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Isolation and characterization of polyribosomes and non-ribosomal ribonucleoprotein particles from the duckweed Spirodela oligorrhiza
Plant Science Letters, 25 (1982) 337--344 Elsevier/North-Holland Scientific Publishers Ltd.
337
ISOLATION AND CHARACTERIZATION OF POLYRIBOSOMES AND NON-RIBOSOMAL RIBONUCLEOPROTEIN PARTICLES FROM THE DUCKWEED SPIR ODELA OLIGORRHIZA
J A N H. V A N E E and R U D I J. P L A N T A Biochemisch Laboratorium der Vrije Universiteit, de Boelelaan 1083, 1081 H V A msterda~ (The Netherlands)
(Received July 8th, 1981) (Revision received December 7th, 1981) (Accepted December 7th, 1981)
SUMMARY
Polyribosomes and non-ribosomal ribonucleoprotein (RNP) particles were isolated from the duckweed Spirodela oligorrhiza. Cytoplasmic polyribosomes from light-grown plants are generally larger than cytoplasmic polyribosomes from dark-grown plants. Non-ribosomal RNP particles from both light-grown and dark-grown plants sediment at about 25 S and have a buoyant density in CsC1 of 1.39 g/cm 3. They show a relative stability in high-salt solutions, do not contain rRNA and show large differences in protein content and composition as compared with ribosomes.
INTRODUCTION
The availability of an isolation procedure for polyribosomes and nonribosomal RNP particles from the duckweed Spirodela oligorrhiza would permit comparative studies on the protein-synthesizing capacity of this plant at different developmental stages. Usually, free and membrane-bound polyribosomes can be isolated rather easily from higher plants, using procedures of the type described by Larkins and coworkers [ 1--5]. Spirodela polysomes, however, could not be isolated in this way, nor by other methods described [6,7], most probably due to tissue characteristics like for instance a high ribonuclease activity and to run off. Therefore, we have developed another isolation procedure, in which we inhibited the RNase activity by adding spermine, spermidine and polyvinylsulphate (PVS) [28] and prevented run off by adding cycloheximide. Abbreviations: Brij 58, polyethyleneglycol monostearylether; DTT, dithiotreitol;PVS, polyvinylsulphate ;RNP, ribonucleoprotein; SDS, sodium dodecyl sulphate. 0304--4211/82/0000--0000/$02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
338 In this paper we describe the isolation and characterization of polyribosomes and non-ribosomal RNP particles from light-grown and dark-grown Spirodela oligorrhiza. MATERIALS AND METHODS
Growth conditions Axenic Spirodela oligorrhiza (duckweed) plantlets were grown on Hutners medium [8] for 10 days at 25°C and 1800 lux of continuous fluorescent light. Dark-grown plants were grown under similar conditions but in the absence of light. Isolation of polyribosomes Plants were collected and washed twice with sterile redistilled water. Approx. 100 g of plants were homogenized at 4°C with a Waxing Blendor, during 3 × 10 s at maximal speed in 3--4 vol. of homogenization buffer containing: 200 mM Tris--HC1 (pH 8.5), 400 mM sucrose, 20 mM KC1, 5 mM MgC12, 0.25 mM spermine, 0.375 mM spermidine, 0.5 mM dithiotreitol (DTT), 5 mM ~-mercaptoethanol, 50 ~g/ml PVS and 100 ~g/ml cycloheximide. The homogenate was filtered through four layers of a 30-pro pore size nylon gauze and subsequently centrifuged for 10 min at 1200 × g to remove plant debris and chloroplasts. The supernatant was recentrffuged during 30 min at 18 000 rev./min and 4°C in a Beckman type 45 Ti rotor. The supernatant was layered upon