidyl transferase center of mammalian mitochondrial ribosomes. The results of these experiments provide a background for continuing studies on the structure and function of mammalian mitochondrial ribosomes, especially on the role of individual ribosomal components in defined functions. Abbreviations. A site, aminoacyl-tRNA binding site; P site, peptidyl-tRNA binding site.
MATERIALS AND METHODS Preparation of Mitochondrial and Cytoplasmic Ribosomes
Mitochondrial and cytoplasmic ribosomes were prepared from partially purified bovine liver mitochondria as described previously 14- 61. To obtain mitochondrial ribosomes free from cytoplasmic ribosomes, some preparations of mitochondria were treated with 0.5 % digitonin between the third and fourth washes [7,8]. Mitochondria were resuspended in buffer A, 10 mM Tris . HC1 pH 7.6,lO mM MgC12, 0.1 M KC1, 5 mM 2-mercaptoethanol and 0.1 mM EDTA and were lysed by the addition of Triton X-100 to 2%. The crude ribosomes were recovered by centrifugation and incubated for 5 min at 37 "C in buffer B, 20 mM MgClz, 100 mM KC1, 20 mM triethanolamine . HCl pH 7.5, 5 mM 2-mercaptoethanol, containing 1 mM puromycin to discharge nascent polypeptides 151. This solution was clarified by centrifugation and the ribosomes were separated by centrifugation in 10- 30 % sucrose density gradients in buffer B [6]. Fractions containing 55-S or 80-S monoribosomes and the mitochondrial small and large subribosomal particles were pooled separately and concentrated by centrifugation. The ribosomes were used immediately in peptidyl transferase assays. On occasion, mitochondrial 39-S subunits were further washed by resuspension in the high salt buffer C, 0.5 M KC1, 5 mM MgC12, 20 mM triethanolamine . HCl pH 7.5 and 5 mM 2-mercaptoethanol and freed from released
442
proteins by sucrose density gradient centrifugation in buffer C.
Preparation of E. coli Ribosomes E. coli ribosomes were prepared from E. coli K-12, strain 1200 Sup by the procedure of Nirenberg 191 with the following modifications : the cells were ruptured by sonication in a Bronwill Biosonik sonicator and no DNase was added. Crude ribosomes were washed by resuspension and centrifugation in the high salt buffer D, 10 mM MgClZ, 1 M NH4C1, 6 niM 2-mercaptoethanol and 10 mM Tris . HCl pH 7.5. Ribosomes were resuspended in buffer E, 10 mM Mg(OAc)z, 0.2 M KCl, 25 mM Tris pH 7.5, and stored frozen at - 70 "C.
Peptidyl Transferase Assay Ac13H]Leu-tRNA was prepared [lo, 111 from E. coli B tRNA. The peptidyl transferase activity was measured at 25 "C by a modified 'fragment reaction' 112,131. The reaction mixture contained: 33 mM Tris . HCl pH 7.5,267 mM KC1, 13.3 mM Mg(OAc)z, 0.66 mM puromycin, 15 - 25 pmol of ribosomes, 9.3 nM AcljHILeu-tRNA (6000- 10000 counts/min) and 33% (v/v) ethanol in a volume of 0.15 ml 141.
Antibiotic Susceptibility of Bovine Mitochondrial Ribosomes Table 1. Peptidyl transferase activity of ribosomes Ribosomes (15-25 pmol) were incubated for 10 min with puromycin and A c [ ~ H ] L ~ u - ~ R N described A ~ s inMaterials and Methods. Activity of 28-S mitochondria1 subribosomal particles is the average value of two experiments; other values are averages of 6-21 experiments Ribosome
counts. min-' . pmol-' Mitochondrial 55-S Mitochondrial 39-S Mitochondrial 28-S E . coli 70-S E. coli 50-S Cytoplasmic 80-S
3
0
RESULTS Bovine mitochondrial 55-S ribosomes and 39-S subribosomal particles show high activities in the transpeptidation reaction. In fact, mitochondrial ribosomes are about as active (80 - 90 %) as E. coli ribosomes in the peptidyl transferase assay (Table 1). This is in marked contrast to the low activity of cytoplasmic ribosomes in this system (Table l), as was noted earlier 141. The kinetics of the peptidyl transferase reaction of 55-S ribosomes are compared to those of E. coli 70-S ribosomes in the Lineweaver-Burk plots in Fig. 1. Under these reaction conditions, we calculate
143 123 1 159 I48 7
I
Materials Puromycin dihydrochloride was obtained from Nutritional Biochemical Corp. and chloramphenicol was purchased from Sigma Chemical Co. LincomycinHCl and celesticetin were obtained from Dr George B. Whitfield, Upjohn & Co. PA1 14A and carbomycin were donated by Dr Nathan Belcher, Pfizer Co., tylosin tartrate was a gift from Dr Robert Hosley, Lilly Research Labs, and vernamycin A was obtained from Ms Barbara Stearns, Squibb Co. E. coli B de-aminoacylated tRNA was purchased from General Biochemicals and 14.5-3H2]leucine, 55 Ci/mmol, from Schwarz-Mann.
