A site of reversible conformational alteration in rat liver ribosomes

BIOCHIMICA ET BIOPHYSICA ACTA

147

BBA 95188

A SITE OF R E V E R S I B L E CONFORMATIONAL ALTERATION IN RAT L I V E R RIBOSOMES T. HULTIB! AND A. SJ~QVIST

Department o/ Cell Physiology, Wenner-Gren Institute, Stockholm (Sweden) (Received November 28th, 1968)

SUMMARY

I. A protein in the larger subunit of rat liver ribosomes, characterized by disc electrophoresis on polyacrylamide gel, has been used as a marker of a reversible conformational alteration in the particles. In normal ribosomes or in ribosomes dissociated by treatment with EDTA in the cold, the protein is relatively resistant to chymotrypsin. It is rapidly transformed by trypsin into a fragment ("T") resistant to further trypsin or chymotrypsin attack. 2. Under several well-defined conditions the marker protein (or its shielded, tryptic fragment) are rendered susceptible to chymotrypsin by a time- and temperature-dependent reaction. Three such conditions are described: (A) incubation of the (dissociated) ribosomes at 35 ° in Mg2+-free media; (B) incubation of the ribosomes with acridine reagents; (C) incubation with media of increased ionic strength. Chymotrypsin transforms the unmasked marker protein or T-fragment into a smaller fragment, C. 3. The unmasking of the marker protein (or its tryptic fragment) by incubation at increased ionic strength is reversible, again by a temperature-dependent reaction. The unmasking and its reversal are not markedly influenced by MgCI~ within the range of 1.5-16. 5 raM. 4. The activity of the ribosomes in endogenous or poly U-directed amino acid incorporation is not seriously impaired by the unmasking or by the proteolytic test reactions.

INTRODUCTION

The initial action of proteolytic enzymes on rat liver ribosomes is to a considerable extent determined by structural factors 1. Because of the compactness of the particles, the proteolytic attack primarily involves proteins with peptide loops of suitable specificity exposed at the ribosomal surface, i.e., outside the range of structural shielding. Proteolytic enzymes can therefore be used as tools not only for indicating the location of individual proteins in relation to the ribosomal surface ~, but also for detecting and specifying such conformational alterations in the particles that may modify the shielding pattern a. The latter possibility is illustrated by a protein in the larger ribosomal subunit a, easily identified by its characteristically low rate of cathodic migration in Biochim. Biophys. Acta, 182 (1969) 147-157

148

T. HULTIN, A. SJOQVIST

polyacrylamide-gel electrophoresis. In intact ribosomes this protein is readily attacked b y trypsin, but is remarkably resistant to chymotrypsin 2. Available data indicate that the main part of the molecule is located inside the particles a, effectively protected against proteolytic attack, while a minor, well-defined loop, containing basic amino acids but deficient in aromatic amino acids 5, reaches the ribosomal surface 2. In the experiments described below we have studied some conditions which lead to the unmasking of a chymotrypsin-sensitive part of this protein by a reversible, time- and temperature-dependent reaction. Although the reaction can be induced by Mg 2+ deficiency, neither the unmasking as such nor its reversal is inhibited by the presence of bivalent cations at concentrations commonly used in ribosomal systems. RNA m a y be involved in the shielding, since this is gradually eliminated by preincubation of the particles with ribonuelease, although at fairly high concentrations. MATERIALS AND METHODS

