Nascent Subribosomal Particles in Tetrahymena pyrqormis

Transcription

1 Eur. J. Biochem. 13 (1970) Nascent Subribosomal Particles in Tetrahymena pyrqormis Vagn LEICK and Jan ENCBERG Biokemisk Institut B, Krabenhavns Universitet Julius EMMERSEN Institut for Molekylax Biologi, Odense Universitet (Received November 21,1969) The metabolism and physical-chemical properties of nascent (precursor) 40 and 60 S subribosomal particles have been studied in exponentially growing Tetrahymena pyrifornzis GL. Analysis of formaldehyde-fixed 40 and 60 S particles in isopycnic CsCl gradients reveals that they have a lower buoyant density than the corresponding 30 and 50 S ribosomal sub-units. Moreover, overall labeling of the cells with [3H]amino acid and [32P]phosphate shows that theprecursor particles attain larger 3H/32P ratios than the corresponding sub-units, indicating that the precursor particles contain extra (accessory) protein that splits off when they are converted into ribosomal sub-units. The extra protein constitutes 10 to 20 /0 of the total structural protein. Experiments in pulse-labeling with radioactive amino acids indicate that 40 and 60 S particles are very rapidly labeled. This suggests that, apart from the RNA moiety, at least part of the structural ribosomal protein moiety associated with the nascent particles is newly synthesized. The possible involvement of 40 and 60 S particles in sub-unit exchange between rounds of translation was investigated by comparing their kinetics of labeling with that of the ribosomal sub-units. The results of the comparison indicate that the rate of recycling of ribosomal sub-units through the pool of 40 and 60 S particles is too slow to account for sub-unit exchange between each round of translation. Subribosomal precursor particles sedimenting around 40 and 60 S and containing 17 S, 25 S and 5 S rrna, respectively, have previously been identified in the cytoplasm of the ciliate protozoon, Tetmhymena pyriformis [l 1. The 40 and 60 S particles are precursors of the 30 and 50s ribosomal subunits, respectively. During exponential growth they assemble in the macronucleus at a rate more than 10 times the one at which a HeLa cell nucleolus assembles ribosomal particles [2]. In both animal cells and Tetrahymena the 40 and 60 S subribosomal precursor particles contain a complement of structural protein [3,2]. Some characteristic accessory proteins were found associated with these particles in L cells [4,5]. In addition to the precursor particles, a pool of free native ribosomal sub-units has been identified in cytoplasmic cell extracts from animal [6, 71 and bacterial sources [8]. It has been suggested that these native subunits participate in sub-unit exchange between rounds of translation [9-11]. The present study was carried out in order to describe the 40 and 60 S subribosomal precursor particles in Tetrahymena in greater detail and to investigate the role of the various particles in polyribosome formation and metabolism. There is evidence that the 40 and 60 S particles probably do not participate in sub-unit exchange and that at least a part of the complement of structural proteins of the 40 and 60 S particles is newly synthesized. Moreover, the nascent particles contain accessory proteins which are split off when the particles are converted into their corresponding ribosomal subunits. A preliminary report on this work was presented previously [ 1 I a]. MATERIALS AND METHODS Organism and Growth Medium Tetrahymena pyriformis GL (amicronucleate) was used in all these experiments. The cultures were grown in siliconized aspirator bottles at 28", aerated by bubbling air through the medium. The medium had the following composition : 0.75O/, proteose peptone (Difco), 0.75O/, yeast extract (Difco), 1.5 /0 glucose, 1 mm MgSO,, 0.05 mm CaCl, and 0.1 mm ferric citrate. The generation time was approx. 2.5 hours. The number of cells was measured with a Coulter counter, Model F. All experiments were carried out at a cell concentration of approx. 2x105 cells/ml

