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Nuclear genome codes for chloroplast ribosomal proteins in Acetabularia
Copyright ,411 rights
0
of
1973 by Academic Press, Inc. reproduction in any form reseroed
Experimental Cell Research 80 (1973) 63-68
NUCLEAR
GENOME CODES FOR CHLOROPLAST
RIBOSOMAL I. Isolation
PROTEINS IN ACETABULARIA
and Characterization
of Chloroplast
Ribosomal Particles
K. KLOPPSTECH and H. G. SCHWEIGER Max-Planck-lnstitut
fiir Zellbiologie,
D 2940 Wilhelmshaven,
BRD
SUMMARY A method is described for the isolation of chloroplast ribosomes from Acetabuluria cells in yields sufficient for the characterization of these particles. Ribosomal particles sedimenting with 7OS, 56S, 44S, and 30s have been obtained. The monoribosome sediments with 70s and dissociates into a larger 44s and a smaller 30s subunit. The sedimentation behaviour of the particles as well as the equilibrium between monoribosomes and their subunits is not influenced by the centrifugation step as could be revealed by formaldehyde fixation.
Although chloroplasts contain DNA and dispose their own genetic information it has not been possible so far to prove that any of the chloroplast proteins are coded by the chloroplast genome. Nuclear dependence of chloroplast protein constituents has been demonstrated for a number of chloroplast proteins such as malic dehydrogenase [19], lactic dehydrogenase [17], aldolase [l], and a number of membrane proteins [2, 12, 151. Similar conclusions have been drawn from genetic
experiments
on
chloroplast
ribo-
somal proteins [16]. The only established role of chloroplast DNA is the coding of chloroplast ribosomal [18, 211 and transfer RNA [22]. From this point of view, it was quite interesting to investigate the problem of where the genetic information for the chloroplast ribosomal proteins comes from by means of the nucleus transplantation technique in Acetabularia. In order to approach this problem it was 5-731803
necessary to characterize the chloroplast ribosomes and standardize the isolation procedures. The techniques involved as well as the ribosome patterns of Acetabuluriu under different conditions are described and ribosome patterns of 5 species of Acetabularia are compared. An early attempt to establish the ribosomal pattern of A. mediterranea has been made by Janowski and co-workers [lo].
MATERIAL
AND
METHODS
Culture conditions. Cells of Acetabularia mediterranea, A. crenulata, A. calyculus, Acicularia schenckii, and A. (Polyphysa) cliftonii were grown in Erd-Schreiber
medium (ESM) under conditions described earlier 16, 201. Axenic cultures of A. mediterrunea were obtained in accordance with Gibor & Izawa [5] and Berger [3]. of chloroplasts. Four hundred to 1000 plants at the stage of initiation of cap formation were washed 3 times in ESM, cut into 5 mm pieces, and homogenized with a teflon pestle in 40 ml of MES buffer at 4°C. The MES buffer contained per 10.05 Isolation
Exptl Cell Res 80 (1973)
64 K. Kloppstech & H. G. Schweiger moles [2-(N-morpholino)ethane-sulfonic acid] adjusted to pH 6.1 with NaOH, 0.02 moles NaCi, 0.33 moles sorbitol, 0.002 moles NaNO,, 0.002 moles EDTA (dipotassium salt), 0.002 moles Na isoascorbate, 0.001 moles MnCl,, 0.001 moles MgCl,, and 0.0005 moles K,HPO, [ll]. The homogenate was passed through two layers of cheese cloth and centrifuged for 2 min at 100 g to remove membranous material. The supernatant was centrifuged for 15 min at 1 000 g, the sediment resuspended by gentle homogenization in 20 ml of MEA buffer and the chloroplasts sedimented by recentrifugation at 4°C. The MEA buffer contained, per liter, 0.1 moles KCI, 0.01 moles Tris buffer, 0.015 moles Mg2+ as acetate and 0.01 moles mercaptoethylamine and was adjusted to pH 7.4. The chloroplast preparation