Preparation and pretrealment o] ribosomes Liver ribosomes were prepared from starved 2o0-3oo-g Sprague-Dawley rats by the method of RENDI AND HULTIN 6, and were stored at --20 °. The particles were normally suspended in an ice-cold medium containing 15o mM sucrose, 75 mM KC1, 5 mM MgC12, I mM mercaptoethanol and 35 mM Tris-HC1 buffer (pH 7.7, measured at 25°). The suspensions were cleared b y centrifuging for 7 min at 15 ooo ×g, and were adjusted to a protein concentration 7 of 2 mg/ml. Trypsin-pretreated ribosomes (T-ribosomes) were prepared by incubating the ribosomal suspensions for 5 rain at 35 ° with i . o # g / m l trypsin. After incubation, IO/~g/ml soybean trypsin inhibitor was added. For dissociation, the ribosomes were suspended in MgC12-free medium containing i mM EDTA, and were dialyzed for 2 h (o °) against lOO-2OO vol. of the same medium. The ionic strength of the ribosomal suspensions was increased by adding calculated amounts of solid KC1, NH4C1 or NaC1. To restore the normal ionic composition, I-ml samples were dialyzed for 3 h with vigorous stirring against 200 vol. of twice-changed, ice-cold suspension medium. The efficiency of the equilibration was verified b y analyses.

Assay o/ structural shielding I-ml samples of pretreated or control ribosomes were incubated with chymotrypsin, usually for IO rain at 35 °, at a concentration of 1.2/,g/ml, or otherwise as specified in the legends. The proteolytic action was stopped b y the addition of 35/,I of 6 M HC1 to the chilled suspension. The ribosomes were extracted twice with cold o.2 M HC1 as described previously 2. The protein extracts were dialyzed for 3-16 h against o.oi M HC1, and urea was added to a final concn, of 1.7 M. Disc electrophoresis was run in a 12.5 % polyacrylamide-gel system (pH 4.3), essentially as described by REISFELD et al. s, but in the presence of 7 M urea. The length of the columns was IOO or i8o ram, the diameter 5 ram. After staining for I h in a solution of I °/o Amidoschwarz in 1.2 M acetic acid, the columns were destained by transversal electrophoresis in 1.2 M acetic acid, and stored in ethylene glycol at --20 °. Biochim. Biophys. Acta, 182 (1969) 147-157

CONFORMATIONAL ALTERATION IN RIBOSOMES

149

The electrophoretic patterns were recorded with a Joyce-Loebl double-beam microdensitometer, provided with a 62I-nm interference filter, and usually operated with an optical wedge of 0-2.

Amino acid incorporation The endogenous and poly U-dependent activity of the ribosomal preparations in amino acid incorporation was determined as described previously 2.

Chemicals Trypsin (EC 3-4.4.4), e-chymotrypsin (EC 3.4.4.5), ATP and GTP were obtained from the Sigma Chemical Co., St. Louis, Mo. Because of the relatively high ribonuclease tolerance of the system, no efforts were made to remove the minor traces of ribonuclease that may contaminate these twice-crystallized preparations. Soybean trypsin inhibitor was obtained from the Serva Entwicklungslabor, Heidelberg, Germany, proflavine from the Mann Research Lab., New York, N.Y., acridine orange from the E. Merck AG, Darmstadt, Germany, poly U from the Miles Chemical Co., Clifton, N.J., and E14Clleucine (34.0 C/mole) and [14Clphenylalanine (44.2 C/mole) from the Radiochemical Centre, Amersham, England.