2 Vol.13, No.2, 1970 V. LEICK, J. ENGBERG, and J. EMMERSEN 239 where the cultures were in the mid-exponential growth phase. After sampling, the cell suspension was rapidly cooled to 0" by violent stirring in a -40" cold or by pouring the suspension over crushed frozen growth medium. The cells were harvested by centrifugation at 3000 to 4000 x g for 2 to 5 min. Preparation of the Crude Cytoplasmic Ribosome Xuspension All operations were carried out at 0 to 4". The cells were washed once in 0.1 M sucrose containing 0.01 M Tris-C1 (ph 7.5), 1 mm MgCl, and 3 mm CaCl,, and were resuspended in the same Trissucrose buffer. One (v/v) Nonidet P40 (Shell) dissolved in the above Tris-sucrose buffer solution was added up to a final concentration of 0.15 to 0.30 /,, which caused immediate lysis of the cells. The lysate was centrifuged at 150OOxg for 10 min and the supernatant represented the crude suspension of cytoplasmic ribosomes. This procedure is a modification of the one introduced by Mita et al. [15]. Preparation of the Polysome Xuspension All these operations were carried out at 0 to 4". The operations from cell sampling to the first sucrose gradient centrifugation were performed within 30 min. The cell suspension was chilled by pouring it over crushed, frozen growth medium and the cells were harvested by centrifugation at 2000 x g for 3-4min. The cell pellet was resuspended in a sucrose-buffer solution containing : 0.1 M sucrose, 0.01 M Tris-C1 (ph 7.2), 1 mm MgCl,, 3 mm CaCl,, 0.5 M NH,Cl, 0.20/, Nonidet P40 and 2 mg Bentonite per ml. The lysate was centrifuged at x g for 10 min. The polysomes contained in the 15000xg supernatant were purified by sucrose gradient centrifugation. The material in the fractions containing polysomes was precipitated by layering 2.5 ml polysome suspension onto 2.5ml 40 /, sucrose (w/v) in 0.02 M triethanolamine-c1 (ph 7.2), 1 mm MgCl,, 0.05 M KC1, (pretreated with Bentonite, 2 mglml) and then centrifuged at 10000O~g for 1 hour. Ribosomes yielded by this method corresponded to about 30 /, of the cellular rrna. Dissociation of Ribosomes into 50 X and 30 X Sub- Units The poly- or monosomes were resuspended in 0.02 M triethanolamine-cl buffer (ph 7.2), 0.05 M KC1 and 1 mm EDTA and incubated at 0" for 1Omin. When fractions were used from sucrose gradients containing mono- or polyribosomes, EDTA was added up to a final concentration of 1 mm. In some experiments ribosomal sub-units were obtained by dialysing suspensions of ribosomes against 0.02 M triethanolamine-c1 buffer (ph 7.2), containing 0.05 M KC1, for 5 to 6 hours. The properties of the sub-units obtained by EDTA treatment and by dialysis were identical when analyzed by sucrose gradient centrifugation. Before the sub-units were used in further analyses (see below), they were usually stabilized by formaldehyde-fixation. Fixing Ribosomal Particles in Formaldehyde The various ribonucleoproteins were fixed for 24 hours in Sol0 formaldehyde, according to Perry and Kelley [12], before analysis in isopycnic CsCl gradients. After fixation, the formaldehyde and sucrose were removed by dialysis against 0.02 M triethanolamine-hc1 buffer (ph 7.2), 0.05 M KC1. When ribosomal particles were reanalyzed by sucrose gradient centrifugation they were usually stabilized by a brief fixation with formaldehyde (30 min). Fixing in formaldehyde did not alter the sedimentation properties of the various particles in sucrose gradients using unfixed particles as markers. Sucrose Gradient Centrifugation of Ribosomal Particles All centrifugations were carried out at 4" in a Spinco ultracentrifuge, model L. The linear sucrose gradients (5-200/, or /,) were of a total volume of either 5ml or 27 ml, and were centrifuged using the rotors SW50 L or SW25, respectively. In all centrifugations but those involving ribosomal subunits, the buffer solution 0.02 M triethanolamine-c1 (ph 7.2), 1 mm MgCl,, and 0.05 M KC1 was used. MgCl, was omitted from the buffer when ribosomal sub-units were analyzed. All solutions were pretreated with 2 mg Bentonite/ml in order to remove traces of ribonuclease. The gradients were collected by puncturing the bottoms of the tubes. Bovine