378

Biochimica et Biophysica Acta, 517 (1978) 378--389 © Elsevier/North-Holland Biomedical Press

BBA 99104

THE COURSE OF THE ASSEMBLY OF RIBOSOMAL SUBUNITS IN YEAST

TIJS KRUISWIJK, RUDI J. PLANTA * and JOHANNES M. KROP

Biochemisch Laboratorium, Vrije Universiteit, de Boelelaan 1085, Amsterdam (The Netherlands) (Received June 22th, 1977)

Summary The course of the assembly of the various ribosomal proteins of yeast into ribosomal particles has been studied by following the incorporation of radioactive individual protein species in cytoplasmic ribosomal particles after pulselabelling of yeast protoplasts with tritiated amino acids. The pool of ribosomal proteins is small relative to the rate of ribosomal protein synthesis, and, therefore, does not affect essentially the appearance of labelled ribosomal proteins on the ribosomal particles. From the labelling kinetics of individual protein species it can be concluded that a number of ribosomal proteins of the 60 S subunit (L6, L7, L8, L9, L l l , L15, L16, L23, L24, L30, L32, L36, L40, L41, L42, L44 anal L45) associate with the ribonucleoprotein particles at a relatively late stage of the ribosomal maturation process. The same was found to be true for a number of proteins of the 40 S ribosomal subunit ($10, $25, $27, $31, $32, $33 and $34). Several members (L7, L9, L24 and L30) of the late associating group of 60-S subunit proteins were found to be absent from a nuclear 66 S precursor ribosomal fraction. These results indicate that incorporation of these proteins into the ribosomal particles takes place in the cytoplasm at a late stage of the ribosomal maturation process.

Introduction

In eukaryotic cells ribosome formation starts in the nucleolus with the transcription of a single large precursor RNA molecule containing one copy of each of the two high molecular weight rRNA species and in addition non-ribosomal sequences [ 1,2 ]. During or immediately after transcription ribosomal as well as non-ribosomal proteins associate with this primary transcription product. The * To w h o m

correspondence should be addressed.

379

resulting precursor ribonucleoprotein particles contain most of the structural ribosomal proteins [3--12]. During the ribosomal maturation process the nonribosomal proteins, together with the non-ribosomal RNA sequences, are removed from the preribosomal particles in a number of discrete steps. In previous studies performed in our laboratory three distinct preribosomal particles were detected in cells of the yeast Saccharomyces carlsbergensis, a primitive eukaryote. These particles have sedimentation constants of approx. 90 S, 66 S and 43 S, respectively [13,14]. From kinetic data it could be concluded that there exists a precursor-product relationship between the 90-S preribosomal particles, containing 37 S precursor rRNA, on the one hand and the 66-S and 43-S preribosomal particles, containing 29 S and 18 S precursor rRNA, respectively, on the other. The 66 S particle is processed to a 60 S ribosomal particle in the nucleus while maturation of the 43 S preribosome into the 40 S ribosomal particle takes place in the cytoplasm [13,14]. Buoyant density measurements revealed that the 66 S particle has a higher protein content than the 60 S cytoplasmic ribosomal subunit while the protein content of the 43 S particle equals that of the 40 S ribosomal subunit [13]. Two-dimensional polyacrylamide gel electrophoretic analysis of the protein composition of mature yeast ribosomes demonstrated that the 40 S and 60 S ribosomal subunits contain 30 and 41 different protein species, respectively [15,16]. In order to determine whether the full complement of these structural ribosomal proteins is already present in the preribosomal particles or is partially incorporated at later stages of the ribosome maturation we studied the labelling kinetics of the ribosomal proteins isolated from cytoplasmic 40 S and 60 S ribosomal subunits. The results show that a limited number of ribosomal proteins of the 60 S subunit and also some protein species of the 40 S subunit are incorporated into newly formed ribosomes only after their appearance in the cytoplasm. Most of the yeast ribosomal proteins, however, appear to be assembled already with the preribosomal particles within the nucleus. Materials and Methods