a 7-ml sucrose cushion (1 M sucrose, 40 mM Tris--HC1 (pH 8.5), 20 mM KC1, 5 mM MgC12) and centrifuged for 70 rain at 60 000 rev./min and 4°C in a Beckman type 60 Ti rotor. The pelleted free cytoplasmic polyribosomes were stored until analysis at -70°C. Membrane-bound polyribosomes were extracted from the 45 Ti-pellet after resuspension in a small volume of homogenization buffer without sucrose, containing 5% (v/v) Triton X-100, which liberates membranebound polyribosomes [7,29] and incubation for at least 2 h at 4°C under gentle shaking. The suspension was centrifuged for 30 min at 18 000 rev./ rain at 4°C in a Beckman type 45 Ti rotor. Then the supernatant was carefully layered upon a 1-ml sucrose cushion and centrifuged during 70 min at 68 000 rev./min and 4°C in a Beckman type 65 rotor. The pelleted membrane-bound poIyribosomes were stored until analysis at -70°C. Isola tion of non-ribosomal RNP particles RNP particles were isolated from the cytoplasmic polyribosomal supernatant (described above) by centrifugation for 4 h at 60 000 rev./min and 4°C in a Beckman type 60 Ti rotor. The RNP particles were purified from the pellet by recentrifugation through 5--20% (w/v) sucrose gradients in 40 mM Tris--HC1 (pH 8.5), 20 mM KC1 and 5 mM MgC12, during 22 h at 27 000 rev./min and 4°C in a Beckman SW 27 rotor. The fractions containing the RNP particles were pooled and recentrifuged. The final pellets were stored until analysis at -70°C.
339
Sucrose gradient analysis Polyribosomes were resuspended in a small volume of 40 mM Tris--HC1 (pH 8.5), 20 mM KC1 and 5 mM MgC12. The suspension was clarified by centrifugation for 5 min at 3000 × g and subsequently centrifuged through 10--40% (w/v) sucrose gradients in 40 mM Tris--HC1 (pH 8.5), 20 mM KC1 and 5 mM MgC12 for 1 h at 40 000 rev./min and 4°C in a Beckman SW 41 rotor. Finally the gradients were analyzed by continuously recording the A260 nm. The S-values were inferred from the position of the peaks in the gradient, relative to that of the 80 S monoribosomes from yeast.
Buoyant density determinations Polyribosomes or RNP particles were resuspended in a small volume of sterile redistilled water and diluted with an equal volume of a solution containing 10% (v/v) formaldehyde, 40 mM triethanolamine (pH 7.8), 100 mM KC1, 2 mM MgC12and 1.0% (w/v) polyethyleneglycol monostearylether (Brij 58). After storage for at least 24 h at 4°C, CsC1 was added till N ~ = 1.3700. Subsequently, the suspension was centrifuged for 50 h at 38 000 rev./min and 20°C in a Beckman type 50 rotor. Finally the A260 nm as well as the refractive index was read against the fraction number. The buoyant density was calculated from the refractive index according to Ifft et al. [9].
RNA extraction and agarose gel electrophoresis Polyribosomal and RNP-RNA was extracted by suspending polysomes or RNP particles in a buffer containing 100 mM Tris--H3BO3 (pH 8.5), 2% (w/v) NaC1 and 2% (w/v) triisopropylnaphtalene sulphonate. Deproteinisation was carried out twice at 65°C by extraction with an equal volume of a 1 : 1 mixture phenol solution (500 g phenol, 0.5 g 8-hydroxyquinoline, 70 ml m-cresol, 150 m1,50 mM Tris--HC1 (pH 8.5)) and chloroform. The nucleic acids were precipitated overnight with 2 vol. of ethanol at 4°C and reprecipitated with ethanol at -20°C, after dissolving the precipitate in 0.15 M sodium acetate (pH 6.0), 0.5% sodium dodecyl sulphate (SDS). Subsequently, the RNA fractions were analyzed on 1.6% agarose gels, according to van Ommen et al. [10].