RESULTS

Unmasking o/ ribosomal proteins in Mg2+-delicient media The normal disc-electrophoretic pattern of rat liver ribosomal proteins under the conditions used in the present experiments is illustrated by Fig. IA. A group of rapidly migrating proteins, designated in the diagram by Roman numerals, is clearly separated from a less rapidly moving group designated by Arabic figures. The protein which we were mainly concerned with in this paper belongs to the latter group, and is referred to as Protein IO. Treatment of intact ribosomes with trypsin or chymotrypsin produced characteristic alterations in the protein pattern, as illustrated by Figs. IB and IC. As has been described previously~, Protein IO is relatively resistant to chymotrypsin, but is rapidly altered by trypsin. A major tryptic fragment °, resistant to further attack, appears in the electrophoretic pattern as a well-defined peak (T) superimposed on Fraction 7. Protein 5 in the same group of proteins is refractory to both enzymes under these conditions. Rat liver ribosomes, treated for 2 h at o ° with a Mg2+-free medium containing I mM EDTA, dissociate almost completely into subunits z. When the dissociated particles were incubated with chymotrypsin at 35 ° in the same medium, the previously resistant Proteins 5 and IO were readily attacked, and a major fragment of Protein io was accumulated near position 6 (Fig. 2A, Peak C). As is shown by Fig. 2B, there was a marked difference between Protein 5 and Protein IO in their responses to the dissociating medium. If the Mg2+ concentration of the EDTA-treated suspension was restored before the incubation with chymotrypsin (or if the Mg2+-free suspension was tested with chymotrypsin at o°), Protein 5 was degraded, while Protein IO remained about as resistant as the controls (cl. Fig. IC). However, only a few minutes preincubation of the Mg~+-deficient ribosomes at 35 ° before the addition of MgC12 was sufficient to uncover chymotrypsin-sensitive parts of Protein io (Figs. 2C and Biochim. Biophys. Mcta, 182 (1969) 147-157

zSO

T. HULTIN, A. SJOQVIST

A

+ I

B

C

2

®

5 9

3

6

V

\ Fig. I. E f f e c t s of t r y p s i n a n d c h y m o t r y p s i n o n r a t liver r i b o s o m a l p r o t e i n s in situ. T h e r i b o s o m e s were s u s p e n d e d in a m e d i u m c o n t a i n i n g 15o m M sucrose, 75 m M KC1, 5 m M MgC1v I m M merc a p t o e t h a n o l a n d 35 m M Tris-HC1 b u f f e r (pH 7.7), a n d were i n c u b a t e d for io m i n (35 °) as specified below. T h e p r o t e i n s were e x t r a c t e d w i t h 0.2 M HC1 a n d were a n a l y z e d b y disc electrophoresis in 12.5 % p o l y a c r y l a m i d e gel (pH 4-3). A. Control ribosomes. ]3. R i b o s o m e s i n c u b a t e d w i t h 0. 4 / ~ g / m l t r y p s i n . C. R i b o s o m e s i n c u b a t e d w i t h 1.2 /~g/ml c h y m o t r y p s i n . I n C u r v e A t h e s h a d e d a r e a i n d i c a t e s t h e p o s i t i o n of t h e c h y m o t r y p s i n - r e s i s t a n t p r o t e i n fraction io. T h e section of t h e electrophoretic p a t t e r n i n c l u d e d in s u b s e q u e n t figures is i n d i c a t e d b y arrows. I n Curves ]3 a n d C (and in t h e s u b s e q u e n t figures) d o w n w a r d arrows indicate loss, a n d u p t u r n e d arrows gain, in a b s o r b a n c e . T h e a m o u n t of e x t r a c t e d p r o t e i n a d d e d to each c o l u m n w a s 95 #g.

2D)*. The dissociation was not reversed by the addition of MgC12 under any of these conditions10, n. It is concluded that in the presence of EDTA the structural shielding of Protein 5 was lost concomitantly with the irreversible ribosomal dissociation, while a more energetic treatment was required to alter the shielding pattern at the site of Protein IO.

Selective unmasking o/Protein zo by acridine reagents The experiments shown in Fig. 3 provide additional evidence that the unmasking of chymotrypsin-sensitive parts of Protein I0 is independent of ribosomal dissociation. When ribosomes (suspended in the normal, MgC12-containing medium) were * T h e s e d i m e n t a t i o n v e l o c i t y of t h e s u b u n i t s in sucrose g r a d i e n t c e n t r i f u g a t i o n was n o t m a r k e d l y r e d u c e d b y this p r e i n c u b a t i o n , b u t s o m e u l t r a v i o l e t - a b s o r b i n g m a t e r i a l a p p e a r e d in t h e regions b e t w e e n a n d a b o v e t h e s u b u n i t peaks.