Culture and labelling conditions. Yeast cells (S. carlsbergensis, N.C.Y.C. 74) were cultured at 29°C in a medium containing 10 g glucose, 5 g bacteriological peptone, 3 g yeast extract and 3 g mal extract per I. Cells were harvested and converted into protoplasts as described by Ret~l and Planta [17]. The protoplasts were suspended at a density of 8 • 107 cells per ml in a synthetic medium (S.M.) containing per 1 : 20 g glucose, 2 g KH2PO4, 1 g MgSO4 • 7H20, 4 g (NH4)2SO4, 6 ml 70% sodium lactate, 120 g mannitol, 0.2 mg calcium panthotenate, 0.2 mg pyridoxine, 0.1 mg thiamine, 0.1/~g biotin, 10 mg glutamic acid, 10 mg tryptophan and 50 mg inositol. After a pre-incubation of the protoplast suspension at 29°C for 30 min, one half of the suspension was labelled at 15°C with a mixture of tritiated amino acids (Radiochemical Centre Amersham, 19 /~Ci/ml). Samples (20 ml) were taken at 15, 30, 45, 75 and 120 min after addition of the label. The other half of the culture was incubated at 15°C during 120 min in the presence of a mixture of 14C-labelled amino acids (Radiochemical Centre Amersham, 1.9/~Ci/ml). At the times indicated incorporation of radioactivity was stopped by pouring the sample into a 5-fold excess of ice-cold

380 citrate buffer (10 mM sodium citrate, pH 7.5) containing 0.66 M mannitol and 10 mM MgC12- 6H20 and subsequently 20 ml of the 14C-labelled protoplast suspension was added. Uniformly labelled yeast cells were obtained by culturing in a synthetic medium containing a mixture of tritiated amino acids (Radiochemical Centre Amersham, 1.1 pCi/ml) for five generation times. Isolation o f cytoplasmic ribosomal particles. The labelled protoplasts were pelleted by centrifugation at 10 000 X g for 10 min at 4°C and fractionated into a nuclear and cytoplasmic fraction according to the m e t h o d of SillevisSmitt et al. [18] as modified by Trapman and Planta [19]. Ribosomal particles were isolated from the cytoplasmic fraction by centrifugation at 230 000 X g for 120 min at 4°C in the Spinco SW 50.1 rotor. The ribosomal pellet was suspended in a Tris • HC1 buffer (10 mM, pH 7.4) containing 10 mM MgC12 • 6H20, 500 mM KC1 and 10 mM dithiothreitol and layered on a linear 15--30% (w/v) sucrose gradient in the same buffer. Separation of the subunits was performed by centrifugation of the gradients at 21 000 rev./min for 16 h at 4°C in the Spinco SW-27 rotor. The 40 S and 60 S ribosomal subunits were precipitated from the appropriate sucrose gradient fractions with 0.7 volume of icecold 96% ethanol. The precipitates were collected by centrifugation at 3000 X g for 30 min at 4°C. Isolation o f 66-S precursor ribosomal particles. A protoplast lysate was centrifuged at 2500 X g for 5 min at 4°C in order to remove unbroken cells. The nuclei were pelleted from the supernatant by centrifugation at 10 000 X g for 10 min at 4°C, suspended in a Tris • HC1 buffer (10 mM, pH 7.4) containing 5 mM MgC12 • 6H20, 10 mM KC1, 0.2% (w/v) Brij-58 and 10 mM dithiothreitol, and subsequently lysed by stirring the suspension for 10 min at 0°C. The lysate was centrifuged at 10 000 X g for 10 min at 4°C and the supernatant containing the preribosomal particles was layered on a 15--30% (w/v) sucrose gradient in a Tris-HC1 buffer (10 mM, pH 7.4) containing 10 mM MgC12" 6H20, 10 mM KCI and 1 mM dithiothreitol. After centrifugation at 18 000 rev./min for 16 h at 4°C in the Spinco SW-27 rotor the sucrose gradient was fractionated into 1-ml fractions. The fractions containing the 66-S precursor particles, located by determination of radioactivity in each fraction, were pooled. The 66-S particles were pelleted from the sucrose solution by centrifugation at 40 000 rev./min for 20 h at 4°C in a Spinco SW-41 rotor. Measurement o f de novo synthesis o f ribosomal proteins. Uniformly labelled yeast cells were converted into protoplasts, resuspended in fresh synthetic medium and conditioned at 29°C during 30 min. After lowering the temperature of the protoplast suspension to 15°C, 10 mg of casein hydrolysate per ml was added. At 0, 30 and 60 min thereafter equal fractions of the culture were poured into a 5-fold excess of citrate buffer in order to stop the chase. The protoplasts were pelleted at 10 000 X g for 10 min at 4°C and resuspended in a Tris • HCI buffer (10 mM, pH 7.4) containing 5 mM MgC12 • 6H20, 10 mM KC1, 0.2% (w/v) Brij-58 and 10 mM dithiothreitol. After stirring for 10 min at 0°C the suspension was centrifuged at 10 000 X g for 15 min at 4°C. The ribosomes were isolated by layering the supernatant on a 15% (w/v) sucrose solution in Tris • HCI buffer (10 mM, pH 7.4) containing 10 mM MgC12 • 6H20, 500 mM KC1 and 1 mM dithiothreitol, followed by centrifugation at 230 000 X g during

