Planta (Berl.) 91,329--335 (1970)
Further Similarities between Chloroplast and Bacterial Ribosomes R. J. Eimis Department of Biochemistry, University of Aberdeen, Aberdeen, Scotland Received January 12, 1970 Summary. Protein synthesis by chloroplasts isolated under aseptic conditions from Phaseolus vulgaris leaves is inhibited by the bacterial antibiotics spectinomycin, lincomycin, and erythromycin; that by chloroplasts from 2Vicotiana tabacum leaves is inhibited by spcctinomycin and lincomycin but not by erythromycin. Protein synthesis by cytoplasmic ribosomes from plants and animals is not inhibited by these compounds, nor is amino acid activation by the soluble fraction from bean chloroplasts. These results suggest that chloroplast ribosomes possess sites which bind several unrelated bacterial antibiotics and support the idea that chloroplasts originated from prokaryotic cells. These antibiotics may be useful in studying the process of chloroplast formation in intact cells.
Introduction The occurrence of two classes of ribosomes in the green cells of higher plants was reported first by Lyttleton (1962). He found an 80 S class in the soluble or cytoplasmic fraction and a 70 S class which was restricted largely to the chloroplast fraction; many studies have confirmed this finding for both higher plants and algae (e.g. Stutz and I~oll, 1967). Chloroplast ribosomes thus resemble those from bacteria and blue-green algae in their sedimentation coefficient and in recent years further similarities between prokaryote and chloroplast ribosomes have been reported. For instance, the sizes of their RNA components (Loening and Ingle, 1967), the use of N-formylmethionine as the initiating amino acid in protein synthesis (Schwartz, Meyer, Eisenstadt and Brawerman, 1967), the occurrence of some acidic proteins in the ribosome (Gualerzi and Cammarano, 1969), the inhibition of protein synthesis by ehloramphenicol (Spencer and Wfldman, 1964) in a stereospeeific manner (Ellis, 1969) - - in all these respects ribosomes from chloroplasts resemble those from bacteria and differ from those found in the cytoplasm of both green and non-green plant cells. I t is hard to believe that these similarities are entirely fortuitous and the old idea that chloroplasts have evolved from ingested prokaryotic cells is currently enjoying a revival (Sagan, 1967).
330
R . J . Ellis:
I t is well established t h a t chloroplasts contain DNA, D N A polymerase and DNA-dependent R N A po]ymerase and consequently enjoy some genetic autonomy; it is equally clear t h a t this autonomy is not complete since some mutations which affect chloroplast structure and function are nuclear in origin (Kirk and Tflney-Bassett, 1967). Much remains to be done to determine the location of both the information and the site of synthesis of the chloroplast components. The only antibiotic which has been shown to inhibit protein synthesis b y bacterial and chloroplast ribosomes, but not b y 80S ribosomes, is chloramphenicol; antibiotics such as pnromycin and tetracycline inhibit both 70S and 80S ribosomes (Vazquez and Monro, 1967). However, there is a difficulty associated with chloramphenicol which makes it desirable to use other antibiotics. This difficulty concerns the absence of 70 S ribosomes resistant to chloramphenico]. Resistance of bacteria to ehloramphenicol resides either in reduced permeability or in detoxification b y acetylation (Weisblum and Davies, 1968); resistance a t the ribosomal level has not been reported. Presumably the integrity of the chloramphenicol-binding site is essential to the functioning of the ribosome. The similarity between chloroplast and bacterial ribosomes as regards chloramphenieol could therefore reflect merely t h a t both are of the 70 S class and not arise because they are related in evolution. A number of antibiotics besides chloramphenicol are known to inhibit bacterial but not 80 S ribosomes, and in some cases mutants resistant at the ribosomal level are known. I t follows t h a t the larger the number of such structurally unrelated antibiotics which can be shown to inhibit protein synthesis by chloroplast ribosomes the stronger becomes the argument for the endosymbiont hypothesis. This paper describes some work on the similarity of action of some antibiotics on chloroplast and bacterial ribosomes which supports the concept of an evolutionary relationship between chloroplasts and prokaryotes, and suggests new antibiotics for selectively inhibiting chloroplast ribosomes in vivo. Materials and Methods Expanding leaves of young plants of tobacco (Nicotiana tabacu~z) and bean (Phaseotus vulgaris) were used as sources of chloroplasts. Leaves were surfacesterilised with hypochlorite, washed in cold sterile water and chopped by hand in sterile Honda medium (2.5% Ficoll, 5% Dextran 40, 0.25 M sucrose, 50 mM tris HC1, 1 mM MgSO4, 5 mM 2-mercaptoethanol, pit 7.8 at 20~ as described by Parenti and Margulies (1967). The homogenate was stained through 8 layers of sterile muslin using plastic gloves and centrifuged at 1,000 g for 10 minutes. The pellet was resuspended either in Honda medium or in Tris Mg medium (25 m ~ tris HC1, 10 mM MgSO4, 5 mM 2-mercaptoethanol, pH 7.8 at 20~ The incorporation of ilC-L-leucine into protein was measured by incubating chloroplasts with 0.4 mM ATP, 0.04 mM GTP, 30 mM KC1, an ATP-generating system, and 2.6 iiM
Chloroplast and Bacterial Ribosomes
331
14C-L-teueine (0.5 [xC) in a final volume of 0.6 ml a~ 25~ Protein was exgraeted as described previously (Ellis and 5Iacdonald, 1967) and counted by liquid scintillation at 60 % efficiency. All glassware and media were sterilised before use.