A~[~H]Leu-puromycin formed
m
400 ~ / [ ~ u r o m y c i n ](FM-~)
J
600
Fig. 1. Kinetic analysis of the peptidyl transferase activity of mitochondrial and E, coli monoribosomes using the Lineweaver-Burk plot. Ribosomes were assayed at a concentration of 0.12 pM, and the concentration of N-AC-[~H]L~U-~RNA was 2.6 nM. The points are averages of duplicate values. ( 0 ) Mitochondrial 55-S ribosomes and (A)E. coli 70-S ribosomes
an apparent Km, puromycin of 170 pM, for mitochondrial ribosomes, and 63 pM for E. coli ribosomes. These values fall within the range of those reported for bacterial ribosomes under a variety of conditions 114- 171. The antibiotic gougerotin acts on both bacterial and eukaryotic cytoplasmic ribosomes 1181. Not surprisingly, we find that gougerotin is also inhibitory to the peptidyl transferase activity of 55-S mitochondrial ribosomes (Table 2). Mitochondrial and cytoplasmic ribosomes have essentially the same susceptibility to this antibiotic, requiring somewhat higher concentrations than E. coli ribosomes to achieve comparable levels of inhibition. However, the differences in the response of the three ribosome types is not pronounced, and it is clear that the binding site for gougerotin is conserved in these diverse ribosome types.
N. D. Denslow and T. W. O’Brien
443 150
1
I
I
I
10-5
0-4
10-3
10-2
I
D
I U ~
10-7
10-5
10-4
10-3
10-7
1 ~ 6
Antibiotic concentration ( M )
Fig. 2. Effect of several antibiotics on the peptidyl transferase activity of mitochondrial 55-S (e), mitochondrial 39-S ( 0 ) E. coli 70-S (A), E. coli 50-S [A) and microsomal 80-S (m) ribosomes. Antibiotics are: (A) chloramphenicol, (B) PA114A, (C) vernamycin A, (D) lincomycin, (E) celesticetin, (F) carbomycin and (G) tylosin. The values are averages of 2-4 experiments Table 2. Fffect of gougerotin on the peptidyl transjerase activity of bovine mitochondrial (5.54) and microsomal (SO-S) ribosomes and E. coli [ 7 0 3 ) ribosomes Assays performed as described in legend of Table 1 ; values are averages of two experiments Gougerotin concn
Ribosome -
E. coli 703 mM
% control
0.01 0.1 1.0
90 60 40
mitochondrial cytoplasmic 55-s 80-S __ -
-
96 60
113 60
Some antibiotic inhibitors of protein synthesis do discriminate between ribosomes of the prokaryotic and eukaryotic classes. In this work we have concentrated on the susceptibility of 55-S ribosomes to a series of inhibitors of prokaryotic specificity that selectively inhibit the peptidyl transferase activity of bacterial ribosomes. Bovine mitochondrial ribosomes
appear to be nearly as sensitive as E. coli ribosomes to antibiotics like chloramphenicol and the streptogramins (Fig. 2A-C and Table 3). In this respect, they resemble typical prokaryotic ribosomes. It appears that the binding site for these antibiotics is highly conserved on the large subunit of bovine mitochondrial ribosomes. The streptogramins bind with high affinity to mitochondrial ribosomes as well as bacterial ribosomes. The 50 % inhibitory levels of the streptogramins that we determined for isolated bovine mitochondria1 ribosomes (Table 3), are close to the K d (0.3 pM) calculated for rat liver mitochondrial ribosomes, using vernamycin A to inhibit protein synthesis within intact mitochondria. These results are in strong contrast to the findings with cytoplasmic ribosomes from the same cells (Fig. 2A-C). Compared to E. coli ribosomes, bovine mitochondrial ribosomes show a diminished susceptibility to the lincosamines, lincomycin and celesticetin (Fig. 2 D, E), and the macrolides, carbomycin and tylosin (Fig. 2F, G). Relatively high concentrations (1 10 mM) of these antibiotics are required to reach 50 % inhibitory levels for mitochondrial ribosomes (Table 3).