Biochim. Biophys. Acta, 182 (1969) 147-157

CONFORMATIONAL ALTERATION IN RIBOSOMES A

B

C

®

)

7 98

151

D

9 6

7

5,

c

i

Fig. 2. U n m a s k i n g of c h y m o t r y p s i n - s e n s i t i v e p a r t s of P r o t e i n IO b y p r e i n c u b a t i o n of dissociated r i b o s o m e s in t h e p r e s e n c e of E D T A . F o r dissociation, t h e r i b o s o m e s were s u s p e n d e d in Mg 2+free m e d i u m , c o n t a i n i n g i m M E D T A , a n d were dialyzed for 2 h (o °) a g a i n s t i o o v o l u m e s of t h e s a m e m e d i u m . A. T h e dissociated r i b o s o m e s were i n c u b a t e d for i o m i n (35 °) in t h e Mg *+free E D T A m e d i u m w i t h 1.2/zg/ml c h y m o t r y p s i n . B. Before t h e i n c u b a t i o n w i t h c h y m o t r y p s i n , 6 m M MgC12 w a s a d d e d to t h e d i s s o c i a t e d ribosomes. C. Similar to B, b u t t h e r i b o s o m a l s u s p e n sion w a s p r e i n c u b a t e d for I m i n (35 °) in t h e Mg2+-free E D T A m e d i u m before t h e a d d i t i o n of 6 m M MgC1 v D. Similar to C, b u t t h e p r e i n c u b a t i o n w a s for 3 m i n (35 °) before t h e MgC12 addition. A

B

C

D 5

E

®

7

9

6

Fig. 3. U n m a s k i n g of c h y m o t r y p s i n - s e n s i t i v e p a r t s of P r o t e i n to b y p r e i n c u b a t i o n of r i b o s o m e s w i t h proflavine. A. T h e r i b o s o m e s were i n c u b a t e d for io rain (35*) w i t h 1.2/~g/ml c h y m o t r y p s i n in n o r m a l s u s p e n s i o n m e d i u m (c]. Fig. 1) c o n t a i n i n g 0.5 m M proflavine. B. Before t h e c h y m o t r y p s i n t e s t t h e a d d e d p r o f l a v i n e w a s largely r e m o v e d f r o m t h e m e d i u m b y dialysis. C. Similar to B, b u t t h e r i b o s o m e s were p r e i n c u b a t e d for 5 m i n (35 °) in t h e p r o f l a v i n e - c o n t a i n i n g m e d i u m before dialysis. D. Similar to A, b u t t h e r i b o s o m e s were i n c u b a t e d for 20 m i n a t o ° w i t h 5/~g/ml c h y m o t r y p s i n in t h e p r e s e n c e of o. 5 rnlVl proflavine. E. Similar to D, b u t t h e r i b o s o m e s were p r e i n c u b a t e d for 5 rain (35 °) in t h e p r o f l a v i n e - c o n t a i n i n g m e d i u m before b e i n g t e s t e d w i t h chym o t r y p s i n at 0%

incubated with chymotrypsin at 35 ° in the presence of o. 5 mM proflavine, Protein IO was readily attacked b y the enzyme, while Protein 5 remained completely resistant (Fig. 3A). Although some dye was bound to the ribosomes in the cold (c/. ref. I2), Protein IO was not affected by chymotrypsin if most of the added proflavine had been removed b y dialysis before the enzyme incubation test (Fig. 3B). A few minutes of preincubation at 35 ° before dialysis, however, was sufficient to induce the chymotrypsin-sensitive state (Fig. 3C). As in the experiments with EDTA, the structural unmasking of Protein IO obviously involved a temperature-dependent mechanism. This is further illustrated b y the fact that the presence of proflavine had no influence on the degradation pattern if the incubation with chymotrypsin was carried out at o ° Biochim. Biophys. Acta, 182 (1969) 147-157