Results 1. Protein Synthesis by Isolated Chloroplasts. The time course of incorporation of leucine into protein b y bean chloroplasts is shown in Fig. 1. Although incorporation is less in Tris Mg than in H o n d a medium and ceases sooner, chloroplasts were routinely incubated in Tris Mg since they lose their outer membranes under these conditions. This ensures t h a t permeability is not a factor in the effects of added compounds. Bean chloroplasts incorporate 0.5 to 1.0 nmoles of 14C-leucine into protein per nag chlorophyll; tobacco chloroplasts give about 10% of this incorporation. These levels of activity are similar to those reported previously b y others (Spencer and Wildman, 1964; Parenti and Margulles, 1967). I t has been established t h a t amino acid incorporation b y tobacco and bean chloroplasts is due to the ribosomes they contain (Boardman, Francki and Wildman, 1965; Ranaletti, Gnanam and Jagendorf, 1969). Table 1 lists the characteristics of leueine incorporation. Both the dependence of incorporation on added A T P and G T P and its sensitivity to ribonuelease indicate t h a t incorporation is not due to contaminating micro-organisms. This is a well-known hazard of chloroplast systems and it is especially important in the present context to ensure t h a t micro-organisms do not contribute to the observed incorporation. Virtually all the labelled protein is soluble in Triton X-100; this rules out both nuclei and bacteria as the agents of incorporation (Parenti and Margulies, 1967).
Table 1. Characteristics o/ leucine incorporation by chloroplasts. Chloroplasts were resuspended in T~is My and incubated /or 60 minutes at 25 ~ with the additions described in Materials and Methods. Ribonuclease and puromyci~ were used at 1 ~g/ml and 200 ~g/ml respectively. Triton soluble refers to the labelled protein that was soluble in 0.2% Triton X-IO0. Incorporation is expressed as a % o/that given by the complete assay mixture
Assay mixture
Complete Complete, zero time Omit ATP and GTP Add ribonuelease Add puromyein Complete, Triton soluble
% Incorporation of l~C-leueine Bean chloroplasts
Tobacco chloroplasts
100 0.5 3 22 27 96
100 1.5 5 20 25 95
332
R . J . Ellis: tOO
-5 0.75 cn
p
8=
0.50
~ d 0.25
0
20
40 60 Minutes
80
100
Fig. 1. Time course of l~C-]eucine incorporation into protein by bean chloroplasts. The 1,000 g pellet was resuspended either in Honda medium (e) or Tris Mg (o) and incubated at 25 ~ with the additions described in Materials and Methods. Speetiaomycin was added at 25 Fg/ml to chloroplasts resuspended in Tris Mg ([]) 2. E/]ects o/ Bacterial Antibiotics on Chloroplast Incorporation. F i v e a n t i b i o t i c s were t e s t e d ; e t a m y c i n a n d l a n k a m y c i n gave n o t m o r e t h a n 15 % i n h i b i t i o n a t 200 ~ g / m l b u t s p e c t i n o m y c i n , l i n c o m y c i n a n d e r y t h r o m y c i n were g o o d inhibitors. Fig. 1 i l l u s t r a t e s t h e t i m e course of inhib i t i o n of b e a n c h l o r o p l a s t i n c o r p o r a t i o n b y s p e e t i n o m y e i n , while Fig. 2 Table 2. Leucine incorporation by cytoplasmic ribosomes. Cytoplasmic ribosomes were prepared /tom sterile red beetroot disks, tobacco leaves, and rat liver, and assayed /or laC-leucine incorporation into protein as described (Ellis and MacDonald, 1967). Results are expressed as a % of the counts incorporated by the complete assay mixture. All antibiotics were used at 200 Fg/ml Assay mixture
Complete Omit ATP and GTP Add spectinomyein Add lincomycin Add erythromyeia Add puromycin
Source of cytoplasmic ribosomes Tobacco leaves
Beetroot Disks
Rat liver
100 10 90 100 93 21
100 7 104 109 104 21
100 8 90 94 93 22
Chloroplast and Bacterial Ribosomes
333
100 =o 80-
~
~ 60o
o 4-0 20
~
,
~
SM i
O
I 50
I 100 l.t,g/ml
I 150
I 200