serum albumin (0.5 mg) and cold trichloroacetic acid (final concentration of 5O/,) were added to each fraction. The precipitate was collected on glass filter discs (Whatman GF/C). The discs were dried for 1.5 hours at 60" and counted in a liquid scintillation counter using a toluene scintillator. Isopycnic CsCl Centrifugation of Ribosomal Particles; Determination of Buoyant Densities The formaldehyde- fixed particles were banded in isopycnic CsCl gradients using the step gradient technique by Brunk and Leick [13]. The fixed material was placed in the upper portion of the step gradient, which usually contained half the amount of CsCl that was in the lower portion. Both upper and lower portions contained 3 ml 0.02 M triethanolamine-buffer (ph 7.2). Centrifugation was performed in a fixed angle 40 Spinco rotor at rev./min for 18 hours. Fractions were collected by puncturing the bottoms of the tubes. Refractive indexes for

3 240 Nascent Particles in Tetrahymena pyriformis Eur. J. Biochem. 20p1 of each fraction were measured by a Zeiss refractometer. From the refractive indexes (corrected for the refractive index of the buffer) calculations of buoyant densities were made according to Ifft, Voet and Vinograd The radioactivity of each fraction was determined as described for sucrose gradients. Isolution of Macronuclear rrna Phenol-sodium dodecyl sulphate extraction of macronuclear RNA was carried out as described earlier [2,18]. The RNA was analyzed in 5 ml 5-20 /, sucrose gradients made up in 0.05 M acetate buffer (ph 5.0), 0.01 M NaC1. Centrifugation was carried out for 3.5 hours at rev./min in the SW 50 L Spinco rotor. gradient (Fig. 1 B), as well as of ribosomal sub-units derived from purified polysomes (Fig. 1 A). The cells were labeled with [3 I]uridine for 8 generations and then pulse-labeled for 12 min with [32P]phosphate. The sucrose gradient reanalysis shows coincidence of 32P- and 3H-labels in Fig. 1 B, indicating that 40 and 60 S particles account for virtually all the rrna sedimenting slower than 80 S inonoribosomes when isolated under the present conditions. In a study of L cells Perry and Kelley showed that a ribonucleoprotein heterogeniety not resolved by sucrose gradient analysis could sometimes be revealed by analysis in isopycnic CsCl gradients [5]. Using this technique to reveal the possible pool of native ribosomal sub-units, the postribosomal fraction of 40 and 60 S particles was analyzed in isopycnic CsCl gradients, as will be described below c E. v) c 0 2s 2..- c.- I c u : 0? 0 Fig. 1. Analysis on sucrose gradients of ribosomal subunits derived from purified polqsomes (A) and of 40 and 60 S particles (B) from cells pulse-labeled for 12 min with [SZP]phosphate. Exponentially growing cells were incubated with [3H]uridine for 8 generations and then pulse-labeled with [32P]phosphate for 12 min. A crude cytoplasmic extract was prepared from the cells and analyzed on a sucrose gradient. (A) Polysomes from fractions in the first sucrose gradient were purified as described in Materials and Methods; derived sub-units were prepared by EDTA-treatment, fixed for 30 min with formaldehyde and analyzed by sucrose gradient centrifugation. (B) Fractions from the 30 to 70 S region in the first sucrose gradient were pooled and fixed for 30 rnin with formaldehyde. An aliquot of the pooled fractions was then reanalyzed on a sucrose gradient. Centrifugation was at rev./min for 14.5 hours using 27 ml /, gradients. 0, ah-radioactivity; 0, 32P-radioactivity RESULTS 40 and 60 S subribosomal precursor particles can be identified by sucrose gradient centrifugation of the crude postmitochondrial fraction from cells pulse-labeled for 6 to 15 min with an RNA precursor ([ 1,2], and Fig. 4 B). The approximate sedimentation values of the particles were determined by using 50 and 30 S ribosomal sub-units as markers. In order to reveal a possible pool of free native subunits apart from the 40 and 60 S precursor particles, various fractions from the initial sucrose gradient of a cytoplasmic extract were reanalyzed by sucrose gradient centrifugation. Fig. I