381

180 min at 4°C in the Spinco SW-50.1 rotor. In all cases the ribosomal pellets were suspended in 2 ml Tris - HCl buffer (10 mM, pH 7.4) containing 0.1 mM MgClz - 6Hz0. Aliquots of this suspension were taken for the determination of radioactivity and protein content. Preparation of an uniformly labelled post-microsomal supernatant. Uniformly labelled yeast cells were suspended in a Tris - HCl buffer (10 mM, pH 7.4) containing 10 mM MgClz - 6Hz0, 10 mM KCl, 0.5% (w/v) Brij-58, 5 mM dithiothreitol and 3 ml macaloid suspension per 1. The suspension was shaken with glass beads in a Braun Shaker for 150 s under cooling with CO* gas. The homogenate was centrifuged at 10 000 X g for 15 min at 4°C and the ribosomes were removed from the supernatant by centrifugation at 170 000 X g for 16 h at 4°C in a Spinco SW-65 rotor. The remaining supernatant was used for the determination of the amount of free ribosomal proteins. Protein extraction ancl gel electrophoretic analysis. Protein extraction was performed with 2-chloroethanol as described previously [ 151. The ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis according to the procedure of Kaltschmidt and Wittmann (ref. 20; see also refs. 15 and 16). Prior to electrophoresis unlabelled ribosomal proteins were added to the radioactive protein preparations to enable us to localize the ribosomal proteins on the slab gel by staining with Coomassie Brilliant Blue. Measurement of protein concentration. Protein concentration of ribosome suspensions was assayed with the biurete method [21] using bovine serum albumin as a standard. Assay for radioactivity. The amount of radioactivity present in ribosomal protein solutions and gradient fractions was determined in a dioxane-based scintillation liquid containing 90 g naphthalene, 7 g PPO and 50 mg POPOP per 1. Radioactivity present in ribosomal protein spots cut from the slab gel was determined after incubation of the gel pieces for 48 h at 40°C in a toluenebased scintillation fluid containing 12.5 g PPO, 125 mg POPOP and 6% (v/v) NCS (Nuclear Chicago Solubilizer). Samples were counted in a Mark II Liquid Scintillation Counter (Nuclear Chicago). Results The labelling kinetics of the individual ribosomal proteins were determined by incubating yeast protoplasts with tritiated amino acids at 15°C. From previous studies carried out in this laboratory [14,19] it appeared that the various steps in ribosome formation are all retarded to about the same extent when the temperature is lowered. At 15°C it takes 14-15 min before newly formed 26 S and 17 S rRNA appear in the cytoplasm (see refs. 14 and 19). In principle, therefore, appearance of newly formed radioactive copies of a particular protein on cytoplasmic ribosomal particles within that period of 15 min would signify relatively late assembly of that protein into the ribosomal structure. In this type of kinetic experiments, however, meaningful1 results can be obtainedonly if the pool of free ribosomal proteins is small relative to the rate of synthesis of ribosomal proteins, and if the pool size of the various individual ribo-