Antibiotic Susceptibility of Bovine Mitochondrial Ribosomes
444 Table 3. Antibiotic susceptibility of E. coli and bovine mitochondrial ribosomes Assay conditions are described in legend of Table 1. Values represent concentrations of antibiotics required to inhibit peptidyl transferase activity by 50 % Antibiotic
E. coli ribosomes
Mitochondria1 ribosomes
703
55-s
393
0.6 0.9 100 1000 2000 700 10000
0.3 0.7 80 200
PM Vernamycin A PA1 14A Chloramphenicol Lincomycin Celesticetin Carbomycin Tylosin
0.1 0.2 40 10 200 1.0 10
A
~-
-
500 -
- 6 . P 4
2
While mitochondrial ribosomes have about the same susceptibility to these lincosamines and macrolides, E. coli ribosomes show a differential response. For example, both celesticetin and lincomycin appear to interact poorly with mitochondrial ribosomes, but lincomycin is a more effective inhibitor than celesticetin for E. coli ribosomes in this system (Fig. 3 D, E). This effect is even more apparent in the case of the macrolides, where the response of bacterial and mitochondrial ribosomes differs by three orders of magnitude (Fig. 2F, G). The resistance of isolated mitochondrial ribosomes to the lincosamines and macrolides may be an intrinsic property of these ribosomes. Alternatively, it may be possible to account for this observation on another basis. For example, the binding site for these antibiotics may be partially obscured or otherwise affected by exogenous proteins that are bound or adsorbed to the ribosome 1201. To test this possibility, assays were also performed using mitochondrial ribosomes that had been washed in a high salt buffer (buffer C) to remove loosely adsorbed proteins. Since the derived subunits obtained by this washing procedure have the same sensitivity to these antibiotics as do the native 39-S subunits of mitochondrial ribosomes (Fig. 2), we conclude that the diminished susceptibility to these inhibitors is an intrinsic property of bovine mitochondrial ribosomes. It can be noted that the mitochondrial ribosomal subunits are somewhat more susceptible (than 55-S ribosomes) to certain antibiotics, especially lincomycin (Fig. 2D). These modest effects are not apparent with bacterial ribosomes. They may reflect partial occlusion of the antibiotic binding site by the small subunit of mitochondrial ribosomes or, perhaps, different conformational states of the large subunits, whether as a free subunit or in a monoribosome couple.