shows the reanalysis on sucrose gradients of pooled fractions from the 30 to 70 S region of the initial sucrose Although conformational differences may be reflected in the density of a macrospecies, the buoyant density of a ribonucleoprotein will, for the most part, be dependent on the RNA/protein ratio. The formaldehyde-fixation, essential for stability of the particles in the strong CsCl solutions 1161, involves a formation of methylene-bridges between primary amino groups in RNA and protein. The cross-linking occurs for RNA and protein within a particle only, as shown in the experiment depicted in Fig.2. Fig.2 shows that 3H-labeled soluble cytoplasmic proteins preincubated with ribosomal particles are not associated with any of these particles when they are reanalyzed by sucrose gradient centrifugation after fixation in the presence of the labeled

4 Vol.13, No.2, 1970 V. LEICK, J. ENaBERa, and J. EMMERSEN s Fig. 2. Sucrose gradient analysis of various 32P-labeled ribosomal particles fixed with formaldehyde in the presence of 8H-labeled proteins. The 32P-labeled particles isolated from sucrose gradients were incubated with 3H-labeled proteins taken from the top fractions of a sucrose gradient such as that shown in Fig.5A. Preincubation was carried out for 30min at 0" followed by fixation with formaldehyde for 24 hours. The fixed particles were reanalyzed on /, sucrose gradients. Centrifugation was for 14.6 hours at rev./min in a 27 ml /, sucrose gradient. 0, 3H-radioactivity; 0, a2p-radioactivity proteins. This also indicates that little or no exchange takes place between ribosomal proteins and the exogenously added 3H-labeled proteins. When centrifuged in conventional, uniformly mixed CsCl gradients, formaldehyde-fixed ribonucleoproteins will sometimes dissociate into heterogeneous material. Thus, fixed 40 S subribosomal particles and 30 S sub-units from Tetrahymem will usually dissociate, whereas the stabler, larger ribosomal particles will not dissociate. In order to determine buoyant densities of the smaller, more fragile ribosomal particles, a gentler and faster technique was developed [13]. The use of a preformed step in CsCl concentration in the centrifuge tube reduces the time required to achieve equilibrium conditions, and makes it possible to band the fixed 40 and 30 S particles, provided they are placed in the upper portion of the step gradient. Pig.3 shows the banding of 40 S particles and of 30 S sub-units. No material can be seen banding in either the top or the bottom of the gradients, indicating that no appreciable dissociation has occurred during the centrifugation Fig. 3. Analysis of 50 S sub-units (A), 30 S sub-units (B) and 40 and 608 subribosomal particles (C) in isopycnic CsCl gradients. The 50 and 30s sub-units were isolated from a sucrose gradient of ribosomal sub-units prepared from cells which had been incubated with [3H]uridine for 8 generations followed by pulse-labeling with [32P]phosphate for 12 min (see Fig. 1A). The particles were fixed with formaldehyde before banding in isopycnic CsCl step gradients. The 40 and 60 S particles were isolated from a sucrose gradient such as that shown in Fig. 1 B. In the present experiment, however, the cells were labeled for 8 generations with [3H]valine and [3zP]phosphate. The gradients in A and B contained 1.7 and 3.8 g CsCl in the upper and lower portions, respectively. The gradient in C contained 1.0 and 3.8 g CsCl in the upper and lower portions, respectively (see Materials and Methods). In A and B: 0, 3H-radioactivity; 0, szpradioactivity; in C: - 0, 82P-radioactivity; 0, 3H-radioactivity