382 somal proteins is more or less equal. Therefore, we determined the pool size of free ribosomal proteins in yeast as described in Table I. The data indicate that only 0.4% of total yeast ribosomal protein is present in a soluble form. As demonstrated by the experiment shown in Table II this pool size is very small compared to the rate of synthesis of ribosomal proteins in yeast protoplasts at 15°C. The specific activity of uniformly labelled ribosomal protein in protoplasts decreases under chase conditions by a b o u t 6% in 30 min. Thus, at 15°C yeast protoplasts synthesize an amount of ribosomal protein per min equalling about half the pool size. The very low amount of radioactivity present in soluble ribosomal protein precluded exact determination of the relative pool sizes of the individual protein species. However, the radioactivity present in ribosomal protein, isolated from the ribosome-free supernatant and separated by two-dimensional electrophoresis, appeared to be distributed equally over the ribosomal protein spots on the gel slab (data not shown). Pool size, thus, is unlikely to be a complicating factor in the interpretation of the labelling kinetics of individual ribosomeb o u n d ribosomal proteins. A schematic representation of the separation pattern of yeast ribosomal proteins is depicted in Fig. 1. The labelling kinetics of the individual ribosomal proteins were measured by following the ratio of 3H pulse label to a '4C steady state label as a function of time (for details see Materials and Methods). In order to be able to compare the labelling kinetics of the various ribosomal proteins directly, the 3H/'4C ratio of each individual protein species after different pulse times was expressed as the percentage of the final 3H/14C ratio after 120 min of 3H labelling. As stated above, under the conditions used ribosomal maturation is essentially completed a b o u t 15 min after the start of transcription of the primary ribosomal transcript. Therefore, the relative 3H/'4C ratio after 15 min of labelling (designated as P (15 min) in Table III) in particular is relevant for discriminating between early and late assembled ribosomal proteins. As can be seen in Table III the P (15 min) values for the proteins of the 60-S

TABLE I D E T E R M I N A T I O N OF T H E P O O L SIZE OF F R E E R I B O S O M A L P R O T E I N IN Y E A S T Y e a s t cells w e r e unifox~nly labelled w i t h a m i x t u r e o f 3 H - l a b e l l e d a m i n o acids, a n d a r i b o s o m e - f r e e cellsap fraction w a s isolated as described in Materials and M e t h o d s . P r o t e i n s w e r e e x t r a c t e d f r o m this f r a c t i o n a n d a k n o w n p a r t w a s m i x e d w i t h unlabelled t o t a l y e a s t r i b o s o m a l protein. T h e p r o t e i n mixtttre w a s separated b y t w o - d i m e n s i o n a l p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s aecol~ling to K a l t s c h m i d t a n d W i t t m a n n [ 2 0 ] . R i b o s o m a l p r o t e i n spots, visualized b y staining, were c u t o u t a n d a s s a y e d f o r 3 H radioactivity. T h e t o t a l a m o u n t o f 3 H radioactivity present in soluble r i b o s o m a l p r o t e i n c a n t h e n be calculated. T h e t o t a l a m o u n t o f 3 H radioactivity present in b o u n d r i b o s o m a l p r o t e i n was d e t e r m i n e d b y assaying t h e p r o t e i n e x t r a c t e d f r o m t h e r i b o s o m a l p e l l e t o b t a i n e d f r o m t h e s a m e c u l t u r e (see Materials a n d M e t h o d s ) . T h e d a t a represent the average o f three separate e x p e r i m e n t s .

Protein f r a c t i o n

Free r i b o s o m a l p r o t e i n Bound ribosomal protein

Total 3H radioactivity (dpm)

%

0 . 0 5 • 106

0.4

1 1 . 0 7 • 106

99.6

383 T A B L E II DE NOVO SYNTHESIS OF RIBOSOMAL PROTEINS Y e a s t cells w e r e u n i f o r m l y l a b e n e d w i t h a m i x t u r e o f t r i t i a t e d a m i n o a c i d s d u z i n g five g e n e r a t i o n t i m e s , c o n v e r t e d i n t o p r o t o p l a s t s a n d c o n d i t i o n e d a t 2 9 ° C f o r 3 0 r a i n . T h e cells w e r e t h e n i n c u b a t e d w i t h a l a r g e e x c e s s o f n o n - l a b e l l e d a m i n o a c i d s a t 1 5 ° C . F r o m s a m p l e s , t a k e n a t 0, 3 0 a n d 6 0 r a i n , r e s p e c t i v e l y , r i b o s o m e s w e r e i s o l a t e d . T h e s p e c i f i c r a d i o a c t i v i t y o f t h e r l b o s o m a l p r o t e i n s in t h e r i b o s o m e s u s p e n s i o n s w a s subsequently determined by the biuxete assay [21] and radioactive measurements. The data represent the average of two separate experiments. Chase period (in r a i n )