152

T. HULTIN, A. SJ()QVIST

o

sos eOT'r~d

TOP BOrTO~

TOP

Fig. 4. S e d i m e n t a t i o n p a t t e r n of ribosomes, p r e i n c u b a t e d u n d e r c o n d i t i o n s l e a d i n g to u n m a s k i n g of P r o t e i n io. A. P r e i n e u b a t i o n for io m i n (35 °) in m e d i u m c o n t a i n i n g 0. 5 m M p r o f l a v i n e (c[. Fig. 3). Before c e n t r i f u g a t i o n m o s t of t h e d y e w a s r e m o v e d b y filtration t h r o u g h S e p h a d e x G-25. B. P r e i n c u b a t i o n for io m i n (35 °) in m e d i u m c o n t a i n i n g 90o m M KC1 (c[. Fig. 5). T h e ionic c o m p o s i t i o n of t h e 9 - 3 6 ~o sucrose g r a d i e n t s w a s t h e s a m e as t h a t of t h e p r e i n e u b a t i o n media. C e n t r i f u g a t i o n w a s for 18o m i n at 39 ooo r e v . / m i n in Spinco r o t o r S W 39. (The slower s e d i m e n t a tion rate in E x p t . B w a s d u e to t h e h i g h e r KC1 c o n c e n t r a t i o n ) .

(Fig. 3D). However, a few minutes of preincubation at 35 ° made Protein IO highly chymotrypsin-sensitive even at low temperatures (Fig. 3E). The ribosomes were not dissociated by the preincubation with proflavine (Fig. 4A). Identical results were obtained with other, commonly used acridine reagents, e.g., acridine orange.

Reversible unmasking o/ Protein zo in media o/high ionic strength As is shown by Fig. 5A, Protein IO was readily attacked by chymotrypsin if the ribosomes were incubated with the enzyme at 35 ° in a medium containing 0. 9 M KC1. At the same time the characteristic chymotryptic fragment C appeared in the electrophoretic pattern. As in the previous experiments, the unmasking was temperature dependent. Protein io remained chymotrypsin resistant if the KC1 concentration was brought back to the normal level (by dialysis in the cold or by dilution) before the test incubation at 35 ° or if the incubation was carried out in the 0. 9 M A

B C

C

D 5

5 .

E 5

/ t] Fig. 5. Reversible u n m a s k i n g of c h y m o t r y p s i n - s e n s i t i v e p a r t s of P r o t e i n IO b y p r e i n c u b a t i o n of r i b o s o m e s in m e d i u m w i t h increased ionic s t r e n g t h . A. T h e r i b o s o m e s were i n c u b a t e d for 3 rain (35 °) w i t h i o / , g / m l c h y m o t r y p s i n in s u s p e n s i o n m e d i u m with i n c r e a s e d KC1 c o n c e n t r a t i o n (900 mM). B. Before t h e i n c u b a t i o n w i t h c h y m o t r y p s i n a t 35 ° t h e excess KC1 w a s r e m o v e d b y dialysis a g a i n s t n o r m a l s u s p e n s i o n m e d i u m . C. Similar to A, b u t t h e r i b o s o m e s were i n c u b a t e d for 20 m i n a t o ° w i t h I o / * g / m l c h y m o t r y p s i n in t h e 90o m M KC1 m e d i u m . D. Similar to B, b u t t h e r i b o s o m e s were p r e i n c u b a t e d for IO m i n (35 °) in t h e 900 m M KC1 m e d i u m before diMysis a g a i n s t n o r m a l s u s p e n s i o n m e d i u m . E. Similar to D, b u t t h e p r e i n c u b a t e d a n d dialyzed r i b o s o m e s were a g a i n p r e i n c u b a t e d for lO m i n (35 °) in t h e n o r m a l m e d i u m before being t e s t e d w i t h c h y m o t r y p s i n (reversal of u n m a s k i n g ) .