Fig. 2. Effects of spectinomyein, lincomycin, and erythromycin on ]eucine incorporation by bean chloroplasts. Bean chloroplasts were resuspended in Tris Mg and incubated at 25~ for 60 minutes with the additions described in Materials and Methods. S M spectinomycin; LM lincomycin; E M erythromyein 100
9
EM
9
80 6o
40 04 20 I 50
I 100 t.Lg/m[
I 150
I 200
Fig. 3. Effects of spectinomycin, lincomycin, and erythromycin on leucine incorporation by tobacco chloroplasts. Details as for Fig. 2 shows the concentration dependence of the inhibition of incorporation of bean chloroplasts b y all three antibiotics. Spectinomycin is the most effective inhibitor followed by lincomycin. A similar graph for tobacco chloroplasts is shown in Fig. 3; in this case spectinomycin and lincomycin inhibit as for bean, but erythromycin does not. 3. E]/ects o/ Bacterial Antibiotics on 8 0 S Ribosomes. All three antibiotics were tested on cytoplasmic ribosomes from plants and animals, including those from tobacco leaves; no significant inhibition was found at 200 Fg/ml (Table 2). None of these compounds inhibited the conversion of leucine to leueyl-s]~NA by the high speed supernatant from bean chloroplasts. 23
Planta (Berl.), Bd. 91
334
R.J. Ellis: Discussion
The data presented in this paper extend still further the area of similarity between ribosomes from chloroplasts and those from prokaryotic cells. Spectinomycin is an aminoglycoside which binds to the 30 S subunit of bacterial ribosomes and may inhibit the transloeation step in protein synthesis (Weisblum and Davies, 1968). The protein that confers sensitivity to spectinomycin has been isolated from the 30S subunit (Bollen, Davies, Ozaki and Mizushima, 1969). Lineomycin binds to the 50 S subunit of bacterial ribosomes and inhibits the formation of the peptide bond; erythromycin also binds to the 508 subunit but inhibits the translocation step (Igarashi, Ishitsuka and Kaji, 1969). These three antibiotics therefore inhibit bacterial protein synthesis at different sites; mutants at the ribosomal level are known for all three compounds. The mode of action of these antibiotics on chloroplast ribosomes is unknown, but the fact that chloroplast ribosomes from bean are sensitive to erythromyein whereas those from tobacco are not (~'igs. 2 and 3) suggests that the erythromyein site is not essential to the functioning of the chloroplast ribosome. This emphasises the point that with antibiotics other than chloramphenieol one may expect to find differences between species. Preliminary experiments have shown that spectinomycin will stop the development of chloroplasts in young leaves of tobacco plants without inhibiting leaf expansion and thus may be of value in determining which chloroplast components are made in vivo on chloroplast ribosomes. I t is known that protein synthesis by yeast mitochondria is inhibited by bacterial antibiotics including chloramphenicol, lincomyein and erythromyein (Lamb, Clark-Walker and Linnane, 1968), and mutants containing mitochondria resistant to erythromyein have been found (Linnane, Lamb, Christodoulou and Lukins, 1968). The intriguing possibility therefore exists that some green plant ceils contain chloroplast ribosomes which are sensitive to one set of bacterial antibiotics and mitochondrial ribosomes sensitive to a different set of such compounds. Such cells would provide excellent material for studying the relation between the development of mitoehondria and chloroplasts. Thanks are due to Drs. J. Ehrlich, W. Keller and D. Suttie for gifts of antibiotics. References Boardman, N. K., Francki, R. I. B., Wildman, S. G. : Protein synthesis by cellfree extracts from ~obacco leaves II. Biochemistry 4, 872--876 (1965). Bollen, A., Davies, J., Ozaki, M., Mizushima, S.: Ribosomal protein conferring sensitivity to the antibiotic spectinomycin in Escherichia coll. Science 165, 85-86 (1969).