contained a contamination of one mitochondrion per 10 chloroplasts as shown by electronmicroscopic techniques. This percentage was by far too small to affect the results on the chloroplast ribosomes. Furthermore there was no indication for the presence of 80s ribosomes or for the corresponding RNA in the chloroplast preparation as judged from the sucrose density gradients. Isolation of ribosomes. In order to isolate the ribosomes the chloroplasts of 400 plants were lysed for 15 min in 12 ml of MEA buffer containing 5 % (v/v) of Triton X-100. During this period the tempera&r& was allowed to increase slowly to about 10°C. Later on all steps were performed at 4°C. Chloroplast membranes were removed bv centrifugation for 15 min at 15 000 g. The resilting supernatant was centrifuged for 2 h at 140000 g. Five such pellets corresponding to 2 000 plants were pooled, homogenized in 12 ml of MEA buffer and the ribosomes recentrifuged as described above. The ribosomal material was resusnended in 0.4 ml of gradient buffer of appropriate M@ concentration and stored in an ice bath over night. The gradient buffer contained 0.1 moles KCI, 0.01 moles Tris per liter and varying concentrations of M@+ and was adiusted to nH 7.4. The resulting suspe&ion was centiifuged S-min at 6 000 g and the clarified supernatant layered upon isokinetic gradients. Gradient centrz’fugation. The gradients (13 ml) were prepared in a system using a closed mixing chamber of 10.5 ml filled with 5 % (w/w) sucrose in gradient buffer while the storage vessel contained about 15 ml of 27 % (w/w) sucrose in the same buffer. In this gradient there was a linear relationship between migration distances and sedimentation coefficients for particles of a density of 1.4 g/cmS. The E. coli 50s ribosomal subunit and the 80 S rat liver ribosome respectively served as a reference. The gradients were centrifuged for 150 min at 40 000 rpm in an SW40 rotor. Analysis of the gradients and calculation of the sedimentation coefficients was performed according to Dillard [4]. Formaldehyde gradients. For fixation the ribosomal nellets were dissolved in aradiem buffer (low2 M Mg2+) in which the Tris b;ffer had been ieplaced bv triethanolamine-HCl buffer. The fixation was performed by the addition of one part of 20 % freshly Exptl Cell Res 80 (1973)
prepared and neutralized formaldehyde to 4 parts of ribosome solution. After 15 min at 2°C the ribosomes were layered on an exponential sucrose density gradient which contained 4 % formaldehyde and again triethanolamine instead of Tris buffer. In these experiments the sedimentation coefficients have been corrected to allow for the higher viscosity due to the formaldehyde concentration in the gradients [7].
RESULTS The sedimentation pattern of the ribosomes isolated from the chloroplasts of A. mediterranea in th? presence of 2 x 1O-3 M Mg2’ is shown in fig. 1. Peaks were found in the 7OS, 56S, 44S, and 30 S regions. The proportions of the peaks differed somewhat from preparation to preparation, especially the 56s peak varied by comparison with the dominant 44s region. The reason for this variation cannot be explained at the moment. The yield in a number of experiments was about 100 ,ug of ribosomal material from 400 plants using the assumption that 1 mg of ribosomes corresponded to 15 absorbance units at 260 nm and 1 cm path length. 50s
E.coli
I
44s
Fin. 1. -, ribosomes fixed with 5 % formaldehyde; ...r... unfixed control. Sedimentation of chloroplast ribosomes of A. mediterranea fixed with formaldehyde. Each orofile represents ribosomes isolated from 400 plants. The sedimentation coefficients of the fixed ribosomes have been corrected proportionate to the reduced sedimentation of fixed E. coli 50s subunits, when compared with the control.