3H dpm/ml ribosome suspension

mg protein/ml ribosome suspension

Specific radioa c t i v i t y (X 1 0 - 6 )

0 30 60

9.82 " 106 9 . 9 5 " 106 9.89 " 106

4.90 5.29 5.52

2.00 1.88 1.79

I00 94 89

ribosomal subunits range from 0 to 20. Obviously, the lowest value is representative for proteins, which associate with the primary transcript immediately after, or even during transcription. A somewhat arbitrary division of the proteins in two groups has been made (Table III). Group B is considered to represent proteins associating at an early stage of ribosome formation. Those listed in group A, most likely associate with the immature 60 S subunit at a relatively late stage of ribosome assembly. In particular, proteins L30, L45, L9, L7 and L6 seem to become incorporated into the large ribosomal subunit at a very late stage of its formation. In order to provide more evidence for this suggestion we determined the extent of labelling of the proteins present in nuclear 66-S preribosomal particles (Table IV). The 3H/14C ratio of the proteins was compared with the average ratio for the proteins L37, L20, L12, L22, L25, L10 and L21. These proteins associate already with the primary rRNA transcript as concluded from ~)2-D ORIC~N

~-o@ I

~)2--0 ORIGIN

l@,-v ~-Oel

io~-o

I 3

5¢

IC~ ~11

4

.....

"1

a c=._=_:_.~,

0 44 I 0451

~10

lj 8

16 ~---~

17 20 21 0

O0

oo

5 ~8 0

70

6 ''~

0 (::3- ~0ooozor-7~

~22

a~ ~'aT~ 028 (::)29

_~.

024

23

0373° 39

40S

O~ (92-D

~000,, 042

60S

043

®2-V

Fig. 1. S c h e m a t i c r e p r e s e n t a t i o n o f t h e t w o - d i m e n s i o n a l s e p a r a t i o n o f t h e r i b o s o m a l p r o t e i n s o f 4 0 - S a n d 80-S ribosomal subunits of yeast. Ribosomal proteins were separated by two-dimensional polyacrylamide gel e l e c t r o p h o r e s i s a c c o r d i n g t o K a l t s e h m i d t a n d W i t t m a n n (ref. 2 0 , see also refs. 1 5 a n d 1 6 ) . T h e f i g u r e is a t r a c i n g o f t h e s p o t s visible o n t h e t w o - d i m e n s i o n a l slab gel a f t e r s t a i n i n g w i t h C o o m a s s i e B r i l l i a n t Blue.

384 T A B L E III L A B E L L I N G K I N E T I C S O F T H E P R O T E I N S O F 60-S R I B O S O M A L S U B U N I T S IN Y E A S T Y e a s t p r o t o p l a s t s w e r e labelled at 15°C w i t h t r i t i a t e d a m i n o acids d u r i n g 15, 30, 45, 75 a n d 120 rain. C y t o p l a s m i c r i b o s o m e s w e r e i s o l a t e d f r o m e a c h pulse-labelled p r o t o p l a s t s u s p e n s i o n a f t e r a d d i t i o n of an e q u a l p o r t i o n o f a p r o t o p l a s t c u l t u r e labelled w i t h 14C-labelled a m i n o acids for 1 2 0 rain. R i b o s o m a l p r o t e i n s w e r e e x t r a c t e d f r o m the s e p a r a t e 40 S a n d 60 S p a r t i c l e s a n d s e p a r a t e d b y t w o - d i m e n s i o n a l p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s as d e s c r i b e d in Materials a n d M e t h o d s . T h e 3 H a n d 14C r a d i o a c t i v i t y in e a c h r i b o s o m a l p r o t e i n s p o t was d e t e r m i n e d . T h e 3 H / 1 4 C r a t i o of a p r o t e i n a f t e r 15, 30, 4 5 a n d 75 rain o f 3 H pulse-labelling w a s e x p r e s s e d as the p e r c e n t a g e o f t h e 3 H / 1 4 C r a t i o o f t h a t p r o t e i n a f t e r 1 2 0 rain o f 3 H labelling ( d e s i g n a t e d as P ) . T h e 3 H a n d 14C r a d i o a c t i v i t y o f t h e s t r o n g l y acidic r i b o s o m a l p r o t e i n s L 4 4 a n d L 4 5 w e r e a s s a y e d a f t e r t w o - d i m e n s i o n a l s e p a r a t i o n o f a p r o t e i n e x t r a c t o b t a i n e d f r o m undissocia t e d c y t o p l a s m i c r i b o s o m e s . T h i s w a s n e c e s s a r y b e c a u s e t h e s e t w o p r o t e i n s are lost w h e n 60-S r i b o s o m a l s u b u n i t s are p r e c i p i t a t e d w i t h e t h a n o l ( u n p u b l i s h e d results). I n this t a b l e o n l y t h e P v a l u e s at 15 rain a n d a t 4 5 r a i n are p r e s e n t e d . T h e d a t a r e p r e s e n t t h e a v e r a g e o f t w o s e p a r a t e e x p e r i m e n t s a n d m a y v a r y to as m u c h as 20%. A