Analysis of polyribosomal and RNP-proteins Polyribosomal and RNP particles were denatured in SDS and/3-mercaptoethanol, essentially as described by Lindberg and Sundquist [11] and subsequently subjected to SDS polyacrylamide gradient gel electrophoresis as described by Laemmli [ 12]. RESULTS
Sedimentation profiles of polyribosomes and RNP particles As shown in Fig. 1 Spirodela oligorrhiza cytoplasmic polyribosomes can be separated in sucrose gradients in distinct size classes. Free polyribosomes
340
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Fig. 1. Sedimentation profiles obtained after centrifugation of cytoplasmic polyribosomes (about 2.0 A=60 nm units in all cases) through 10--40% sucrose gradients. A: free cytoplasmic polyribosomes from light-grown plants. B: membrane-bound polyribosomes from light-grown plants. C: free cytoplasmic polyribosomes from clark-grown plants. D: membrane-bound polyribosomes from dark-grown plants. The horizontal arrow indicates the direction of sedimentation.
from light-grown plants have sizes up to octamers (Fig. 1A), while membrane-bound polysomes are present up to heptamers (Fig. 1B). The respective polysome populations from dark-grown plants seem to be significantly smaller, with pentamers (Fig. 1C) and trimers (Fig. 1D) as largest polysomes. Incubation of the polysomal structures in the presence of EDTA causes an almost complete dissociation into 40 S and 60 S ribosomal subunits (Fig. 2). Treatment with pancreatic RNase (substrate/enzyme, 20 : 1 (w/w) in 10 mM Tris--HCl (pH 7.6) for 0.5 h at 37°C) reduces the maximal size from octamers to trimers, while the amount of monosomes (80 S) is greatly increased (Fig. 2). These results clearly demonstrate that the rapid-sedimenting structures are polysomes rather than ribosomal aggregates. Figure 3 shows the sedimentation behaviour of RNP particles from both light-grown and dark-grown Spirodela plants. These particles sediment with a sedimentation coefficient of approx. 25 S, as was calculated from their position in the sucrose gradient according to the method of McEwen [ 30].
341 06,
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, =
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2 4 6 8 1012 !41618 20 22 2~12628 30 32 34 fraction number
* ~ •) on cytoplasmic Fig. 2. The effect of EDTA ( . . . . . ) and pancreatic RNAase ( ). The horizontal arrow indicates the direction of sedimentation. polyribosomes (
Buoyant density of polyribosomes and RNP particles Both polyribosomes and RNP particles were fixed with formaldehyde and centrifuged in CsCl
o
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/
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Fig. 3. Sedimentation of non-ribosomal R N P particles from either light-grown or darkgrown plants through 5-20% sucrose gradients.
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Fig. 4. Absorbance scans of RNP particles and polyribosomes obtained after centrifugation in density gradients. A: formaldehyde-fixed ribonucleoprotein particles in CsCI. B: unfixed RNP particles in Cs=SO,. C: formaldehyde-fixed polyribosomes in CsCI. Fig. 5. 1.6% agarose gel electrophoresis of polyribosomal-RNA and RNP-RNA from light-grown plants relative to 25 S, 23 S, 18 S and 16 S ribosomal RNA. (1) Membranebound polyribosomal RNA (0.8 ug); (2) free cytoplasmic polyribosomal RNA (2.0 ug); (3) RNP-RNA (0.5 ~g).
density of the unfixed particles remains approx. 1.4 g/cm 3. Therefore, these particles cannot be considered as artificial RNA-protein complexes. Polyribosomes band at a density of 1.55 g/cm 3 (Fig. 4C), which is a characteristic of ribosomes [22--24] and to a much lower extent at 1.45 g/cm% which indicates the presence of some polyribosomal ribonucleoprotein particles [24]. R N A and protein components of polyribosomes and R N P particles
Figure 5 shows the resultsof a 1.6% agarose slab gel electrophoresisof R N A isolated from membrane-bound polyribosomes (lane 1), free polyribosomes (lane 2) and non-ribosomal R N P particles(lane 3) allfrom lightgrown plants.As can be seen,the R N A moiety of R N P particlesconsists of a heterogeneous population ranging in sizefrom 4 S and 16 S, which differs quite significantlyfrom the polyribosonml R N A components, that range from 4 S and 25 S. This observation suggests that the RNP particles represent
343 67.000 94.000 J
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B Fig. 6. Densitometric representation of an 8--20% SDS polyacrylamide gradient electrophoresis of RNPs (A) and polyribosomal proteins (B) from light-grown plants, relative to phosphorylase b (94 000), albumin (67 000), ovalbumin (43 000), carbonic anhydrase (30 000), trypsin inhibitor (20 200) and ~-lactalbumin (14 000).