Nuclear genome and chloroplast ribosomal proteins. I
65
The conclusion is supported by data from experiments in which the ribosomal pellet from chloroplasts was treated with 0.1 ,ug of pancreatic ribonuclease at 0°C for 10 min before the gradient centrifugation. Under 50s E.coli these conditions the yield of 70s particles was increased (fig. 3) at the expense of “LJL polyribosomes which up to the pentamer 50s E.coli are displayed in the sedimentation pattern ‘A under appropriate conditions (fig. 3). The conversion of polysomes into 70s monosomes under the influence of RNase indicates that 70s ribosomes are constituents of the polysomes and that in the chloroplasts as in other protein synthesizing systems the polysomes are the functional units involved in protein synthesis. Fig. 2. Chloroplast ribosomes of A. mediterranea at The evidence for the conclusion that the concentrations. A, lo-” M Mg2+; different Mg2+ B, 2 x 10-a M Mg2+;C, 5 x 1O-4M Mg2+; D, 2 x 1O-4 705 particles represent the chloroplast monoM Mgz+. Each graph representsribosomesof 400 ribosomes is enhanced by the following excells.The isokineticgradients(13 ml) contain from 5 to 27% sucrosein gradient buffer, Mg2+ as inperiment (fig. 4): dicated. Centrifugation was performed at 40 000 rpm The 70s particles were isolated in the for 150 min in an SW40 rotor. The meniscus of the gradients is marked by the line at the right. presence of 1O-2 M Mg2+, collected by centrifugation and subjected to a second Formaldehyde-fixed ribosomal particles gradient centrifugation in the presence of sedimented with the same sedimentation coefficients as did the unfixed ribosomal particles. The proportion of 70s particles to 44s particles in the formaldehyde gradient did not vary when compared with the unfixed control (fig. 1). In order to characterize the chloroplast monoribosomes as well as the subunits of these particles sedimentation patterns at different Mg2+ concentrations were compared (fig. 2A, B, C, 0). It is evident that in the presence of 100 mM KCl, even at a conFig. 3. -, untreated sample; . ...... sample treated centration of 2 x 1O-3 M Mg2+, the 70 S with 0.1 pg/ml of pancreatic ribonuclease for 10 min tl particle had almost completely disappeared at 2°C prior to centrifugation. Polyribosomes, ribosomes, and ribosomal particles (fig. 2B), whereas other particles, especially isolated from the chloroplasts of A. mediterranea. in the 56s region, remained intact down to a The ribosomal pellet from 400 cells was suspended in 0.2 ml of a buffer containing 0.1 M KCI; 0.05 M concentration of 2 x 1O-4M Mg2+ (fig. 2 C, 0). Tris-HCl, pH 7.4; 0.01 M Mg (CH,COO), and layered This experiment suggeststhat the chloro- on 5 ml linear gradients from 0.3 to 1.3 M sucrose in the same buffer. Centrifugation was performed in plast monoribosome sediments with 70s. an SW 39 rotor at 35 000 rpm for 75 min Exptl Cell Res 80 (1973)
66
K. Kloppstech & H. G. Schweiger 50s
E.coli
50s
E.coli
50s
E.coli
(44s
Fig. 4. Dissociation products of 70s ribosomal particles of the chloroplasts of Acetabuluria. The particles corresponding to the 70 S region of a 1O-2 M Mg2+ gradient (fig. 2A) were collected by centrifugation (2 h at 50 000 rpm in an SW50.1 rotor), suspended in gradient buffer with 2 x lO-s M Mge+, and separated again on an isokinetic gradient as described under Methods. The profile corresponds to about 20 pg of ribosomes.
2 x 1O-3 M Mg2+. Under these conditions the main portion of the material sedimented at positions corresponding to 44s and 30s. These particles are regarded as the large and the small subunits of the monoribosome respectively. Similar conclusions can be drawn from uridine incorporation experiments in which the 44 S and 30s particles were labelled more rapidly than the 56s and 70s particles. The significance of the 56s particle thus far remains unclear. Its nature will be discussed in detail later on. The sedimentation patterns of chloroplast ribosomes from five species of Acetabularia, namely from A. mediterranea, A. calyculus, Acicularia schenckii, A. crenulata, and A. cliftonii were compared. The patterns from A. crenulata and A. cliftonii together with A. mediterranea are compared in fig. 5. No significant differences could be detected between these species. Although we were not able to detect substantial amounts of microorganisms on the purified plants the possibility that the ribosomal material might have derived from microorganisms bound to the cell wall was excluded in the following way. Axenic cells of A. mediterranea were Exptl Cell Res 80 (1973)
50s
E.coli
144s
Fig. 5. Ribosomal particles isolated from the chloroplasts of Acetabularia. A, A. mediferranea: B, A. crenulata; C, A. (Polyphysu) cliftonii. The 140000 g pellet of a 15 000 g chloroplast lysate supematant from 400 plants (about 100 pg of ribosomal material) was suspended in gradient buffer with 2 x 10” M Mg and centrifuged over 13 ml isokinetic gradients as described under Methods.
incubated in the presence of 14C02 for 7 days and the chloroplasts of these plants isolated together with those of 400 unlabelled cells grown under non-axenic conditions. After sucrose density gradient centrifugation the radioactivity coincided with that of the UV absorption curve. In contrast to the axenic cells non axenic plants which had been incubated with 14C02 exhibited substantial incorporation in the 50s region besides the 44s peak but there was no peak in the UV absorption profile. From this result it can be concluded that ribosomes from microorganisms did not interfere with our unlabelled ribosomal preparations to any significant extent.