B

Protein

P ( 1 5 rain)

P ( 4 5 rain)

Protein

P (15 min)

P ( 4 5 rain)

L30 L45 L9 L7 L6 L42 L44 L24 L41 L40 L32 L15 L16 L23 Lll L36 L8

20 12 11 10 9 8 S 6 6 5 5 3 3 3 3 3 3

55 51 46 45 35 30 30 30 26 23 20 19 19 19 19 17 17

L13 L18 L35 L34 L4 L3 L43 L31 L39 L33 L26 L29 L3S L17 L19 L5 L2 L37 L20 L12 L22 L25 LIO L21

1--2 1--2 1 1 1 1 1 1 1 1 1 1 1 1 1 0--1 0--1 0 0 0 0 0 0 0

19 10 11 13 12 12 12 11 9 10 12 10 7 11 S 11 10 10 10 9 11 8 8 7

their labelling kinetics (see the value of P (15 min) in Table III). Therefore, they are assumed to be present on every 66 S particle. Consequently, the 3H/ '4C ratios for these proteins can be used as a guide to detect proteins, which are clearly underrepresented in the 66-S precursor particles. As may be concluded from Table IV the 66 S fraction is deficient in several proteins previously shown to have a relatively high P (15 min) value (Table III) being indicative of a relatively late incorporation into ribosomal particles. This is especially true for proteins L30, L9, L7 and L24 and to a lesser extent for L6 and L15. Since the 66-S particles are always contaminated to some extent (maximally 7%) with cytoplasmic 60-S subunits [19], the first four protein species may even be completely absent from these nuclear precursor particles. Therefore, the conclusion seems to be warranted that proteins L30,

385 TABLE IV L A B E L L I N G C H A R A C T E R I S T I C S OF R I B O S O M A L P R O T E I N S IN N U C L E A R 66-S P R E R I B O S O M A L PARTICLES Yeast p r o t o p l a s t s w e r e labelled w i t h a m i x t u r e o f tritiated a m i n o acids at 1 5 ° C d u r i n g 45 vain. Nuclear 66-S p r e r i b o s o m a l particles w e r e isolated as described in Materials and M e t h o d s . T h e proteins extracted from these particles were separated by t w o - d i m e n s i o n a l p o l y a c r y l a m i d e gel electrophoresis [ 2 0 ] . P r i o r to electrophoresls 60-S subunit p r o t e i n s isolated f r o m yeast p r o t o p l a s t s labelled w i t h 14C-labelling a m i n o acids d u r i n g 1 2 0 rain w e r e a d d e d to the 3 H pulsc-labelled p r o t e i n s . Urflabelled 60-S r i b o s o m a l proteins were also a d d e d in order to be able to localize the r i b o s o m a l proteins o n the e l e c t r o p h e r o g r a m b y staining. The p r o t e i n s p o t s were c u t o u t from the slab gel and the 3 H / 1 4 C ratio in each s p o t w a s determined. Protein

L30 L45 L9 L7 L6 L42 L44 L24 L41 L40 L32 L15 L16 L23 Lll L36 L8

3H/I 4C (45 min) 1.2 n.d. * 0.6 0.6 1.7 2.7 n.d. * 1.1 3.6 3.7 3.6 2.1 3.0 "3.0 3.1 4.0 4.5

Protein

3 H/I 4 C ( 4 5 min)

L13 L18 L35 L34 L4 L3 L43 L31 L39 L33 L26 L29 L38 L17 L19 L5 L2 L37 L20 L12 L22 L25 LIO L21

3.5 5.0 4.3 3.5 3.4 3.6 4.9 3.8 3.7 4.7 4.0 3.3 3.9 5.1 3.5 3.3 3.4 5.5 3.6 3.5 3.5 4.7 4.3 4.0

* n.d., n o t determined.