5 242 Nascent Particles in Tetrahymena pyriformis Eur. J. Bioehem. The greater stability of the larger ribosomal particles compared to the smaller ones is also revealed when both are manipulated in unfixed conditions. Thus, during isolation and analysis in sucrose gradients 40 S and 30 S particles usually are much more fragile than the corresponding larger particles. A similar observation was made in HeLa cells, where 30 S subunits were more sensitive to treatment with salt solutions than 50 S sub-units [17]. The approximate sedimentation values of the rapidly-labeled 40 and 60 S subribosomal particles were determined in sucrose gradients, using 50s and 30 S ribosomal sub-units as markers [I]. In order to (a) evaluate if the higher sedimentation values of the subribosomal particles were due to differences in macromolecular composition or to differences in ribosomal sub-units, could be explained by a higher protein content in the subribosomal precursor particles. This interpretation was confirmed when 3H/32P ratios were measured for the various particles isolated from cells incubated for 8 generations with [3H]amino acid and [32P]phosphate. 3H/32P ratios were measured for the two amino acids ~-[~H]valine and ~-[3H]lysine. The results shown in Table 2 indicate that the subribosomal precursor particles have a larger 3H/32P ratio than their corresponding ribosomal sub-units. Table I shows how protein content can be calculated from the buoyant density of various particles, assuming buoyant densities of 1.9 for RNA and 1.3 for protein. The calculations indicate that the 60s particle contains 15O/, more protein than the corresponding 50 S sub-unit and Table 1. Buoyant densities of various formaldehyde-fixed ribosomal particles in CsCl gradients The buoyant densities were determined by measuring refractive indexes of the fractions in the CsCl gradients (see footnote) Buoyant Number of Protein in particle Particle density determi- calculated from nations buovant densitv' wt. "@ Polysome Monosome f S particle j, S sub-unit S particle & S sub-unit f a Weight protein = 100 x s-q+, where e is the measured density of the particles in CsCl and en and ep are the densities of RNA and protein assumed to be 1.9 and 1.3, respectively. conformation and (b) reveal a possible pool of native sub-units, the various particles were banded in isopycnic CsCl gradients after fixation with formaldehyde. Fig.3 and Table I show the results of the CsCl bandings of various formaldehyde-fixed subribosomal precursor particles and ribosomal subunits. Fig. 3 gives a clear indication of a significantly higher buoyant density for the ribosomal sub-units than for their corresponding precursor particles. The finding (Table I) of very similar buoyant densities for the 30 S and 50 S sub-units and the monosomes and polysomes indicates that ribosomal sub-units derived from mono- and polysomes are modified very little from their native state. The precursor subribosomal particles and their corresponding sub-units possess identical rrna complements [18]. The finding of higher sedimentation values in sucrose gradients combined with the buoyant density of the subribosomal precursor particles being lower than that of the corresponding Table 2. sh/32p ratios of the various ribosomal particles isolated from cells which were incubated for 8 generations with [S2P]- phosphate and [SH]amino acid The various particles were prepared as described in Materials and Methods and were isolated and purified in sucrose gradients as in Fig. 1 ['HIValine [JHILysine Ribosomal Comparison Comparison particle srp ratio of precursor ratio of precursor and product and product particles particles 'H an A - in A - in a/,, =P -P of sub-unit of sub-unit 60 S particle S sub-unit S particle 30 S sub-unit that the 40 S particle contains 20 /, more protein than 30 S sub-units. Somewhat similar figures were obtained when the protein content was measured by the isotope method, using r3h]valine as the radioactive amino acid (Table 2). However, the values obtained with r3h]lysine are higher, indicating perhaps that the extra protein attached to the precursor particles is rich in the basic amino acid, lysine. Analysis of 40 and 60 S particles in isopycnic CsCl gradients (Fig. 3) emphasizes the homogeneous character of the two species of particles, first observed by sedimentation in sucrose gradients (Fig. 1 B). In contrast to evidence provided by CsCl gradient analysis of the post-ribosomal fraction from L cells [5], there was no indication whatsoever of t8he presence of native sub-units in crude extract.