Nuclear genome and chloroplast ribosomal proteins. I DISCUSSION In this paper the ribosomal particles of the chloroplasts were investigated in 5 species of Acetabularia by means of isokinetic sucrose gradient centrifugation technique. Four classes of particles were found which sedimented with 7OS, 56S, 44S, and 30s. Very similar sedimentation coefficients were obtained after fixing the ribosomal particles with formaldehyde and sedimenting on sucrose gradients containing formaldehyde. From the formaldehyde experiments it is apparent that the S values for the different particles are not influenced by the gradient centrifugation step for instance by hydrostatic pressure
Ef31. In an earlier paper ribosomal particles of whole Acetabuluriu cells had been attributed to material sedimenting with 82S, 65 S, and 48s by linear gradient centrifugation [9]. In a more recent paper these values had been corrected to 7OS, 5OS, and 30s [IO]. Based on the results with different Mg2+ concentrations and on the effect of gentle RNase treatment the 70s particles are considered to be the monoribosomes which dissociate into 44s and 30s subunits at about 2 x lO-3 M Mg2+. The difference in the sedimentation coefficient of the heavy subunit as estimated by Janowski et al. [lo] and in our laboratory might be explained by difficulties in calibrating linear gradients for the determination of sedimentation coefficients. The substantially higher percentage of 70s ribosomes in our ribosomal preparations as compared to an earlier report might be due to the addition of mercaptoethylamine to the isolation buffer [16]. Another point worth mentioning is the fact that we succeeded in increasing the yield of ribosomal particles from a given number of Acetabzduriu cells by a factor of
61
about 50. This might be due to the large volume of buffer in which the plants were homogenized and to the composition of the buffer as could be shown in control experiments. Furthermore, the volume of the buffer which was used for lysing a given amount of chloroplasts turned out to be critical for the completeness of the lysis. Up to now the nature of the 56s particle, which has not been described earlier, is not fully understood. Its electrophoretic protein pattern [13] as well as the sedimentation behaviour of its RNA [14] seems to be identical with that of the 70s ribosomes. A remarkable property of the 56s particle is that it is stable down to a M@+ concentration of 2 x 1O-4 M. The possibility that the 56s particles originate from 70s ribosomes by dissociation is excluded by the fact that the dissociation products of the 70s particles are the 44 S and 30 S subunits. Preliminary experiments indicate that a high yield of 56s particles is obtained from plants which are growing under unfavorable conditions. These results suggest that the 56s particle consists of a partially unfolded 70s particle. It might be interesting to test these particles for their activity in protein synthesis. We thank Dr Sigrid Berger for electron microscopical control of the chloroplast preparations and Mrs H. Stelzer for excellent technical assistance.
REFERENCES 1. Anderson, L E & Levin, D A, Plant physiol 46 (1970) 819. 2. Apel, K & Schweiger, H G, Eur j biochem 25 (1972) 229. 3. Berger, S, Doctoral thesis, University of Cologne (1967). 4. Dillard, W, cited by Rahmsdorf, J, Doctoral thesis, Freie UniversitLt Berlin (1970). 5. Gibor, A & Izawa, M, Proc natl acad sci US 50 (1963) 1164. 6. HPmmerling, J, Ann rev plant physiol 14 (1963) 65. 7. Huxley, H E & Zubay, G, J mol biol 2 (1960) 10. Exptl Cell Res 80 (1973)
68 K. Kloppstech & H. G. Schweiger 8. Infante, A A & Baierlein, R, Proc natl acad sci US 68 (1971) 1780. 9. Janowski, M, Life sci 5 (1966) 2113. 10. Janowski, M, Bonotto, S & Boloukhere, M, Biochim biophys acta 174 (1969) 525. 11. Jensen, R G & Bassham, J A, Proc natl acad sci US 56 (1966) 1095. 12. Kawashima, N & Wildman, S G, Biochim biophys acta 262 (1972) 42. 13. Kloppstech, K & Schweiger, H G, Exptl cell res 80 (1973) 69. 14. Kloppstech, K. Unpublished results. 15. Kung, S D, Thomber, J P & Wildman, S G, FEBS letters 24 (1972) 185. 16. Mets, L J & Bogorad, L, Science 174 (1971) 707. 17. Reuter, W & Schweiger, H G, Protoplasma 68 (1969) 357.