Biochim. Biophys. Acta, 182 (I969) 147-157

153

CONFORMATIONAL ALTERATION IN RIBOSOMES

KC1 medium at o ° (Figs. 5B and 5C). However, only a few minutes of preincubation at 35 ° in the high KC1 medium made Protein IO chymotrypsin sensitive under these conditions, and the unmasking was not reversed by dialysis in the cold or by dilution (Fig. 5D). The KCl-induced unmasking of Protein IO was readily (although not quantitatively) reversed when the preincubated and dialyzed ribosomes were again incubated for a few min at 35 ° in the normal medium before the chymotrypsin test (Fig. 5E). At the same time, the formation of the chymotryptic fragment C in the test system was suppressed. (To demonstrate the reversibility of the KC1 effect, whether the test incubation with chymotrypsin was carried out at o ° or 35 ° was of minor importance, since no reversal could be observed in the presence of the enzyme.) A

B

c

D

i

5

E

,

g ® ,

987

+

Fig. 6. Mg 2+ independence of KCl-induced u n m a s k i n g of Protein io. The incubation with chym o t r y p s i n (5.o/,g/ml) was for 4 min (35 °) at the following KC1 concentrations: A, 85o mM; B, C, 75 ° mM; D, E, 65 ° mM. The MgC12 concentrations were 16.5 mM (Curves A, B, D) or 1.5 mM (Curves C, E), as compared to t h e 5 mM MgC12 in the normal suspension medium.

The sensitization of Protein IO to chymotrypsin by incubation at 35 ° in high KC1 media occurred in the concentration range of 0.6-0. 9 M (Figs. 6A, 6C and 6E). The same result was obtained when the ionic strength was increased b y the addition of NH4C1 or NaC1 (not illustrated). The unmasking, as well as its reversal, were not markedly influenced by variations in the Mg *+ concentration (Fig. 6), and the reactions were not inhibited b y spermine or cycloheximide at concentrations which strongly interfere with amino acid incorporation (not illustrated). The ribosomes were only slightly dissociated by incubation for IO min (35 °) in the 0. 9 M KC1 medium (Fig. 4B), and their activity of endogenous and poly Udirected amino acid incorporation was not seriously impaired 3 (Table I).

Unmasking o] the shielded tryptic ]ragment o/ Protein Io These experiments were carried out with ribosomes in which Protein IO had been transformed into fragment T by tryptic digestion ("T-ribosomes"). As is shown b y Fig. 7A, the T-fragment showed the same resistance to chymotrypsin as Protein io in the intact particles (c/. Fig. IC). The resistance was not lost when the T-ribosomes were dissociated by treatment with E D T A in the cold (Fig. 7B). However, a few minutes preincubation of the dissociated particles at 35 ° before the addition of MgC12 made the shielded fragment chymotrypsin sensitive, and chymotryptic fragment C appeared in the electrophoretic pattern (Figs. 7 C and 7D). Biochim. Biophys. Acta, 182 (1969) I47-157