Chloroplast and Bacterial Ribosomes
335
Ellis, 1~. J. : Chloroplast ribosomes: stereospecificity of inhibition by chloramphenicol Science 158, 477---478 (1969). -Macdonald, I. R. : Activation of protein synthesis by mierosomes from aging beet disks. Plant Physiol. 42, 1297--1302 (1967). Gualerzi, C., Cammarano, P. : Comparative electrophoretic studies on the protein of chloroplast and cytoplasmic ribosomes of spinach leaves. Biochim. biophys. Acta (Amst.) 190, 170--186 (1969). Igarashi, K., Ishitsuka, H., Kaji, A.: Comparative studies on the mechanism of action of lincomycin, streptomycin and erythromyein. Biochem. biophys. Res. Commun. 87, 499--504 (1969). Kirk, J. T. 0., Tilney-Bassett, R. A . E . : The plastids. London: Freeman & Co. 1967. Lamb, A. J., Clark-Walker, G. D., Linnane, A. W. : The biogenesis of mitochondria IV. Biochim. biophys. Aeta (Amst.) 161, 415--427 (1968). Linnane, A. W., Lamb, A. J., Christodoulou, G., Lukins, I-I. B.: The biogenesis of mitochondria VI. Proe. nat. Acad. Sci. (Wash.) 59, 1288--1293 (1968). Loening, U. E., Ingle, J.: Diversity of RNA components in green plant tissues. Nature (Lond.) 215, 363--367 (1967). Lyttleton, J. W. : Isolation of ribosomes from spinach chloroplasts. Exp. Cell Res. 2 6 , 312--317 (1962). Parenti, F., Margulies, M. M. : In vitro protein synthesis by plastids of Phaseolus vulgaris I. Plant Physiol. 42, 1179--1186 (1967). Ranaletti, M., Gnanam, A., Jagendorf, A. T.: Amino acid incorporation by isolated chloroplasts. Biochim. biophys. Acta (Amst.) 186, 192--204 (1969). Sagan, L.: On the origin of mitosing cells. J. theoret. Biol. 14, 225--274 (1967). Schwartz, J. H., Meyer, R., Eisenstadt, J.M., Brawerman, G.: Involvement of N-formylmethionine in initiation of protein synthesis by cell-free extracts of Euglena gracilis. J. molec. Biol. 25, 571--574 (1967). Spencer, D., Wildman, S. G.: The incorporation of amino acids into protein by cell-free extracts of tobacco leaves. Biochemistry 8, 954--959 (1964). Stutz, E., Noll, H. : Characterisation of cytoplasmic and chloroplast polysomes in plants: evidence for three classes of ribosomal I~NA in nature. Proc. nat. Acad. Sei. (Wash.) 57, 774--781 (1967). Vazquez, D., Monro, R. E. : Effects of some inhibitors of protein synthesis on the binding of aminoacyl-tRNA to ribosomal subunits. Biochim. biophys. Acta (Amst.) 142, 155--173 (1967). Weisblum, B., Davies, J.: Antibiotic inhibitors of the bacterial ribosome. Bact. Rev. 82, 493--528 (1968). R. J. Ellis Department of Biochemistry University of Aberdeen, Aberdeen AB 91 AS, Scotland
28*
Further Similarities between Chloroplast and Bacterial Ribosomes R. J. Eimis Department of Biochemistry, University of Aberdeen, Aberdeen, Scotland Received January 12, 1970 Summary. Protein synthesis by chloroplasts isolated under aseptic conditions from Phaseolus vulgaris leaves is inhibited by the bacterial antibiotics spectinomycin, lincomycin, and erythromycin; that by chloroplasts from 2Vicotiana tabacum leaves is inhibited by spcctinomycin and lincomycin but not by erythromycin. Protein synthesis by cytoplasmic ribosomes from plants and animals is not inhibited by these compounds, nor is amino acid activation by the soluble fraction from bean chloroplasts. These results suggest that chloroplast ribosomes possess sites which bind several unrelated bacterial antibiotics and support the idea that chloroplasts originated from prokaryotic cells. These antibiotics may be useful in studying the process of chloroplast formation in intact cells.