6 Vol.13, No.2, 1970 V. LEICK, J. ENGBERG, and J. EMMERSEN 243 Bound 60 S subribosomal precursor particles in crude extracts of pulse-labeled ribosomes and polysomes from Tetrahymena can be liberated by lowering the magnesium concentration by dialysis [18]. Purified polysomes were prepared (Fig. 4A) in order to determine whether bound 60 S particles are constituents of short-living ribosomes containing one 60s and one 30s sub-unit. When these polysomes were dissociated into sub-units no 60 S particles were found, as seen in Fig. 1 A. The absence of 60 S particles in purified polysomes suggests that the 60 S particle loses its extra protein when it is ncorporated into a polysome. The procedure used to solate purified polysomes gives rather low yields in the cytoplasm. Accordingly, since the subribosoma1 particles are assembled from the macromolecular constituents in the macronucleus [Z], a nascent ribosomal protein must migrate to the nucleus, be incorporated into a ribosomal particle, and then reappear in this form in the cytoplasm. With this background it was surprising to find that the 40 and 60s particles were very rapidly labeled by radioactive amino acids. Fig.5A shows a sucrose gradient analysis of a crude cytoplasmic extract from exponentially growing cells which had been incubated with [32P]phosphate for 8 generations, and then pulse-labeled with [3H]leucine for 10 min. From this sucrose gradient, polysomal sub- A E Fig.4. Sucrose gradient analysis of purified polysomes from cells labeled for 8 generations with L3H]uridine and then pulse-labeled for 12min with (32P]phosphate. (A) Centrifugation for 1.5 hours at rev./min. (B) Centrifugation for 6 hours at rev./min. The gradients consisted of 27 ml of 5-20 o/o sucrose. 0, 3H-radioactivity; 0, 32P-radioactivity (30 /0) and requires the presence of 0.5 M NH,CI which does not affect the stability of free 40 and 60 S precursor particles (Fig. 4B). It is, however, uncertain whether the previously reported bound 60 S particles are procedural artefacts or biologically significant particles. It cannot be excluded that 60 S subribomosal particles are present, at some stage, in the form of membrane-bound particles, and that these larger entities sediment in the polysome region of a sucrose gradient. The study of the synthesis and metabolism of ribosomal proteins is a more difficult task than the study of rrna metabolism. First, ribosomal proteins are numerous and no methods are yet available to distinguish them from the other proteins of the cell. Secondly, there is still the question of where the synthesis of the ribosomal proteins takes place in the cell. Since the macronuclei of Tetrahymena contain few, if any, ribosomes [2], it is most probable to assume that the ribosomal proteins are synthesized units were prepared and reanalyzed on a new sucrose gradient (Fig. 5B). The fractions from the gradient (Fig. 5A) containing 60 S and 40 S precursor particles were also reanalyzed by sucrose gradient centrifugation (Fig. 5C and D, respectively). The experiment shows that 40 and 60 S particles have an approx. 10-fold larger 3H/32P ratio than the corresponding derived sub-units. This indicates that at least part of the complement of structural protein associated with the nascent particles is newly synthesized and, accordingly, must be present in free form as rather small pools in the cell. At present, it cannot be determined if the extra (accessory) protein associated with the precursor particles is selectively labeled at a fast rate. The possible role of native sub-units in the initiation of protein synthesis has recently received much attention. In bacteria there is evidence that the ribosomal sub-units dissociate between rounds of translation and presumably recycle through a

7 244 Nascent Particles in Tetrahymena pyriformis Eur. J. Biochem. I I s 0 50 S c < lo 2 z Y w..ḍ-.k e u 9 u 4 la5 $ v 9 1.o D Fig.5. Incorporation of [3H]leucine into 50 and 30 8 subunits and into 40 and 60 X subribosomal particles. Exponentially growing cells were incubated with [32P]phosphate for 6 generations, followed by incubation with [3H]leucine for 10 min. (A) A crude cytoplasmic extract was prepared from the cells and analyzed on a sucrose gradient. Centrifugation same as Fig.4B. (B) Ribosomal sub-units were prepared from pooled fractions in gradient A and were reanalyzed on a sucrose gradient after fixation with formaldehyde for 30 min. (C) Reanalysis on a sucrose gradient of 60 S particles taken from the 60 S region of gradient A and then fixed for 30 min. (D) Reanalysis on a sucrose gradient of 40 S particles taken from the 40 S region of gradient A and then fixed with formaldehyde for 30 min. Centrifugation in B, C and D was at rev./min for 14 hours in 27 ml Ol0 sucrose gradients. 