lo
tl
6
nl "0
0.6 0.2 0.4 Inhibitor concentration (mM)
1.0
Fig. 3. Analysis of the peptidyl transferase activity of ribosomes in Henderson [26] plots. The data in Fig.2 are plotted using the equation u o / v i = m[I], 1 where: 00 is the velocity of the reaction in the absence of inhibitor, u i is the velocity in the presence of inhibitor, m, the slope, = Z ( N i / & ) / D , an expression containing terms that relate to the mechanism of the reaction and to the interaction of the inhibitor with the enzyme, Ki,and [I], is the concentration of the inhibitor. Bovine mitochondrial 55-S (0)and 3 9 3 (0),E. coli 7 0 3 (A) and 5 0 3 (A), and bovine cytoplasmic 80-S (I ribosomes ) were tested with (A) chloramphenicol, (B) lincomycin and (C) carbomycin
+
Because of the diminished susceptibility of mitochondrial ribosomes to these lincosamines and macrolides, it is possible to identify concentrations of these antibiotics which are clearly inhibitory to bacterial ribosomes, but which are without effect on mitochondrial or cytoplasmic ribosomes. However, since mitochondrial ribosomes are susceptible to these antibiotics, higher concentrations will discriminate the three types of ribosomes from each other. For example, the peptidyl transferase activity of E. coli ribosomes is reduced to 50% or less by a 10 pM concentration of lincomycin or carbomycin, levels which are not
445
N. D. Denslow and T. W. O'Brien
Table 4. lnhibiror constants (Ki) and apparent dissociation constants (Ki) f o r the interaction of chloraniphenicol, lincomycin nnd carbomycin with mitochondrial and E. coli ribosomes 55-S Ribosomes calculated from
Antibiotic
70-S Ribosomes calculated from
-~
-
~~
Scatchard plot
Henderson plot ~
Ki ~
~~
~~
Scatchard plot
Ki ~~
Henderson plot
~-
.~
Ki
Ki
~
~
Ki
Ki
~~
-~
~
~
PM ~~
100 390 -
Chloramphenicol Lincomycm Carbomycm ~
~
20.4" 80" - 390
21 ~
500
~
~
~
29 5.1 -
2 5" 0.49"- 5.7b
29
-
1.9'
-
~
~
+
For competitive inhibition: K ; = KA/[I ([S]/K,,,)],where Ki is the inhibitor constant, KA is the apparent dissociation constant, obtained frpm the slope of the Scatchard plot, [S] is the puromycin concentration and K,,, is the Michaelis constant for puromycin. For non-competitive inhibition: Ki = KA. For non-competitive inhibition: Ki = l / m in the Henderson plot, Fig. 3. a
inhibitory to mitochondrial or cytoplasmic ribosomes (Fig. 2D, F). On the other hand, discriminatory levels of these antibiotics (1 mM, for example) which inhibit mitochondrial ribosomes by 50 %, are without effect on cytoplasmic ribosomes, while completely suppressing the activity of E. coli ribosomes (Fig. 2D, Fj. This level of lincomycin susceptibility observed for isolated bovine mitochondrial ribosomes appears to be characteristic for mitochondrial ribosomes of mammals [4,21,22] and insects (231. In fact, the KA (250 pM) calculated for lincomycin from studies on the inhibition of protein synthesis in intact rat liver mitochondria 1221 is in the range of that found for isolated bovine mitochondrial ribosomes (Table 4). On the other hand, the susceptibility observed for mammalian mitochondrial ribosomes to carbomycin depends on the system used. Mitochondria1 ribosomes isolated from bovine liver (Fig.2), rat liver 1241 and even from Neurospora [24], appear to have the same susceptibility to carbomycin. These results reflect intrinsic properties of the isolated mitochondrial ribosomes and not secondary effects, as are observed in studies with intact mitochondria [22] which appear able to concentrate carbomycin 1241. It should be noted that ribosomes isolated from yeast mitochondria are an exception to this pattern, in that they appear as susceptible to lincomycin and carbomycin as bacterial ribosomes 1251.
Linear Transformation of the Inhibition Data To better compare the response of the different kinds of ribosomes to representative antibiotics from each group, the inhibition data is analyzed in Fig. 3 using a linear transformation (Henderson plot) of the rate equation for inhibited reactions 1261. One effect of this transformation is to amplify the apparent differences between bacterial and mitochondrial ribosomes, especially when their susceptibilities differ by
more than a factor of 10. As a consequence, the mitochondrial ribosomes tend to resemble cytoplasmic ribosomes moreso than prokaryotic ribosomes in their response to lincomycin (Fig. 3 Bj and especially to carbomycin (Fig. 3C). A major advantage of this transformation is that the slope of the linear plot obtained is proportional to the inhibitor constant, Ki. Since the relationship between Ki and the slope of such plots depends upon the mechanism of inhibition, inhibitor constants can be calculated for those antibiotics whose mechanism of action is known. For competitive inhibitors of puromycin in the peptidyl transferase assay,
K i = - (1 .