Introduction The occurrence of two classes of ribosomes in the green cells of higher plants was reported first by Lyttleton (1962). He found an 80 S class in the soluble or cytoplasmic fraction and a 70 S class which was restricted largely to the chloroplast fraction; many studies have confirmed this finding for both higher plants and algae (e.g. Stutz and I~oll, 1967). Chloroplast ribosomes thus resemble those from bacteria and blue-green algae in their sedimentation coefficient and in recent years further similarities between prokaryote and chloroplast ribosomes have been reported. For instance, the sizes of their RNA components (Loening and Ingle, 1967), the use of N-formylmethionine as the initiating amino acid in protein synthesis (Schwartz, Meyer, Eisenstadt and Brawerman, 1967), the occurrence of some acidic proteins in the ribosome (Gualerzi and Cammarano, 1969), the inhibition of protein synthesis by ehloramphenicol (Spencer and Wfldman, 1964) in a stereospeeific manner (Ellis, 1969) - - in all these respects ribosomes from chloroplasts resemble those from bacteria and differ from those found in the cytoplasm of both green and non-green plant cells. I t is hard to believe that these similarities are entirely fortuitous and the old idea that chloroplasts have evolved from ingested prokaryotic cells is currently enjoying a revival (Sagan, 1967).
330
R . J . Ellis:
I t is well established t h a t chloroplasts contain DNA, D N A polymerase and DNA-dependent R N A po]ymerase and consequently enjoy some genetic autonomy; it is equally clear t h a t this autonomy is not complete since some mutations which affect chloroplast structure and function are nuclear in origin (Kirk and Tflney-Bassett, 1967). Much remains to be done to determine the location of both the information and the site of synthesis of the chloroplast components. The only antibiotic which has been shown to inhibit protein synthesis b y bacterial and chloroplast ribosomes, but not b y 80S ribosomes, is chloramphenicol; antibiotics such as pnromycin and tetracycline inhibit both 70S and 80S ribosomes (Vazquez and Monro, 1967). However, there is a difficulty associated with chloramphenicol which makes it desirable to use other antibiotics. This difficulty concerns the absence of 70 S ribosomes resistant to chloramphenico]. Resistance of bacteria to ehloramphenicol resides either in reduced permeability or in detoxification b y acetylation (Weisblum and Davies, 1968); resistance a t the ribosomal level has not been reported. Presumably the integrity of the chloramphenicol-binding site is essential to the functioning of the ribosome. The similarity between chloroplast and bacterial ribosomes as regards chloramphenieol could therefore reflect merely t h a t both are of the 70 S class and not arise because they are related in evolution. A number of antibiotics besides chloramphenicol are known to inhibit bacterial but not 80 S ribosomes, and in some cases mutants resistant at the ribosomal level are known. I t follows t h a t the larger the number of such structurally unrelated antibiotics which can be shown to inhibit protein synthesis by chloroplast ribosomes the stronger becomes the argument for the endosymbiont hypothesis. This paper describes some work on the similarity of action of some antibiotics on chloroplast and bacterial ribosomes which supports the concept of an evolutionary relationship between chloroplasts and prokaryotes, and suggests new antibiotics for selectively inhibiting chloroplast ribosomes in vivo. Materials and Methods Expanding leaves of young plants of tobacco (Nicotiana tabacu~z) and bean (Phaseotus vulgaris) were used as sources of chloroplasts. Leaves were surfacesterilised with hypochlorite, washed in cold sterile water and chopped by hand in sterile Honda medium (2.5% Ficoll, 5% Dextran 40, 0.25 M sucrose, 50 mM tris HC1, 1 mM MgSO4, 5 mM 2-mercaptoethanol, pit 7.8 at 20~ as described by Parenti and Margulies (1967). The homogenate was stained through 8 layers of sterile muslin using plastic gloves and centrifuged at 1,000 g for 10 minutes. The pellet was resuspended either in Honda medium or in Tris Mg medium (25 m ~ tris HC1, 10 mM MgSO4, 5 mM 2-mercaptoethanol, pH 7.8 at 20~ The incorporation of ilC-L-leucine into protein was measured by incubating chloroplasts with 0.4 mM ATP, 0.04 mM GTP, 30 mM KC1, an ATP-generating system, and 2.6 iiM
Chloroplast and Bacterial Ribosomes
331
14C-L-teueine (0.5 [xC) in a final volume of 0.6 ml a~ 25~ Protein was exgraeted as described previously (Ellis and 5Iacdonald, 1967) and counted by liquid scintillation at 60 % efficiency. All glassware and media were sterilised before use.