Exptl Cell Res 80 (1973
18. Schweiger. H G & Berger. S. Biochim bionhvs _ _ acta 87-(1964) 533. - ’ ’ 19. Schweiger, H G, Master, R W P & Werz, G, Nature 216 (1967) 554. 20. Schweiger. H G, Curr top microbial immunol 50 (1969) i. . 21. Scott, N S, Munns, R, Graham, D & Smillie, R M, Autonomy and biogenesis of mitochondria and chloroplasts (ed N K Boardman, A W Linnane & R M Smillie) p. 383. North-Holland, Amsterdam, London (1971). 22. Weil, J H. Personal communication.
Received December 12, 1972
0
of
1973 by Academic Press, Inc. reproduction in any form reseroed
Experimental Cell Research 80 (1973) 63-68
NUCLEAR
GENOME CODES FOR CHLOROPLAST
RIBOSOMAL I. Isolation
PROTEINS IN ACETABULARIA
and Characterization
of Chloroplast
Ribosomal Particles
K. KLOPPSTECH and H. G. SCHWEIGER Max-Planck-lnstitut
fiir Zellbiologie,
D 2940 Wilhelmshaven,
BRD
SUMMARY A method is described for the isolation of chloroplast ribosomes from Acetabuluria cells in yields sufficient for the characterization of these particles. Ribosomal particles sedimenting with 7OS, 56S, 44S, and 30s have been obtained. The monoribosome sediments with 70s and dissociates into a larger 44s and a smaller 30s subunit. The sedimentation behaviour of the particles as well as the equilibrium between monoribosomes and their subunits is not influenced by the centrifugation step as could be revealed by formaldehyde fixation.
Although chloroplasts contain DNA and dispose their own genetic information it has not been possible so far to prove that any of the chloroplast proteins are coded by the chloroplast genome. Nuclear dependence of chloroplast protein constituents has been demonstrated for a number of chloroplast proteins such as malic dehydrogenase [19], lactic dehydrogenase [17], aldolase [l], and a number of membrane proteins [2, 12, 151. Similar conclusions have been drawn from genetic
experiments
on
chloroplast
ribo-
somal proteins [16]. The only established role of chloroplast DNA is the coding of chloroplast ribosomal [18, 211 and transfer RNA [22]. From this point of view, it was quite interesting to investigate the problem of where the genetic information for the chloroplast ribosomal proteins comes from by means of the nucleus transplantation technique in Acetabularia. In order to approach this problem it was 5-731803
necessary to characterize the chloroplast ribosomes and standardize the isolation procedures. The techniques involved as well as the ribosome patterns of Acetabuluriu under different conditions are described and ribosome patterns of 5 species of Acetabularia are compared. An early attempt to establish the ribosomal pattern of A. mediterranea has been made by Janowski and co-workers [lo].
MATERIAL
AND
METHODS
Culture conditions. Cells of Acetabularia mediterranea, A. crenulata, A. calyculus, Acicularia schenckii, and A. (Polyphysa) cliftonii were grown in Erd-Schreiber
medium (ESM) under conditions described earlier 16, 201. Axenic cultures of A. mediterrunea were obtained in accordance with Gibor & Izawa [5] and Berger [3]. of chloroplasts. Four hundred to 1000 plants at the stage of initiation of cap formation were washed 3 times in ESM, cut into 5 mm pieces, and homogenized with a teflon pestle in 40 ml of MES buffer at 4°C. The MES buffer contained per 10.05 Isolation
Exptl Cell Res 80 (1973)
64 K. Kloppstech & H. G. Schweiger moles [2-(N-morpholino)ethane-sulfonic acid] adjusted to pH 6.1 with NaOH, 0.02 moles NaCi, 0.33 moles sorbitol, 0.002 moles NaNO,, 0.002 moles EDTA (dipotassium salt), 0.002 moles Na isoascorbate, 0.001 moles MnCl,, 0.001 moles MgCl,, and 0.0005 moles K,HPO, [ll]. The homogenate was passed through two layers of cheese cloth and centrifuged for 2 min at 100 g to remove membranous material. The supernatant was centrifuged for 15 min at 1 000 g, the sediment resuspended by gentle homogenization in 20 ml of MEA buffer and the chloroplasts sedimented by recentrifugation at 4°C. The MEA buffer