m
Km
IS]
+Km
)
'
where m is the slope, K , is the Michaelis constant for puromycin and [S] is the puromycin concentration [26]. Since chloramphenicol is a competitive inhibitor of the puromycin reaction for bacterial ribosomes 1271, we can evaluate the chloramphenicol inhibitor constant Ki from the above relationship. This value, calculated for E. coli ribosomes in our system as 2.9 pM, compares well with the published values (in the range of 2.2 pM) for the chloramphenicol dissociation constant at 0°C [17,28]. It is reasonable to assume that chIoramphenico1 acts as a competitive inhibitor for mitochondrial ribosomes as well. In this case, the competitive inhibition constant calculated for mitochondrial 55-S ribosomes is 27 pM. This value, about 10-fold higher than that for E. coli ribosomes in this system, reflects the difference noted earlier in the inhibitor-response profiles in Fig. 2A. Carbomycin and lincomycin inhibit the binding of substrates to both the aminoacyl-tRNA binding (A) site and the peptidyi-tRNA binding (P) site [29,30] of E. coli ribosomes, complicating the calculation of inhibitor constants from Henderson plots 1261. However, the relative affinity of mitochondrial and bac-
446
Antibiotic Susceptibility of Bovine Mitochondria1 Ribosomes I
I
7
I
I
1
A
1.6 -
6
i
E
. 2.
4
2
0
40
t
t -0
B
Q2
06
0.4
0.8
1.0
I
Fig. 4. Analysis of the inhibition of the peptidyl transferase activity of mitochondrial and E. coli monoribosomes by lincomycin, using a (n/KJ. The symbols modified Scatchard plor: i/(I/ = - (i/KJ used are: i, the fraction of inhibition of the peptidyl transferase reaction effected by a concentration of inhibitor [I]; n, the number of binding sites per ribosome affecting the measured activity and K & the apparent dissociation constant for the inhibitor. Ki is equal to Ki [(K,,, [S])/K,,,] for competitive type inhibitors and to K i for non-competitive type inhibitors. The intercept on the x-axis is a direct measure of n, the number of binding sites. ( 0 ) 55-S rnitochondrial ribosomes, and (A)7 0 6 E. coli ribosomes
+
+
terial ribosomes for these antibiotics can be ascertained directly from the slopes of the Henderson plots (Fig. 3B, C). Thus it appears that E. coli ribosomes have a 50-fold higher affinity for lincomycin, and a 250-fold higher affinity for carbomycin, than do bovine mitochondrial ribosomes. Assuming that carbomycin acts mainly as a noncompetitive inhibitor in this reaction [27], we calculate inhibitor constants for carbomycin of 1.9 pM for E. coli ribosomes, and 500 pM for mitochondrial ribosomes. The lincomycin inhibition data (Fig. 2D) can be plotted in a Scatchard format (Fig.4) to provide an estimate of the apparent dissociation constant K& as well as the number of ribosomal binding sites at which lincomycin inhibits the peptidyl transferase reaction. The affinity of lincomycin for mitochondrial ribosomes is about 65-fold lower than for E.coli ribosomes (Table 4). Nevertheless, we find 0.7 lincomycin binding site/mitochondrial ribosome, a value close to that found for E. coli ribosomes in this system, 0.8 site/ ribosome (Fig. 4). Analysis of the chloramphenicol inhibition data (Fig.2A) in a Scatchard plot shows that mitochondrial ribosomes, like bacterial ribosomes, contain one
Fig. 5. Analysis of the inhibition of the peptidyl transferase activity of mitochondria1 and E. coli monoribosomes by chloramphenicol, using a modified Scatchard plot, as in Fig. 4
binding site at which chlorarnphenicol inhibits peptidyl transferase activity (Fig. 5). Apparent dissociation constants for chloramphenicol calculated from these Scatchard plots show that E. coli ribosomes have a slightly higher affinity for chloramphenicol than mitochondrial ribosomes (Table 4). The inhibitor constants calculated from these apparent dissociation constants agree well with those calculated for mitochondrial and bacterial ribosomes from the Henderson plots (Table 4) and also with the literature values available for E. coli [31].
DISCUSSION We have used the peptidyl transferase assay to examine the susceptibility of bovine mitochondrial ribosomes to several antibiotics. With the exception of gougerotin, which inhibits all types of ribosomes, the antibiotics used in this study are selective inhibitors of bacterial ribosomes. We find that all of these antibiotics are inhibitory to isolated bovine mitochondrial ribosomes and, by this criterion alone, it would appear that these mitochondrial ribosomes are of the prokaryotic type. Ribosomes from E. coli and bovine mitochondria are equally susceptible to chloramphenicol and the streptogramins, PA-1 14A and vernamycin A (Table 3). However, bovine mitochondrial ribosomes are much less sensitive than E. coli ribosomes to the lincosamine and macrolide antibiotics tested, requiring 100- 1000-fold higher levels of these antibiotics to achieve comparable inhibition.