154

T. HULTIN, A. SJOQVIST

TABLE I ENDOGENOUS I0

UNMASKED

AND POLY U-DEPENDENT OR CHYMOTRYPSIN

AMINO ACID INCORPORATION

BY RIBOSOMES

V*~ITH P R O T E I N

DEGRADED

R i b o s o m e s , s u s p e n d e d in a m e d i u m of o.15 1Y[ sucrose, 75 m M KC1, 5 m M MgC12, i m M m e r c a p t o e t h a n o l a n d 35 m M Tris-HC1 b u f f e r (pH 7-7), were p r e i n c u b a t e d for io rain (35 °) as indicated. i - m l s a m p l e s of t h e i n c u b a t e d s u s p e n s i o n s were d i l u t e d w i t h 3 m l m e d i u m a n d were placed on t o p of c e n t r i f u g e t u b e s c o n t a i n i n g 7-5 m l m e d i u m of h i g h e r d e n s i t y (o. 3 M sucrose). A f t e r cent r i f u g i n g for 6o m i n a t 15o ooo × g, t h e a c t i v i t y of t h e s e d i m e n t e d particles w a s d e t e r m i n e d in a n a m i n o acid i n c o r p o r a t i n g s y s t e m c o n t a i n i n g io m M p h o s p h o e n o l p y r u v a t e , I m M A T P , o.2 m M G T P , 2o p g / m l p y r u v a t e kinase, 23 # M L- Ex*CJleucine or 17. 5/uM L- [a4C]phenylalanine, i o o m M KC1, 3 ° m M Tris-HC1 b u f f e r (pH 7.7), 8 m g / m l cell-sap p r o t e i n a n d p r e i n c u b a t e d r i b o s o m e s at a final conch, of i.o m g R N A p e r ml. P o l y U, w h e n added, was 300 p g / m l . T h e i n c o r p o r a t i o n w a s m e a s u r e d a t d i f f e r e n t MgC1, c o n c e n t r a t i o n s . T h e i n d i c a t e d v a l u e s refer to MgC12 c o n c e n t r a t i o n s of 7 m M ( e n d o g e n o u s incorporation) a n d 8 - 9 m M (poly U - d e p e n d e n t incorporation), which g a v e o p t i m u m activity. T h e v a l u e s are e x p r e s s e d as c o u n t s / m ~ n p e r ioo # g r i b o s o m a l R N A . B a c k g r o u n d values, o b t a i n e d in t h e a b s e n c e of r i b o s o m e s (27, 52 a n d 7 ° c o u n t s / m i n , respectively), h a v e been s u b t r a c t e d .

Additions to preincubation system

Incorporation Leucine

None C h y m o t r y p s i n (1.2/zg/ml) KC1 (0.9 M) KC1 plus c h y m o t r y p s i n P r o f l a v i n e (0. 5 mM) P r o f l a v i n e plus c h y m o t r y p s i n

A

B

Phenylalanine

747 647 72o 673 503 427

C

Without poly U

With poly U

553 484 535 47 ° 371 323

1865 1961 1957 1946 1671 1132

D

C

65

~

®

Fig. 7. U n m a s k i n g of t h e t r y p t i c f r a g m e n t T of P r o t e i n io b y p r e i n c u b a t i o n of t r y p s i n - t r e a t e c t r i b o s o m e s (T-ribosomes) in t h e presence of E D T A . T - r i b o s o m e s were p r e p a r e d b y t r e a t i n g ribosomes, for 5 m i n (35 °) with. I.O p g / m l t r y p s i n in n o r m a l s u s p e n s i o n m e d i u m (4. Fig. IB). A f t e r t h e a d d i t i o n of io p g / m l of s o y b e a n t r y p s i n inhibitor, a c o n t r o l s a m p l e (A) w a s w i t h d r a w n f r o m t h e s u s p e n s i o n , while t h e r e s t of t h e m a t e r i a l w a s dissociated b y t h e a d d i t i o n of io m M E D T A a n d dialysis for 2 h (o °) a g a i n s t 20o vol. of Mg~+-free m e d i u m c o n t a i n i n g i m M E D T A . A. Tr i b o s o m e s i n c u b a t e d for 5 m i n (35 °) w i t h 2.5 Ng/ml c h y m o t r y p s i n in n o r m a l s u s p e n s i o n m e d i u m . B. Dissociated T-ribosomes, to w h i c h 6 m M MgC12 w a s a d d e d before t h e t e s t i n c u b a t i o n w i t h c h y m o t r y p s i n . C. Similar to B, b u t t h e s u s p e n s i o n of d i s s o c i a t e d T - r i b o s o m e s w a s p r e i n c u b a t e d for i m i n (35 °) in t h e E D T A - c o n t a l n i n g , Mg2+-free m e d i u m before t h e a d d i t i o n of 6 m M MgC1v D. Similar to C, b u t t h e p r e i n c u b a t i o n w a s for 4 m i n (35 °) before t h e MgCI~ addition.