contained, per liter, 0.1 moles KCI, 0.01 moles Tris buffer, 0.015 moles Mg2+ as acetate and 0.01 moles mercaptoethylamine and was adjusted to pH 7.4. The chloroplast preparation contained a contamination of one mitochondrion per 10 chloroplasts as shown by electronmicroscopic techniques. This percentage was by far too small to affect the results on the chloroplast ribosomes. Furthermore there was no indication for the presence of 80s ribosomes or for the corresponding RNA in the chloroplast preparation as judged from the sucrose density gradients. Isolation of ribosomes. In order to isolate the ribosomes the chloroplasts of 400 plants were lysed for 15 min in 12 ml of MEA buffer containing 5 % (v/v) of Triton X-100. During this period the tempera&r& was allowed to increase slowly to about 10°C. Later on all steps were performed at 4°C. Chloroplast membranes were removed bv centrifugation for 15 min at 15 000 g. The resilting supernatant was centrifuged for 2 h at 140000 g. Five such pellets corresponding to 2 000 plants were pooled, homogenized in 12 ml of MEA buffer and the ribosomes recentrifuged as described above. The ribosomal material was resusnended in 0.4 ml of gradient buffer of appropriate M@ concentration and stored in an ice bath over night. The gradient buffer contained 0.1 moles KCI, 0.01 moles Tris per liter and varying concentrations of M@+ and was adiusted to nH 7.4. The resulting suspe&ion was centiifuged S-min at 6 000 g and the clarified supernatant layered upon isokinetic gradients. Gradient centrz’fugation. The gradients (13 ml) were prepared in a system using a closed mixing chamber of 10.5 ml filled with 5 % (w/w) sucrose in gradient buffer while the storage vessel contained about 15 ml of 27 % (w/w) sucrose in the same buffer. In this gradient there was a linear relationship between migration distances and sedimentation coefficients for particles of a density of 1.4 g/cmS. The E. coli 50s ribosomal subunit and the 80 S rat liver ribosome respectively served as a reference. The gradients were centrifuged for 150 min at 40 000 rpm in an SW40 rotor. Analysis of the gradients and calculation of the sedimentation coefficients was performed according to Dillard [4]. Formaldehyde gradients. For fixation the ribosomal nellets were dissolved in aradiem buffer (low2 M Mg2+) in which the Tris b;ffer had been ieplaced bv triethanolamine-HCl buffer. The fixation was performed by the addition of one part of 20 % freshly Exptl Cell Res 80 (1973)
prepared and neutralized formaldehyde to 4 parts of ribosome solution. After 15 min at 2°C the ribosomes were layered on an exponential sucrose density gradient which contained 4 % formaldehyde and again triethanolamine instead of Tris buffer. In these experiments the sedimentation coefficients have been corrected to allow for the higher viscosity due to the formaldehyde concentration in the gradients [7].
RESULTS The sedimentation pattern of the ribosomes isolated from the chloroplasts of A. mediterranea in th? presence of 2 x 1O-3 M Mg2’ is shown in fig. 1. Peaks were found in the 7OS, 56S, 44S, and 30 S regions. The proportions of the peaks differed somewhat from preparation to preparation, especially the 56s peak varied by comparison with the dominant 44s region. The reason for this variation cannot be explained at the moment. The yield in a number of experiments was about 100 ,ug of ribosomal material from 400 plants using the assumption that 1 mg of ribosomes corresponded to 15 absorbance units at 260 nm and 1 cm path length. 50s
E.coli
I
44s
Fin. 1. -, ribosomes fixed with 5 % formaldehyde; ...r... unfixed control. Sedimentation of chloroplast ribosomes of A. mediterranea fixed with formaldehyde. Each orofile represents ribosomes isolated from 400 plants. The sedimentation coefficients of the fixed ribosomes have been corrected proportionate to the reduced sedimentation of fixed E. coli 50s subunits, when compared with the control.