N.D. Denslow and T. W. O’Brien
It is noteworthy that bovine mitochondrial ribosomes are sensitive to all of the antibacterial antibiotics used, especially in view of the fact that cytoplasmic ribosomes from the same cells are refractory to these inhibitors. This observation shows that binding sites for these antibiotics are conserved, albeit to different degrees, in the mitochondrial ribosomes, but not in the cytoplasmic ribosomes. This observation is of special interest, if ribosomal proteins comprise antibiotic binding sites 132,331, since the proteins which confer this ‘prokaryotic’ aspect to mitochondrial ribosomes are themselves, like cytoplasmic ribosomal proteins, encoded in the nuclear genome and synthesized on cytoplasmic ribosomes. Despite their common biosynthetic origin the mitochondrial ribosomal proteins are nevertheless very different from those of cytoplasmic ribosomes, on the basis of their 2-dimensional electrophoretic properties [34,35] (and T. W. O’Brien, unpublished observation). More significantly, the mitochondrial ribosomal proteins also differ extensively from those of bacterial ribosomes [34] (T. W. O’Brien, unpublished observation), suggesting that only selected sequences are conserved in the mitochondrial ribosomal proteins. From studies on the interaction of antibiotics with ribosomes, it appears that several functional groups on the ribosome may contribute to the actual binding site for a given antibiotic 1361. In the case of E. coli ribosomes, at least three separate groups on the ribosome appear to be involved in binding chloramphenicol 136,371. Operationally, these functional groups are located within the A site of the peptidyl transferase center, where chloramphenicol blocks the entry of the 3’-terminal fragment of aminoacyl-tRNA [30]. However, the exact nature of the actual binding site for chloramphenicol is unknown. The various groups comprising this binding site may be contributed by one 1381 or more proteins 1391 and perhaps even by a region of the ribosomal RNA. The fact that mitochondrial and bacterial ribosomes bind chloramphenicol with comparable affinities (Table 4), suggests that the chloramphenicol-binding site on mitochondrial ribosomes contains equivalent functional groups, in the same spatial orientation, as those on bacterial ribosomes. A similar situation appears to exist for the binding sites of PA-114A and vernamycin A, since both kinds of ribosomes are sensitive to very low concentrations (0.1 - 1 pM) of these antibiotics. In E. coli, the ribosomal binding site of the streptogramins overlaps that of chloramphenicol ; the streptogramins can inhibit the binding of the 3’ terminus of aminoacyl-tRNA, as well as that of chloramphenicol [40,41]. Thus, the same functional groups that interact with chloramphenicol in the A site, conserved in the mitochondrial ribosome, may also be involved in binding the streptogramins. In E. coli ribosomes, the binding
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domain for streptogramins extends into the P site of the peptidyl transferase center [40], where additional groups may be involved in binding these antibiotics. These extra interactions may account for the higher affinity of bacterial ribosomes for the streptogramins, compared to chloramphenicol [42] (Fig. 2). Since mitochondrial ribosomes have an equally high affinity for the streptogramins (Fig. Z), the additional groups involved are probably conserved in mitochondrial ribosomes. We interpret the diminished susceptibility of mitochondrial ribosomes (relative to E. coli ribosomes) to the lincosamines (Fig. 2D, E) and the macrolides (Fig. 2F, G), as resulting from the absence or improper positioning, on mitochondrial ribosomes, of one or more of the equivalent functional groups that are involved in binding these antibiotics to bacterial ribosomes. The binding domain for these antibiotics extends into both the A and the P site regions of the peptidyl transferase center of bacterial ribosomes [29,30]. The binding sites for these antibiotics also overlap that for chloramphenicol [41] suggesting the involvement of common functional groups at the A site. Since these