Results 1. Protein Synthesis by Isolated Chloroplasts. The time course of incorporation of leucine into protein b y bean chloroplasts is shown in Fig. 1. Although incorporation is less in Tris Mg than in H o n d a medium and ceases sooner, chloroplasts were routinely incubated in Tris Mg since they lose their outer membranes under these conditions. This ensures t h a t permeability is not a factor in the effects of added compounds. Bean chloroplasts incorporate 0.5 to 1.0 nmoles of 14C-leucine into protein per nag chlorophyll; tobacco chloroplasts give about 10% of this incorporation. These levels of activity are similar to those reported previously b y others (Spencer and Wildman, 1964; Parenti and Margulles, 1967). I t has been established t h a t amino acid incorporation b y tobacco and bean chloroplasts is due to the ribosomes they contain (Boardman, Francki and Wildman, 1965; Ranaletti, Gnanam and Jagendorf, 1969). Table 1 lists the characteristics of leueine incorporation. Both the dependence of incorporation on added A T P and G T P and its sensitivity to ribonuelease indicate t h a t incorporation is not due to contaminating micro-organisms. This is a well-known hazard of chloroplast systems and it is especially important in the present context to ensure t h a t micro-organisms do not contribute to the observed incorporation. Virtually all the labelled protein is soluble in Triton X-100; this rules out both nuclei and bacteria as the agents of incorporation (Parenti and Margulies, 1967).
Table 1. Characteristics o/ leucine incorporation by chloroplasts. Chloroplasts were resuspended in T~is My and incubated /or 60 minutes at 25 ~ with the additions described in Materials and Methods. Ribonuclease and puromyci~ were used at 1 ~g/ml and 200 ~g/ml respectively. Triton soluble refers to the labelled protein that was soluble in 0.2% Triton X-IO0. Incorporation is expressed as a % o/that given by the complete assay mixture
Assay mixture
Complete Complete, zero time Omit ATP and GTP Add ribonuelease Add puromyein Complete, Triton soluble
% Incorporation of l~C-leueine Bean chloroplasts
Tobacco chloroplasts
100 0.5 3 22 27 96
100 1.5 5 20 25 95
332
R . J . Ellis: tOO
-5 0.75 cn
p
8=
0.50
~ d 0.25
0
20
40 60 Minutes
80
100
Fig. 1. Time course of l~C-]eucine incorporation into protein by bean chloroplasts. The 1,000 g pellet was resuspended either in Honda medium (e) or Tris Mg (o) and incubated at 25 ~ with the additions described in Materials and Methods. Speetiaomycin was added at 25 Fg/ml to chloroplasts resuspended in Tris Mg ([]) 2. E/]ects o/ Bacterial Antibiotics on Chloroplast Incorporation. F i v e a n t i b i o t i c s were t e s t e d ; e t a m y c i n a n d l a n k a m y c i n gave n o t m o r e t h a n 15 % i n h i b i t i o n a t 200 ~ g / m l b u t s p e c t i n o m y c i n , l i n c o m y c i n a n d e r y t h r o m y c i n were g o o d inhibitors. Fig. 1 i l l u s t r a t e s t h e t i m e course of inhib i t i o n of b e a n c h l o r o p l a s t i n c o r p o r a t i o n b y s p e e t i n o m y e i n , while Fig. 2 Table 2. Leucine incorporation by cytoplasmic ribosomes. Cytoplasmic ribosomes were prepared /tom sterile red beetroot disks, tobacco leaves, and rat liver, and assayed /or laC-leucine incorporation into protein as described (Ellis and MacDonald, 1967). Results are expressed as a % of the counts incorporated by the complete assay mixture. All antibiotics were used at 200 Fg/ml Assay mixture
Complete Omit ATP and GTP Add spectinomyein Add lincomycin Add erythromyeia Add puromycin
Source of cytoplasmic ribosomes Tobacco leaves
Beetroot Disks
Rat liver
100 10 90 100 93 21
100 7 104 109 104 21
100 8 90 94 93 22
Chloroplast and Bacterial Ribosomes
333
100 =o 80-
~
~ 60o
o 4-0 20
~
,
~
SM i
O
I 50
I 100 l.t,g/ml
I 150
I 200