Nuclear genome and chloroplast ribosomal proteins. I
65
The conclusion is supported by data from experiments in which the ribosomal pellet from chloroplasts was treated with 0.1 ,ug of pancreatic ribonuclease at 0°C for 10 min before the gradient centrifugation. Under 50s E.coli these conditions the yield of 70s particles was increased (fig. 3) at the expense of “LJL polyribosomes which up to the pentamer 50s E.coli are displayed in the sedimentation pattern ‘A under appropriate conditions (fig. 3). The conversion of polysomes into 70s monosomes under the influence of RNase indicates that 70s ribosomes are constituents of the polysomes and that in the chloroplasts as in other protein synthesizing systems the polysomes are the functional units involved in protein synthesis. Fig. 2. Chloroplast ribosomes of A. mediterranea at The evidence for the conclusion that the concentrations. A, lo-” M Mg2+; different Mg2+ B, 2 x 10-a M Mg2+;C, 5 x 1O-4M Mg2+; D, 2 x 1O-4 705 particles represent the chloroplast monoM Mgz+. Each graph representsribosomesof 400 ribosomes is enhanced by the following excells.The isokineticgradients(13 ml) contain from 5 to 27% sucrosein gradient buffer, Mg2+ as inperiment (fig. 4): dicated. Centrifugation was performed at 40 000 rpm The 70s particles were isolated in the for 150 min in an SW40 rotor. The meniscus of the gradients is marked by the line at the right. presence of 1O-2 M Mg2+, collected by centrifugation and subjected to a second Formaldehyde-fixed ribosomal particles gradient centrifugation in the presence of sedimented with the same sedimentation coefficients as did the unfixed ribosomal particles. The proportion of 70s particles to 44s particles in the formaldehyde gradient did not vary when compared with the unfixed control (fig. 1). In order to characterize the chloroplast monoribosomes as well as the subunits of these particles sedimentation patterns at different Mg2+ concentrations were compared (fig. 2A, B, C, 0). It is evident that in the presence of 100 mM KCl, even at a conFig. 3. -, untreated sample; . ...... sample treated centration of 2 x 1O-3 M Mg2+, the 70 S with 0.1 pg/ml of pancreatic ribonuclease for 10 min tl particle had almost completely disappeared at 2°C prior to centrifugation. Polyribosomes, ribosomes, and ribosomal particles (fig. 2B), whereas other particles, especially isolated from the chloroplasts of A. mediterranea. in the 56s region, remained intact down to a The ribosomal pellet from 400 cells was suspended in 0.2 ml of a buffer containing 0.1 M KCI; 0.05 M concentration of 2 x 1O-4M Mg2+ (fig. 2 C, 0). Tris-HCl, pH 7.4; 0.01 M Mg (CH,COO), and layered This experiment suggeststhat the chloro- on 5 ml linear gradients from 0.3 to 1.3 M sucrose in the same buffer. Centrifugation was performed in plast monoribosome sediments with 70s. an SW 39 rotor at 35 000 rpm for 75 min Exptl Cell Res 80 (1973)
66
K. Kloppstech & H. G. Schweiger 50s
E.coli
50s
E.coli
50s
E.coli
(44s
Fig. 4. Dissociation products of 70s ribosomal particles of the chloroplasts of Acetabuluria. The particles corresponding to the 70 S region of a 1O-2 M Mg2+ gradient (fig. 2A) were collected by centrifugation (2 h at 50 000 rpm in an SW50.1 rotor), suspended in gradient buffer with 2 x lO-s M Mge+, and separated again on an isokinetic gradient as described under Methods. The profile corresponds to about 20 pg of ribosomes.
2 x 1O-3 M Mg2+. Under these conditions the main portion of the material sedimented at positions corresponding to 44s and 30s. These particles are regarded as the large and the small subunits of the monoribosome respectively. Similar conclusions can be drawn from uridine incorporation experiments in which the 44 S and 30s particles were labelled more rapidly than the 56s and 70s particles. The significance of the 56s particle thus far remains unclear. Its nature will be discussed in detail later on. The sedimentation patterns of chloroplast ribosomes from five species of Acetabularia, namely from A. mediterranea, A. calyculus, Acicularia schenckii, A. crenulata, and A. cliftonii were compared. The patterns from A. crenulata and A. cliftonii together with A. mediterranea are compared in fig. 5. No significant differences could be detected between these species. Although we were not able to detect substantial amounts of microorganisms on the purified plants the possibility that the ribosomal material might have derived from microorganisms bound to the cell wall was excluded in the following way. Axenic cells of A. mediterranea were Exptl Cell Res 80 (1973)
50s
E.coli
144s
Fig. 5. Ribosomal particles isolated from the chloroplasts of Acetabularia. A, A. mediferranea: B, A. crenulata; C, A. (Polyphysu) cliftonii. The 140000 g pellet of a 15 000 g chloroplast lysate supematant from 400 plants (about 100 pg of ribosomal material) was suspended in gradient buffer with 2 x 10” M Mg and centrifuged over 13 ml isokinetic gradients as described under Methods.
incubated in the presence of 14C02 for 7 days and the chloroplasts of these plants isolated together with those of 400 unlabelled cells grown under non-axenic conditions. After sucrose density gradient centrifugation the radioactivity coincided with that of the UV absorption curve. In contrast to the axenic cells non axenic plants which had been incubated with 14C02 exhibited substantial incorporation in the 50s region besides the 44s peak but there was no peak in the UV absorption profile. From this result it can be concluded that ribosomes from microorganisms did not interfere with our unlabelled ribosomal preparations to any significant extent.