a specific class of particles rather than artificial ribosomes or ribosomal subunits. This suggestion was confirmed by Fig. 6, which shows the absorbance scans of an 8--20% SDS polyacrylamide gradient gel electrophoresis of the proteins isolated from RNP particles and polyribosomes from light-grown plants. From the differences, which are most striking in the low molecular weight region, it was concluded that RNP particles do not contain ribosomal proteins and consequently cannot be the product of ribosome degradation. DISCUSSION
As compared with polyribosomes, isolated from other higher plants [1--7], all profiles of Spirodela oligorrhiza polysomes isolated by our procedure show a rather high percentage of monosomes and ribosomal subunits (Fig. 1 ). Although this still might be the result of some degradation and/or run off, it might also reflect the in vivo situation, since these types of sedimentation profiles have also been found for polyribosomes of Lemna gibba L.G-3, another member of the duckweed family [25]. Analysis of the postribosomal supernatant revealed the presence of RNP complexes, which sediment through sucrose gradients as a rather broad band with a mean sedimentation coefficient of 25 8. According to their buoyant density in CsC1 of 1.39 g/cm 3 (Fig. 4A), their relative stability in high salt solutions (Fig. 4B), the differences in RNA composition (Fig. 5) and the differences in protein composition as compared with that of ribosomes {Fig. 6), these complexes apparently represent the so called non-ribosomal ribonucleoprotein particles [ 13--20, 12, 26,27]. The availability of a good isolation procedure for Spirodela polysomes and RNP particles now permits
344 c o m p a r a t i v e studies o n t h e p r o t e i n - s y n t h e s i z i n g c a p a c i t y o f m R N A e x t r a c t e d from polyribosomes and RNP particles of duckweed plants at different d e v e l o p m e n t a l stages. ACKNOWLEDGEMENTS T h e a u t h o r s are v e r y g r a t e f u l t o Mr. R. Kuijer a n d Mr. H. Lustig f o r t h e i r assistance a n d t h e p e r f o r m a n c e o f s o m e o f t h e e x p e r i m e n t s . REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
B.A. Larkins and E. Davies, Plant Physiol., 52 (1973) 655. A.O. Jackson and B.A. Larkins, Plant Physiol., 57 (1976) 5. E. Davies, B.A. Larkins and R.H. Knight, Plant Physiol., 50 (1972) 581. E. Davies and B.A. Larkins, Plant Physiol., 52 (1973) 339. B.A. Larkins and E. Davies, Plant Physiol., 55 (1975) 749. C.J. Leaver and J.A. Dyer: Biochem, J., 144 (1974) 165. H. Smith, Eur. J. Biochem., 65 (1976) 161. H.S. Hutner, in: W.E. Loomis (Ed.), Growth and Differentiation in Plants, Iowa State College Press, Ames, Iowa, 1953. J.B. Ifft, D.H. Voet and J. Vinograd, J. Phys. Chem., 65 (1961) 1138. G.