L9, L7 and L24 associate with the precursor of the large ribosomal subunit only after its entry into the cytoplasm. Proteins L6 and L15 probably associate with the 66 S particle in the nucleus. The remaining proteins originally assigned to group A (cf. Table III) have 3H/14C ratios comparable to those of the reference proteins L37, L20, etc. and, thus, appear to be assembled into the immature ribosomal particle before its conversion into the 66 S particle. No conclusion with respect to the relative amounts of proteins L44 and L45, which also exhibit high P (15 rain) values (Table III), in the 66 S fraction are possible. Due to the diverging electrophoretic behaviour of these proteins [16] and their comigration with several other (non-ribosomal) proteins in this analysis, we were unable to determine their 3H/14C ratio in this fraction. Fig. 2 depicts the labelling kinetics of some ribosomal proteins isolated from cytoplasmic ribosomal particles in more detail. Newly formed copies of the proteins L30, L9, L7, L6, L44 and L45 (see e.g. L30 in Fig. 2) appear on cytoplasmic particles with a higher rate than the newly formed copies of the

386

E

E

0 100

o

L 30

80 o

~

o

a

k) 6O ~-~ I o~

L 42

L 32 40

~ .

L

18~ B

L31J 2O

15

30

45

75

pulse - ti me (rain)

Fig. 2. L a b e l l i n g kinetics of s o m e r i b o s o m a l p r o t e i n s i s o l a t e d f r o m 60-S c y t o p l a s m i c s u b u n i t s . T h e 3 H / 14C r a t i o of t h e r i b o s o m a l p r o t e i n s a f t e r a pulse-labelling w i t h t r i t i a t e d a m i n o acids d u r i n g 15, 30, 45, 75 a n d 1 2 0 r a i n w e r e d e t e r m i n e d (see l e g e n d to T a b l e I I I a n d Materials a n d M e t h o d s ) . T h e 3 H / 1 4 C r a t i o o f a r i b o s o m a l p r o t e i n a t a given pulse t i m e w a s p l o t t e d as t h e p e r c e n t a g e o f t h e 3 H / 1 4 C r a t i o a f t e r 1 2 0 rain o f labelling v e r s u s pulse t i m e . A a n d B r e f e r t o t h e r e s p e c t i v e g r o u p s o f r i b o s o m a l p r o t e i n s i n d i c a t e d in T a b l e III.

TABLE V L A B E L L I N G K I N E T I C S O F T H E P R O T E I N S O F T H E 40-S R I B O S O M A L S U B U N I T S I N Y E A S T T h e s a m e e x p e r i m e n t a l p r o c e d u r e as d e s c r i b e d in t h e l e g e n d t o T a b l e I I I w a s f o l l o w e d (see also Materials a n d M e t h o d s ) . T h e d a t a r e p r e s e n t t h e a v e r a g e of t w o s e p a r a t e e x p e r i m e n t s a n d m a y v a r y t o as m u c h as 20%. A

B

Protein

P (15 min)

P (45 min)

Protein

P (15 rain)

P (45 rain)