Nuclear genome and chloroplast ribosomal proteins. I DISCUSSION In this paper the ribosomal particles of the chloroplasts were investigated in 5 species of Acetabularia by means of isokinetic sucrose gradient centrifugation technique. Four classes of particles were found which sedimented with 7OS, 56S, 44S, and 30s. Very similar sedimentation coefficients were obtained after fixing the ribosomal particles with formaldehyde and sedimenting on sucrose gradients containing formaldehyde. From the formaldehyde experiments it is apparent that the S values for the different particles are not influenced by the gradient centrifugation step for instance by hydrostatic pressure
Ef31. In an earlier paper ribosomal particles of whole Acetabuluriu cells had been attributed to material sedimenting with 82S, 65 S, and 48s by linear gradient centrifugation [9]. In a more recent paper these values had been corrected to 7OS, 5OS, and 30s [IO]. Based on the results with different Mg2+ concentrations and on the effect of gentle RNase treatment the 70s particles are considered to be the monoribosomes which dissociate into 44s and 30s subunits at about 2 x lO-3 M Mg2+. The difference in the sedimentation coefficient of the heavy subunit as estimated by Janowski et al. [lo] and in our laboratory might be explained by difficulties in calibrating linear gradients for the determination of sedimentation coefficients. The substantially higher percentage of 70s ribosomes in our ribosomal preparations as compared to an earlier report might be due to the addition of mercaptoethylamine to the isolation buffer [16]. Another point worth mentioning is the fact that we succeeded in increasing the yield of ribosomal particles from a given number of Acetabzduriu cells by a factor of
61
about 50. This might be due to the large volume of buffer in which the plants were homogenized and to the composition of the buffer as could be shown in control experiments. Furthermore, the volume of the buffer which was used for lysing a given amount of chloroplasts turned out to be critical for the completeness of the lysis. Up to now the nature of the 56s particle, which has not been described earlier, is not fully understood. Its electrophoretic protein pattern [13] as well as the sedimentation behaviour of its RNA [14] seems to be identical with that of the 70s ribosomes. A remarkable property of the 56s particle is that it is stable down to a M@+ concentration of 2 x 1O-4 M. The possibility that the 56s particles originate from 70s ribosomes by dissociation is excluded by the fact that the dissociation products of the 70s particles are the 44 S and 30 S subunits. Preliminary experiments indicate that a high yield of 56s particles is obtained from plants which are growing under unfavorable conditions. These results suggest that the 56s particle consists of a partially unfolded 70s particle. It might be interesting to test these particles for their activity in protein synthesis. We thank Dr Sigrid Berger for electron microscopical control of the chloroplast preparations and Mrs H. Stelzer for excellent technical assistance.
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68 K. Kloppstech & H. G. Schweiger 8. Infante, A A & Baierlein, R, Proc natl acad sci US 68 (1971) 1780. 9. Janowski, M, Life sci 5 (1966) 2113. 10. Janowski, M, Bonotto, S & Boloukhere, M, Biochim biophys acta 174 (1969) 525. 11. Jensen, R G & Bassham, J A, Proc natl acad sci US 56 (1966) 1095. 12. Kawashima, N & Wildman, S G, Biochim biophys acta 262 (1972) 42. 13. Kloppstech, K & Schweiger, H G, Exptl cell res 80 (1973) 69. 14. Kloppstech, K. Unpublished results. 15. Kung, S D, Thomber, J P & Wildman, S G, FEBS letters 24 (1972) 185. 16. Mets, L J & Bogorad, L, Science 174 (1971) 707. 17. Reuter, W & Schweiger, H G, Protoplasma 68 (1969) 357.
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18. Schweiger. H G & Berger. S. Biochim bionhvs _ _ acta 87-(1964) 533. - ’ ’ 19. Schweiger, H G, Master, R W P & Werz, G, Nature 216 (1967) 554. 20. Schweiger. H G, Curr top microbial immunol 50 (1969) i. . 21. Scott, N S, Munns, R, Graham, D & Smillie, R M, Autonomy and biogenesis of mitochondria and chloroplasts (ed N K Boardman, A W Linnane & R M Smillie) p. 383. North-Holland, Amsterdam, London (1971). 22. Weil, J H. Personal communication.
Received December 12, 1972