-J.B. van Ommen, P.H. Boer, G.S.P. Groot, M. de Haan, E. Roosendaal, L.A. Grivell, A. Haid and R.J. Schweyer, Cell, 20 (1980) 173. U. Lindberg and B. Sundquist, J. Mol. Biol., 86 (1974) 451. U.K. Laemmli, Nature, 227 (1970) 680. A.S. Spirin, in: L. Bosch (Ed.), The Mechanism of Protein Synthesis and its Regulation, Elsevier/North-Holland, Amsterdam, 1972, p. 515. A.S. Spirin, Mol. Biol. Rep., 5 (1979) 53. A.S. Spirin, Eur. J. Biochem., 10 (1969) 20. M.A. Ajtkhozkin, Kh. J. Doschanov and A.U. Akhanov, FEBS Lett., 66 (1976) 124. A. Ferret, M. Delseny and Y. Guitton, Plant Sci. Lett., 14 (1979) 31. H.V. Tan and G. Schapira, Eur. J. Biochem., 85 (1978) 271. D. Baltimore and A.S. Huang, J. Mol. Biol., 47 (1970) 263. V.L. Leytin, O.V. Podobed and M.I. Lerman, J. Mol. Biol., 51 (1970) 727. J.R. Greenberg, J. Mol. Biol., 108 (1977) 403. P. Cammarano, A. Romeo, M. Gentile, A. Felsani and C. Gualerzi, Biochim. Biophys. Acta, 281 (1972) 597. M.A. Ajtkhozkin, A.U. Akanov and Kh.J. Doschanov, FEBS Lett., 31 (1973) 104. A. Burny, G. Huez, G..Marbaix and H. Chantrenne, Biochim. Biophys. Acta, 190 (1969) 228. E.M. Tobin, Proc. Natl. Aead. Sei. U.S.A., 75 (1978) 4749. M.A. Ajtkhozkin and U.A. Akhapov, FEBS Lett., 41 (1974) 275. E. SiIverstein, Biochemistry, 12 (1973) 951. W.H. Mager, Ph.D. Thesis, Vri|e Universiteit, Amsterdam, 1976. C.J. Leaver and J.L. Key, J. Mol. Biol., 49 (1970) 671. C.R. McEwen, Anal. Biochem,, 20 (1967) 114.
337
ISOLATION AND CHARACTERIZATION OF POLYRIBOSOMES AND NON-RIBOSOMAL RIBONUCLEOPROTEIN PARTICLES FROM THE DUCKWEED SPIR ODELA OLIGORRHIZA
J A N H. V A N E E and R U D I J. P L A N T A Biochemisch Laboratorium der Vrije Universiteit, de Boelelaan 1083, 1081 H V A msterda~ (The Netherlands)
(Received July 8th, 1981) (Revision received December 7th, 1981) (Accepted December 7th, 1981)
SUMMARY
Polyribosomes and non-ribosomal ribonucleoprotein (RNP) particles were isolated from the duckweed Spirodela oligorrhiza. Cytoplasmic polyribosomes from light-grown plants are generally larger than cytoplasmic polyribosomes from dark-grown plants. Non-ribosomal RNP particles from both light-grown and dark-grown plants sediment at about 25 S and have a buoyant density in CsC1 of 1.39 g/cm 3. They show a relative stability in high-salt solutions, do not contain rRNA and show large differences in protein content and composition as compared with ribosomes.