Fig. 2. Effects of spectinomyein, lincomycin, and erythromycin on ]eucine incorporation by bean chloroplasts. Bean chloroplasts were resuspended in Tris Mg and incubated at 25~ for 60 minutes with the additions described in Materials and Methods. S M spectinomycin; LM lincomycin; E M erythromyein 100
9
EM
9
80 6o
40 04 20 I 50
I 100 t.Lg/m[
I 150
I 200
Fig. 3. Effects of spectinomycin, lincomycin, and erythromycin on leucine incorporation by tobacco chloroplasts. Details as for Fig. 2 shows the concentration dependence of the inhibition of incorporation of bean chloroplasts b y all three antibiotics. Spectinomycin is the most effective inhibitor followed by lincomycin. A similar graph for tobacco chloroplasts is shown in Fig. 3; in this case spectinomycin and lincomycin inhibit as for bean, but erythromycin does not. 3. E]/ects o/ Bacterial Antibiotics on 8 0 S Ribosomes. All three antibiotics were tested on cytoplasmic ribosomes from plants and animals, including those from tobacco leaves; no significant inhibition was found at 200 Fg/ml (Table 2). None of these compounds inhibited the conversion of leucine to leueyl-s]~NA by the high speed supernatant from bean chloroplasts. 23
Planta (Berl.), Bd. 91
334
R.J. Ellis: Discussion
The data presented in this paper extend still further the area of similarity between ribosomes from chloroplasts and those from prokaryotic cells. Spectinomycin is an aminoglycoside which binds to the 30 S subunit of bacterial ribosomes and may inhibit the transloeation step in protein synthesis (Weisblum and Davies, 1968). The protein that confers sensitivity to spectinomycin has been isolated from the 30S subunit (Bollen, Davies, Ozaki and Mizushima, 1969). Lineomycin binds to the 50 S subunit of bacterial ribosomes and inhibits the formation of the peptide bond; erythromycin also binds to the 508 subunit but inhibits the translocation step (Igarashi, Ishitsuka and Kaji, 1969). These three antibiotics therefore inhibit bacterial protein synthesis at different sites; mutants at the ribosomal level are known for all three compounds. The mode of action of these antibiotics on chloroplast ribosomes is unknown, but the fact that chloroplast ribosomes from bean are sensitive to erythromyein whereas those from tobacco are not (~'igs. 2 and 3) suggests that the erythromyein site is not essential to the functioning of the chloroplast ribosome. This emphasises the point that with antibiotics other than chloramphenieol one may expect to find differences between species. Preliminary experiments have shown that spectinomycin will stop the development of chloroplasts in young leaves of tobacco plants without inhibiting leaf expansion and thus may be of value in determining which chloroplast components are made in vivo on chloroplast ribosomes. I t is known that protein synthesis by yeast mitochondria is inhibited by bacterial antibiotics including chloramphenicol, lincomyein and erythromyein (Lamb, Clark-Walker and Linnane, 1968), and mutants containing mitochondria resistant to erythromyein have been found (Linnane, Lamb, Christodoulou and Lukins, 1968). The intriguing possibility therefore exists that some green plant ceils contain chloroplast ribosomes which are sensitive to one set of bacterial antibiotics and mitochondrial ribosomes sensitive to a different set of such compounds. Such cells would provide excellent material for studying the relation between the development of mitoehondria and chloroplasts. Thanks are due to Drs. J. Ehrlich, W. Keller and D. Suttie for gifts of antibiotics. References Boardman, N. K., Francki, R. I. B., Wildman, S. G. : Protein synthesis by cellfree extracts from ~obacco leaves II. Biochemistry 4, 872--876 (1965). Bollen, A., Davies, J., Ozaki, M., Mizushima, S.: Ribosomal protein conferring sensitivity to the antibiotic spectinomycin in Escherichia coll. Science 165, 85-86 (1969).
Chloroplast and Bacterial Ribosomes
335
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