functional groups appear to be conserved at the A site of mitochondrial ribosomes (above), the diminished susceptibility of mitochondrial ribosomes observed for the lincosamines and macrolides suggest that the additional groups required for stabilizing the. binding of these antibiotics to the P site are missing, or improperly oriented, on mitochondrial ribosomes. Thus, the disposition of functional groups within the A site of mitochondrial ribosomes appears to be more conserved, relative to bacterial ribosomes, than that of analogous groups in the P site. It is expected that essential structural features of the peptidyl transferase locus would be shared by prokaryotic and eukaryotic ribosomes alike, since both kinds of ribosomes interact with the common substrates, aminoacyl-tRNA and peptidyl-tRNA. This intrinsic property of the peptidyl transferase center is apparently responsible for the binding of antibiotics like puromycin and the 4-aminohexose pyrimidine nucleosides, including gougerotin [18]. In this sense, these antibiotics probably bind to some of the same functional groups in the A site [18] that are required for the binding and manipulation of the natural substrates by either kind of ribosome. We find that bovine mitochondrial ribosomes are just as susceptible to gougerotin as are bacterial and eukaryotic cytoplasmic ribosomes (Table 2). This result suggests that the components of the gougerotin binding site that are conserved in these three structurally dissimilar ribosomes, are absolutely essential for this central mechanism of protein synthesis. In addition to this universal ribosomal domain, there exists another set of functional groups at the
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N. D. Denslow and T. W. O’Brien: Antibiotic Susceptibility of Bovine Mitochondria] Ribosomes
ribosomal A site, common to the prokaryotic type of ribosomes. These groups, responsible for the susceptibility of mitochondrial ribosomes to the antibacterial antibiotics (above), delineate a structural domain which is fundamentally different in ribosomes of the prokaryotic and eukaryotic types. In this respect, it is most interesting that mitochondrial ribosomes have not lost their susceptibility to chloramphenicol, as have eukaryotic ribosomes, since mitochondrial ribosomes not only comprise a most heterogeneous group [I], but they also appear to be evolving more rapidly than other ribosomes [43- 461. This work was supported by grant GM-15438 from the United States Public Health Service, National Institutes of General Medical Sciences. We are grateful for the technical assistance of Warren Clark and Mark Critoph.
REFERENCES 1. O’Brien, T. W. (1977) in International Cell Biology (Brinkley, B. R. & Porter, K. R., eds) pp. 245-255, Rockefeller University Press, New York. 2. OBrien, T. W. & Matthews, D. E. (1976) in Handbook qf Genetics (King, R. C., ed.) vol. 5, pp. 535-580, Plenum Publishing Co., New York. 3. O’Brien, T. W. (1976) in Protein Synthesis (McConkey, E., ed.) vol. 2, pp. 249-307, Marcel Dekker, New York. 4. Denslow, N. D. & O’Brien, T. W. (1974) Biochem. Biophys. Res. Commun. 57, 9-16. 5. Hamilton, M. G. & O’Brien, T. W. (1974) Biochemistry, 13, 5400 - 5403. 6. O’Brien, T. W. (1971) J . Bid. Chenz. 246, 3409-3417. 7. Schnaitman, C., Erwin, V. G. & Greenawalt, J. W. (1967) J . Cell Biol. 32, 719-735. 8. Loewenstein, J., Scholte, H. R. & Wit-Peeters, E. M. (1970) Biochim. Biophys. Acta, 223, 432-436. 9. Nirenberg, M. W. (1963) Methods Enzymol. 6, 17-23. 10. Nishizuka, Y., Lipman, F. & Lucas-Lenard, J. (1968) Methods Enzymol. 12B, 708 - 721. 11. Haenni, A. L. & Chapeville, F. (1966) Biochim. Biophys. Acta, 114, 135-148. 12. de Vries, H., Agsteribbe, E. & Kroon, A. M. (1971) Biochim. Biophys. Acta, 246, 11 1 - 122. 13. Miskin, R., Zamir, A. & Elson, D. (1970) J. Mol. Biol. 54, 355- 378. 14. Pestka, S. (1972) Proc. Natl Acad. Sci. U.S.A. 65, 624-628. 15. Fahncstock, S., Neumann, H., Shashoua, V. & Rich, A. (1970) Biochemistry, 5, 2477 - 2483. 16. Fico, R. & Coutsogeorgopoulos, C. (1972) Biochem. Biophys. Res. Commun. 47,645-651. 17. Fernandez-Muiioz, R. & Vazquez, D. (1973) Mol. Biol. Rep. I , 75-79.