INTRODUCTION
The availability of an isolation procedure for polyribosomes and nonribosomal RNP particles from the duckweed Spirodela oligorrhiza would permit comparative studies on the protein-synthesizing capacity of this plant at different developmental stages. Usually, free and membrane-bound polyribosomes can be isolated rather easily from higher plants, using procedures of the type described by Larkins and coworkers [ 1--5]. Spirodela polysomes, however, could not be isolated in this way, nor by other methods described [6,7], most probably due to tissue characteristics like for instance a high ribonuclease activity and to run off. Therefore, we have developed another isolation procedure, in which we inhibited the RNase activity by adding spermine, spermidine and polyvinylsulphate (PVS) [28] and prevented run off by adding cycloheximide. Abbreviations: Brij 58, polyethyleneglycol monostearylether; DTT, dithiotreitol;PVS, polyvinylsulphate ;RNP, ribonucleoprotein; SDS, sodium dodecyl sulphate. 0304--4211/82/0000--0000/$02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
338 In this paper we describe the isolation and characterization of polyribosomes and non-ribosomal RNP particles from light-grown and dark-grown Spirodela oligorrhiza. MATERIALS AND METHODS
Growth conditions Axenic Spirodela oligorrhiza (duckweed) plantlets were grown on Hutners medium [8] for 10 days at 25°C and 1800 lux of continuous fluorescent light. Dark-grown plants were grown under similar conditions but in the absence of light. Isolation of polyribosomes Plants were collected and washed twice with sterile redistilled water. Approx. 100 g of plants were homogenized at 4°C with a Waxing Blendor, during 3 × 10 s at maximal speed in 3--4 vol. of homogenization buffer containing: 200 mM Tris--HC1 (pH 8.5), 400 mM sucrose, 20 mM KC1, 5 mM MgC12, 0.25 mM spermine, 0.375 mM spermidine, 0.5 mM dithiotreitol (DTT), 5 mM ~-mercaptoethanol, 50 ~g/ml PVS and 100 ~g/ml cycloheximide. The homogenate was filtered through four layers of a 30-pro pore size nylon gauze and subsequently centrifuged for 10 min at 1200 × g to remove plant debris and chloroplasts. The supernatant was recentrffuged during 30 min at 18 000 rev./min and 4°C in a Beckman type 45 Ti rotor. The supernatant was layered upon a 7-ml sucrose cushion (1 M sucrose, 40 mM Tris--HC1 (pH 8.5), 20 mM KC1, 5 mM MgC12) and centrifuged for 70 rain at 60 000 rev./min and 4°C in a Beckman type 60 Ti rotor. The pelleted free cytoplasmic polyribosomes were stored until analysis at -70°C. Membrane-bound polyribosomes were extracted from the 45 Ti-pellet after resuspension in a small volume of homogenization buffer without sucrose, containing 5% (v/v) Triton X-100, which liberates membranebound polyribosomes [7,29] and incubation for at least 2 h at 4°C under gentle shaking. The suspension was centrifuged for 30 min at 18 000 rev./ rain at 4°C in a Beckman type 45 Ti rotor. Then the supernatant was carefully layered upon a 1-ml sucrose cushion and centrifuged during 70 min at 68 000 rev./min and 4°C in a Beckman type 65 rotor. The pelleted membrane-bound poIyribosomes were stored until analysis at -70°C. Isola tion of non-ribosomal RNP particles RNP particles were isolated from the cytoplasmic polyribosomal supernatant (described above) by centrifugation for 4 h at 60 000 rev./min and 4°C in a Beckman type 60 Ti rotor. The RNP particles were purified from the pellet by recentrifugation through 5--20% (w/v) sucrose gradients in 40 mM Tris--HC1 (pH 8.5), 20 mM KC1 and 5 mM MgC12, during 22 h at 27 000 rev./min and 4°C in a Beckman SW 27 rotor. The fractions containing the RNP particles were pooled and recentrifuged. The final pellets were stored until analysis at -70°C.
339
Sucrose gradient analysis Polyribosomes were resuspended in a small volume of 40 mM Tris--HC1 (pH 8.5), 20 mM KC1 and 5 mM MgC12. The suspension was clarified by centrifugation for 5 min at 3000 × g and subsequently centrifuged through 10--40% (w/v) sucrose gradients in 40 mM Tris--HC1 (pH 8.5), 20 mM KC1 and 5 mM MgC12 for 1 h at 40 000 rev./min and 4°C in a Beckman SW 41 rotor. Finally the gradients were analyzed by continuously recording the A260 nm. The S-values were inferred from the position of the peaks in the gradient, relative to that of the 80 S monoribosomes from yeast.