0, 32P-radioactivity; 0, 3H-radioactivity pool of free sub-units [10,9]. Moreover, in animal cells, pulse-chase labeling experiments indicate that the majority of free native sub-units found in the cytoplasm are not newly synthesized [6,7]. Provided that these free sub-units are not procedural artefacts, the findings imply that ribosomes dissociate into subunits at some stage in their Life cycle. Since our experiments on Tetrahymena did not reveal any pools of native sub-units like those found in other systems, the possible involvement of 40 and 60s particles in sub-unit exchange between rounds of translation was investigated. For this purpose, straightforward kinetics of labeling of the various particles was carried out using [3H]uridine and [3H]lysine. Using a radioactive RNA precursor and E. coli cells, the labeling kinetics followed in native versus derived sub-units revealed virtually identical kinetics between the two, indicating rapid exchange between native and derived sub-units [lo]. In contrast, the experimental results shown in Fig. 6 indicate that the recycling of Tetrahymena ribosomal subunits through the pool of 40 and 60 S particles must be rather slow-if it occurs at all. In the experiment in Fig. 6, exponentially growing cells were overall labeled with [32P]phosphate and then incubated with [3H]uridine (Fig.6A) or [3H]lysine (Fig.6B). In the cells where [3H]uridine was used as the radioactive precursor (Fig. 6A), the specific radioactivity (3H/3aP ratio) was measured in nuclear rrna, 40 and 60 S particles, and in ribosomal sub-units. In the cells where [SHIlysine was used as precursor, the 3H/32P ratio was measured in 40 and 60 S particles, and in ribosomal sub-units. Fig.6A shows that the specific radioactivity curve of the subribosomal particles

8 Vol. 13, No. 2, 1970 V. LEIGH, J. ENQBERQ, and J. EMMERSEN 245 the flow-rate of ribosomal sub-units back to the pool of subribosomal particles, must be rather slow. The recycling problem will be further discussed in the following section. I W Time (min) Time (nin) Fig.6. (A) Kinetics of labeling with [3H]uridine of nuclear rrna, subribosomal particles and ribosomal sub-units during exponential growth. Exponentially growing cells were incubated with [32P]phosphate for 8 generations followed by [3H]uridine. Samples were taken at the times indicated, and mean specific activities ( 3H/32P) were determined for nuclear rrna (A), subribosomal particles (0) and ribosomal subunits (0). Nuclear rrna was isolated and purified as described in Materials and Methods. Subribosomal particles and ribosomal sub-units were isolated as in Fig. 1. (B) Kinetics of labeling with [3H]lysine of subribosomal particles and of ribosomal subunits during exponential growth. Exponentially growing cells were incubated with [32P]phosphate for 6 generations, followed by incubation with [3H]- lysine. Samples were taken at the times indicated and mean 3H/32P values were determined for subribosomal particles and ribosomal sub-units. The various particles were isolated as described in Fig.4. Subribosomal particles, o ; ribosomal sub-units, H approximates that of nuclear rrna and not the ribosomal sub-units. Although the experiment using radioactive amino acid (Fig.6B) is more difficult to interpret than the one using [3H]uridine, the general conclusion is the same. During exponential growth, I DISCUSSION The present paper deals with the physicalchemical and metabolic properties of free 40 and 60 S cytoplasmic subribosomal particles, precursors of the small and large ribosomal sub-units, respectively, in Tetrahymena. The two species of subribosomal particles account for virtually all the rrna (approx. 20/,) sedimenting slower than 80s monoribosomes in a sucrose gradient when they are reanalyzed by sucrose gradient centrifugation (Fig. 1 B) ; this is further confirmed by their analysis in isopycnic CsCl gradients after fixation with formaldehyde (Fig.3C). The analysis in CsCl gradients reveais that the subribosomal particles have lower buoyant densities than the corresponding ribosomal sub-units (Table 1). Moreover, when cells are overall-labeled with [SHIamino acid and [3zP]- phosphate, the subribosomal particles attain larger 3H/32P ratios than the corresponding sub-units (Table 2). These findings suggest that the 40 and 60 S particles contain more protein than the corresponding ribosomal sub-units and that this extra protein is split off when precursor particles are converted into ribosomal sub-units. From the figures for buoyant densities the amount of extra (accessory) protein has been determined at approx. 200/, in the 40 S particle compared to the protein in 30 S sub-units, and at approx. 