$34 $32 $27 $31 $10 $33 $25

8 5" 4 4 2 2 2

35 25 24 24 22 21 21

S19 S12 $28 S14/15 $4 $30 $22 $20 S2 S18 $21 $16 $7 $26 Sll S6 S17 S13 $24 $5 $3 $29

1 1 1 0--1 0--1 0--1 0--1 0---1 0 0 0 0 0 0 0 0 0 0 0 0 0 0

17 16 11 10 10 11 11 11 11 11 11 10 9 12 10 II 9 11 8 11 8 10

387

remaining 60-S subunit proteins assigned to group A (see e.g. L42 and L32 in Fig. 2). The immediate appearance of newly formed copies of the former proteins (L30, L9, L7, etc.) on cytoplasmic particles can only be explained if we assume that these proteins are exchanged against their unlabelled counterparts on mature ribosomes. Apparently these proteins are located near or on the surface of the mature 60 S subunit which would be consistent with our conclusion that they are assembled at a late stage of the formation of this subunit. The proteins assigned to group B (see e.g. L18 and L31 in Fig. 2) associate with the preribosomal particle with a rate comparable to the non-exchanging proteins of group A (e.g. L42 and L32) though at a later time. The labelling kinetics (expressed as P values) of the ribosomal proteins of the 40 S subunit are presented in Table V. Again a division can be made into two groups of proteins on the basis of these P values. In analogy with the argumentation given above for the proteins of the 60 S subunit, we consider group A to comprise proteins associating at a relatively late stage of the formation of the 40 S subunit, whereas group B represents proteins assembled at a relatively early stage of ribosome formation. Unfortunately it was impossible to isolate precursor particles of the 40 S subunit sufficiently pure to ascertain the presence on or absence of particular proteins from these particles. Discussion The labelling kinetic data presented in this paper clearly show that newly formed copies of a number of ribosomal proteins appear on yeast cytoplasmic ribosomal particles within about 15 min after the start of labelling (Tables III and V), the approximate time required for completion of a yeast ribosome under the conditions used. The translation of these data into the relative time of assembly of particular protein species is somewhat complicated by the fact that newly formed copies of several of the rapidly appearing 60-S subunit proteins (L30, L45, L9, L7, L6 and L44) apparently are capable of being exchanged against their counterparts already present on mature ribosomes (cf. refs. 22 and 23). Study of the labelling kinetics of the ribosomal proteins in the nuclear 66-S preribosomes, however, revealed that the 66-S particles lack completely or almost completely the proteins L30, L9, L7 and L24, and, in addition, are deficient in the amount of proteins L6 and L15 (Table IV). The first four proteins, thus, very likely associate with the immature yeast 60 S subunit very late in the maturation process. Probably, the same holds true for the proteins L44 and L45, of which the presence or absence on 66-S precursor particles could not be established. On the other hand, proteins L6 and L15 most probably associate with the immature large subunit at the level of the 66 S particle. As to the remaining 60-S and 40-S subunit proteins a more or less continuous range of P values is observed (Tables III and V). Apparently, these proteins are assembled into the preribosomal structures at an early stage of the maturation process. Because of the relative inaccuracy in the determination of the P values (see legends to Tables III and V), however, it is difficult to draw any conclusion with respect to the order of assembly of these ribosomal protein species.

388 The late assembly of several proteins in yeast resembles the situation in higher eukaryotes: studies on the assembly of ribosomes in rat liver [10,24] and HeLa cells [12,25--28] also revealed the existence of a number of ribosomal protein species that associate with the immature ribosome at a very late stage of ribosome biosynthesis. Because yeast is a primitive eukaryote it is also interesting to compare our findings with respect to ribosome formation with the large body of data assembled on this process in prokaryotic cells. When the two-dimensional gel electrophoretic pattern of yeast 60-S subunit proteins [15,16] is compared with that of the proteins isolated from 50-S subunits of Escherichia coli [29] it can be seen that the E. coli proteins L6 and L l l migrate at approximately the same position as yeast proteins L7 and L9. Pichon et al. [30] have shown that in E. coli proteins L6 and L l l , which possibly are involved in protein biosynthesis [31,32], are among the last species assembled into the large ribosomal subunit, whereas our results show the same to be true for the yeast proteins L7 and L9. A certain structural as well as a functional homology might thus exist between these two pairs of proteins. The same argument applies to the proteins L44 and L45 from yeast on the one hand and proteins L7 and L12 from E. coli on the other. In both organisms these strongly acidic proteins associate with the immature large subunit at a late stage of its formation (Table III and ref. 30). Therefore, L44 and L45, just like similar strongly acidic ribosomal proteins from mammalian cells [33], may be structurally related to the E. coli proteins L7 and L12, which are required for ribosomal function in protein biosynthesis [34]. Finally, studies on the phosphorylation and methylation of yeast ribosomal proteins revealed that these types of modification are almost completely confined to the late assembling ribosomal proteins (unpublished results), suggesting that modification of these ribosomal proteins does not play an essential role in the process of ribosome assembly.

Acknowledgements The present study was supported in part by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). The authors are indebted to Dr. W.H. Mager for stimulating discussions. We thank Dr. H.A. Raue for critically reading the manuscript.

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