Buoyant density determinations Polyribosomes or RNP particles were resuspended in a small volume of sterile redistilled water and diluted with an equal volume of a solution containing 10% (v/v) formaldehyde, 40 mM triethanolamine (pH 7.8), 100 mM KC1, 2 mM MgC12and 1.0% (w/v) polyethyleneglycol monostearylether (Brij 58). After storage for at least 24 h at 4°C, CsC1 was added till N ~ = 1.3700. Subsequently, the suspension was centrifuged for 50 h at 38 000 rev./min and 20°C in a Beckman type 50 rotor. Finally the A260 nm as well as the refractive index was read against the fraction number. The buoyant density was calculated from the refractive index according to Ifft et al. [9].
RNA extraction and agarose gel electrophoresis Polyribosomal and RNP-RNA was extracted by suspending polysomes or RNP particles in a buffer containing 100 mM Tris--H3BO3 (pH 8.5), 2% (w/v) NaC1 and 2% (w/v) triisopropylnaphtalene sulphonate. Deproteinisation was carried out twice at 65°C by extraction with an equal volume of a 1 : 1 mixture phenol solution (500 g phenol, 0.5 g 8-hydroxyquinoline, 70 ml m-cresol, 150 m1,50 mM Tris--HC1 (pH 8.5)) and chloroform. The nucleic acids were precipitated overnight with 2 vol. of ethanol at 4°C and reprecipitated with ethanol at -20°C, after dissolving the precipitate in 0.15 M sodium acetate (pH 6.0), 0.5% sodium dodecyl sulphate (SDS). Subsequently, the RNA fractions were analyzed on 1.6% agarose gels, according to van Ommen et al. [10].
Analysis of polyribosomal and RNP-proteins Polyribosomal and RNP particles were denatured in SDS and/3-mercaptoethanol, essentially as described by Lindberg and Sundquist [11] and subsequently subjected to SDS polyacrylamide gradient gel electrophoresis as described by Laemmli [ 12]. RESULTS
Sedimentation profiles of polyribosomes and RNP particles As shown in Fig. 1 Spirodela oligorrhiza cytoplasmic polyribosomes can be separated in sucrose gradients in distinct size classes. Free polyribosomes
340
Q4.
C
A
80S
SOS
J
03
Q2
01 0 i=
l
O.4 o
D
eo .a o
Q3-
Q2.
0.1,
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Fig. 1. Sedimentation profiles obtained after centrifugation of cytoplasmic polyribosomes (about 2.0 A=60 nm units in all cases) through 10--40% sucrose gradients. A: free cytoplasmic polyribosomes from light-grown plants. B: membrane-bound polyribosomes from light-grown plants. C: free cytoplasmic polyribosomes from clark-grown plants. D: membrane-bound polyribosomes from dark-grown plants. The horizontal arrow indicates the direction of sedimentation.
from light-grown plants have sizes up to octamers (Fig. 1A), while membrane-bound polysomes are present up to heptamers (Fig. 1B). The respective polysome populations from dark-grown plants seem to be significantly smaller, with pentamers (Fig. 1C) and trimers (Fig. 1D) as largest polysomes. Incubation of the polysomal structures in the presence of EDTA causes an almost complete dissociation into 40 S and 60 S ribosomal subunits (Fig. 2). Treatment with pancreatic RNase (substrate/enzyme, 20 : 1 (w/w) in 10 mM Tris--HCl (pH 7.6) for 0.5 h at 37°C) reduces the maximal size from octamers to trimers, while the amount of monosomes (80 S) is greatly increased (Fig. 2). These results clearly demonstrate that the rapid-sedimenting structures are polysomes rather than ribosomal aggregates. Figure 3 shows the sedimentation behaviour of RNP particles from both light-grown and dark-grown Spirodela plants. These particles sediment with a sedimentation coefficient of approx. 25 S, as was calculated from their position in the sucrose gradient according to the method of McEwen [ 30].