15O/,, in the 60 S particle compared to the protein in 50 8 sub-units (Table 1). The extra protein in the precursor particles is presumably rich in lysine, because an extraordinarily large amount of 3H-counts is incorporated into the precursor particles when [3H]lysine is used as labeled amino acid instead of valine (Table 2). The biological function of the accessory proteins is presently unknown. When Tetrahymenu cells are incubated with a radioactive amino acid, the label is incorporated into 40 and 60s precursor particles at nearly the same rate as when the cells are incubated with an RNA precursor (Fig. 5 and 6). This observation stresses the precursor character of the subribosomal particles. Moreover, it indicates that apart from the rrna complement at least some of the complement of structural protein in the subribosomal precursor particles is very newly synthesized and, accordingly, the free pool of these ribosomal proteins is rather small. When crude cytoplasmic extracts of cells from various bacterial and animal sources are analyzed on sucrose gradients, a pool of native ribosomal subunits which are not newly formed have been observed

9 246 V. LEICK, J. ENGBERG, and J. EMMERSEN: Nascent Particles in Tetrahymena pyriformis Eur. J. Biochem. [5,8,9]. It has been suggested that these native sub-units are involved in sub-unit exchange between rounds of translation [6,7,10,11]. Ribosomal subunits in bacteria and yeast have been shown to be stable during growth and to be continuously recycled through ribosomes [19,20]. These results were interpreted as indicating that ribosomes dissociate into sub-units between successive rounds of protein synthesis. Since no pool of native 30 and 50 S subunits whatsoever could be found in Tetrahymena when ribosomes were preparated by the methods used in this study, the possible role of 40 and 60 S subribosomal particles in sub-unit exchange during protein synthesis was investigated. For this purpose straightforward labeling kinetics were measured for the various particles during exponential growth. The results shown in Fig. 6, indicate that it is rather unlikely that 40 and 60 S particles are involved in sub-unit exchange between each round of translation. Provided that all ribosomes are active in protein synthesis during exponential growth, an average ribosome in vivo incorporates I0 amino acids into protein per second [21], i.e. a polypeptide of mol.wt is completed within 40 sec. This implies that during growth there should exist a rapid rate of exchange between free sub-units and derived subunits. The experiments shown in Pig.6 indicate that the specific radioactivity of the rrna moiety of the subribosomal particles approximates that of nuclear rrna, rather than that of ribosomal sub-units-even 213 generation (I00 min) after the addition of [3H]uridine. This implies that a recycling of subunits through the pool of subribosomal particles, if occurring at all, proceeds rather slowly. It is still unclear why there were no native ribosomal subunits detected in Tetrahymena, but the possibility exists that aggregation of such native sub-units occurs when the crude cell extracts are prepared. Mangiarotti and Schlessinger postulated that single ribosomes in bacteria do not exist in vivo and that all ribosomes dissociate into sub-units at the end of a round of translation [9]. However, evidence for the existence of monoribosomes which are not degradation products of polyribosomes is accumulating for both bacterial and animal cells [11, Moreover, evidence has been provided that monoribosomes in bacteria must react with a dissociation factor in order to split into sub-units [25]. The data so far provided by Tetrahymena favour the scheme that 80 S monosomes are the final product of translation and that 80 S ribosomes, not 30 S and 50 S sub-units, accumulate. It now remains to be shown that 80s ribosomes dissociate before a new round of translation is initiated. This work was supported by EL grant from Carlsbergfondet. The authors wish to thank Dr. Hans Klenow and Dr. Paul Plesner for providing them with good working conditions and for their continuous interest in this work. The technical assistance of Mrs Marianne Brieg is gratefully acknowledged. REFERENCES 1. Leick, V., and Plesner, P., Biochim. Biophys. Acta, 169 (1968) Leick, V., Eur. J. Biochem. 8 (1969) Warner, J., J. Mol. Biol. 19 (1966) Perry, R. P., Progr. ivucleic Acid Res. Illol. Biol. 6 (1967) Perry, R. P., and Kelley, D. E., J. Mol. Biol. 35 (1968) -- "1. 6. Vaughan, M. H., Warner, J. R., and Darnell, J. E., J. Mol. Biol. 25 (1967) Hogan, B. L. M., and Korner, A., Biochim. Biophys. Acta, 169 (1968) Green, H. M., and Hall, B. D., Biophys. J. 1 (1961) Mangiarotti, G., and Schlessinger, G., J. Mol. Biol. 20 (1966) Mangiarotti. G.. and Schlessincrer. G.. J. Mol. Biol. 29 " I, (1567) 395. ' 11. Colombo. B., Vesco, C., and Baglioni, C., Proc. Natl. Amd. hi' U. 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