18. Barbacid, M. & Vazquez, D . (1974) Eur. J . Biochem. 44, 445-453. 19. Dixon, H., Kellerman, G. M., Mitchell, C. H., Towers, N. H. & Linnane, A. W. (1971) Biochem. Biophys. Res. Commun. 43, 780 - 786. 20. Spirin, A. S. & Asatryan, L. S. (1976) FEBSLett. 70, 101- 104. 21. Kroon, A. M. & de Vries, H. (1971) in Autonomy and Biogenesis of Mitochondria and Chloroplasts (Boardman, N. K., Linnane, A. W. & Smillie, R. M., eds) pp. 318 - 327, American Elsevier, New York. 22. Towers, N. R., Kellerman, G . M. & Linnane, A. W. (1973) Arch. Biochem. Biophys. 155, 159- 166. 23. Williams, K. L. & Birt, L. M. (1976) FEBS Lett. 66, 120- 123. 24. de Vries, H., Arendzen, A. J. & Kroon, A. M. (1973) Biochim. Biophys. Acta, 331, 264-275. 25. Grivell, L. A., Netter, P., Borst, P. & Slonimski, P. P. (1973) Biochim. Biophys. Acta, 312, 358 - 367. 26. Henderson, P. J. F. (1972) Biochern. J. 127, 321-333. 27. Pestka, S. (1970) Arch. Biochem. Biophys. 136, 80-88. 28. Pestka,S. (1975) in Antibiotics (Corcoran, J. W. & Hahn,F.E., eds) vol. 3, pp. 370- 395, Springer-Verlag, New York. 29. Celma, M. L., Monro, R. E. & Vazquez, D. (1970) FEBS Lett. 6, 273 -277. 30. Celma, M. L., Monro, R. E. & Vazquez, D. (1971) FEBS Lett. 13, 247-251. 31. Fernandez-Muiioz, R., Monro, R. E., Torres-Pinedo, R. & Vazquez, D. (1971) Eur. J. Biochem. 23, 185-193. 32. Pellegrini, M. & Cantor, C. R. (1977) in Molecular Mechanisms of’ Protein Synthesis (Weissbach, H. & Pestka, S., eds) pp. 468 - 555, Academic Press, New Y ork. 33. Vazquez, D., Barbacid, M. & Fernandez-Mufioz, R. (1975) in Topics in Infectious Diseases (Drews, J. & Hahn, F. E., eds) vol. 1, pp. 193-216, Springer-Verlag, New York. 34. Van den Bogert, C. & de Vries, H. (1976) Biochim. Biophys. Acta, 442, 227-238. 35. Leister, D. E. & Dawid, I. B. (1974) J . Biol. Chem. 245, 510851 18. 36. Hahn, F. E. & Gund, P. (1975) in Topics in Infectious Diseases (Drews, J. & Hahn, F. E., eds) vol. 1, pp. 245 - 266, SpringerVerlag, New York. 37. Cheney, B. V. (1974) J . Med. Chem. 17, 590-599. 38. Pongs, O., Bald, R. & Erdmann, V. A. (1973) Proc. Natl Acad. Sci. U.S.A. 70, 2229-2233. 39. Sonenberg, N., Wilchek, M. & Zamir, A. (1973) Proc. Natl Acad. Sci. U.S.A. 70, 1423-1426. 40. Ennis, H. L. & Duffy, K . E. (1972) Biochim. Biophys. Acra, 281,93- 102. 41. Vazquez, D. (1966) Biochim. Biophys. Acta, 114, 277-288. 42. Contreras, A. & Vazquez, D. (1977) Eur. J . Biochem. 74, 539 - 547. 43. Matthews, D. E., Hessler, R. A. & O’Brien, T. W. (1 978) FEBS Lett. 86, 76-80. 44. Groot, G. S. P., Flavell, R. A. & Sanders, J. P. M. (1975) Biochim. Biophys. Acta, 378, 186- 194. 45. Jakovcic, S., Casey, J. & Rabinowitz, M. (1975) Biochemistry, 14, 2043 - 2050. 46. Leister, D. E. & Dawid, I. B. (1975)J. Mol. Biol. Y6, 119-123.
N. D. Denslow and T. W. OBrien, Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box J-245, J. Hillis Miller Health Center, Gainesville, Florida, U.S.A. 32610