341 06,
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, =
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* ~ •) on cytoplasmic Fig. 2. The effect of EDTA ( . . . . . ) and pancreatic RNAase ( ). The horizontal arrow indicates the direction of sedimentation. polyribosomes (
Buoyant density of polyribosomes and RNP particles Both polyribosomes and RNP particles were fixed with formaldehyde and centrifuged in CsCl
o
05 t
/ 255
~
/
" 20°/°
04. ~
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(11, 5%
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Fig. 3. Sedimentation of non-ribosomal R N P particles from either light-grown or darkgrown plants through 5-20% sucrose gradients.
342
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3
Fig. 4. Absorbance scans of RNP particles and polyribosomes obtained after centrifugation in density gradients. A: formaldehyde-fixed ribonucleoprotein particles in CsCI. B: unfixed RNP particles in Cs=SO,. C: formaldehyde-fixed polyribosomes in CsCI. Fig. 5. 1.6% agarose gel electrophoresis of polyribosomal-RNA and RNP-RNA from light-grown plants relative to 25 S, 23 S, 18 S and 16 S ribosomal RNA. (1) Membranebound polyribosomal RNA (0.8 ug); (2) free cytoplasmic polyribosomal RNA (2.0 ug); (3) RNP-RNA (0.5 ~g).
density of the unfixed particles remains approx. 1.4 g/cm 3. Therefore, these particles cannot be considered as artificial RNA-protein complexes. Polyribosomes band at a density of 1.55 g/cm 3 (Fig. 4C), which is a characteristic of ribosomes [22--24] and to a much lower extent at 1.45 g/cm% which indicates the presence of some polyribosomal ribonucleoprotein particles [24]. R N A and protein components of polyribosomes and R N P particles
Figure 5 shows the resultsof a 1.6% agarose slab gel electrophoresisof R N A isolated from membrane-bound polyribosomes (lane 1), free polyribosomes (lane 2) and non-ribosomal R N P particles(lane 3) allfrom lightgrown plants.As can be seen,the R N A moiety of R N P particlesconsists of a heterogeneous population ranging in sizefrom 4 S and 16 S, which differs quite significantlyfrom the polyribosonml R N A components, that range from 4 S and 25 S. This observation suggests that the RNP particles represent
343 67.000 94.000 J
i
30.000 43000 a
e
i
14.400
20:100 i
i •
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--
B Fig. 6. Densitometric representation of an 8--20% SDS polyacrylamide gradient electrophoresis of RNPs (A) and polyribosomal proteins (B) from light-grown plants, relative to phosphorylase b (94 000), albumin (67 000), ovalbumin (43 000), carbonic anhydrase (30 000), trypsin inhibitor (20 200) and ~-lactalbumin (14 000).
a specific class of particles rather than artificial ribosomes or ribosomal subunits. This suggestion was confirmed by Fig. 6, which shows the absorbance scans of an 8--20% SDS polyacrylamide gradient gel electrophoresis of the proteins isolated from RNP particles and polyribosomes from light-grown plants. From the differences, which are most striking in the low molecular weight region, it was concluded that RNP particles do not contain ribosomal proteins and consequently cannot be the product of ribosome degradation. DISCUSSION
As compared with polyribosomes, isolated from other higher plants [1--7], all profiles of Spirodela oligorrhiza polysomes isolated by our procedure show a rather high percentage of monosomes and ribosomal subunits (Fig. 1 ). Although this still might be the result of some degradation and/or run off, it might also reflect the in vivo situation, since these types of sedimentation profiles have also been found for polyribosomes of Lemna gibba L.G-3, another member of the duckweed family [25]. Analysis of the postribosomal supernatant revealed the presence of RNP complexes, which sediment through sucrose gradients as a rather broad band with a mean sedimentation coefficient of 25 8. According to their buoyant density in CsC1 of 1.39 g/cm 3 (Fig. 4A), their relative stability in high salt solutions (Fig. 4B), the differences in RNA composition (Fig. 5) and the differences in protein composition as compared with that of ribosomes {Fig. 6), these complexes apparently represent the so called non-ribosomal ribonucleoprotein particles [ 13--20, 12, 26,27]. The availability of a good isolation procedure for Spirodela polysomes and RNP particles now permits
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