Biochim. Biophys. Acta, 182 (1969) 147-157

CONFORMATIONAL ALTERATION IN RIBOSOMES A

B

C

155

D

5

5

5

'l 6c

Fig. 8. Unmasking of the tryptic fragment T of Protein lO by preincubation of T-ribosomes in media with increased ionic strength, or in the presence of proflavine. A. T-ribosomes, prepared as described in Fig. 7, were treated in the cold with medium containing 9oo mM KC1 (c[. Fig. 5B). The excess KC1 was removed by dialysis against normal suspension medium before the test incubation for 3 min (35 °) with io/.~g/ml chymotrypsin. B. Similar to A, but the suspension of T-ribosomes in the 900 mM KC1 medium was preincubated for io min (35 °) before dialysis against normal suspension medium. C. Similar to B, but the preincubated and dialyzed T-ribosomes were again incubated for Io min (35*) in normal medium before the chymotrypsin incubation test (partial reversal of unmasking). D. The T-ribosomes were preincubated for 5 min (35 °) in suspension medium with 0. 5 mM proflavine. The particles were dialyzed against normal medium before the chymotrypsin incubation test.

Transformation of fragment T into fragment C by chymotryptic cleavage was also observed when T-ribosomes were treated with media of increased ionic strength (Figs. 8A and 8B), or with acridine reagents (Fig. 8D). However, as with intact ribosomes the treatment was only effective at increased temperature. The unmasking was partially reversed when T-ribosomes, treated with high KC1 medium at 35 ° and subsequently dialyzed against normal medium, were reincubated for a few min at 35 ° before being submitted to the chymotrypsin test (Fig. 8C).

E]]ect o] ribonuclease on the shielding pattern When rat liver ribosomes were preincubated for IO min at 35 ° with ribonuclease at different concentrations, the resistance of Protein IO to chymotrypsin gradually disappeared (Fig. 9). For complete unshielding of this protein fairly high ribonuclease concentrations (c/. ref. 13) were needed (50/~g/ml). The structural shielding of Protein 5 was also successively eliminated by ribonuclease, although with somewhat greater difficulty (Figs. 9B and 9C).

Activity o] ribosomes in amino acid incorporation alter chymotryptic degradation o/ Protein xo The activity of ribosomes in endogenous or poly U-directed phenylalanine incorporation was only moderately reduced by preincubation for io min at 35 ° in media containing either 0. 9 M KC13, 1.2/,g/ml chymotrypsin 2 or 0.5 mM proflavine. (In the presence of 0.5 mM proflavine, the functions of rat liver ribosomes are strongly inhibited13,14.) In view of the marked potentiation of the chymotryptic attack in high KC1 media (Figs. 5, 6 and 8) or in the presence of acridine dyes (Figs. 3 and 8) it seemed of interest to measure the incorporating activity of the particles after preincubation Biochim. Biophys. Acta, 182 (1969) I47-I57

156

T. HULTIN, A. SJOQVIST 2

c

t

2t

Y

l

r Fig. 9. Unmasking of ribosomal proteins by ribonuclease. The ribosomes were preincubated for io min (35 °) with ribondclease at the concentrations indicated below. After the addition of 1.2/~g/ml chymotrypsin the incubation was continued for another io min. Ribonuclease concentrations: A, 5/~g/ml; B, 25/~g/ml; C, 50/,g/ml.

for IO rain at 35 ° with chymotrypsin in the fortified test systems with o. 9 M KC1 or 0.5 mM proflavine. Despite the complete disappearance of Protein Io under these conditions, there was only a limited further reduction in the activity of endogenous or poly U-dependent amino acid incorporation (Table I). DISCUSSION

The method used in this investigation for studying induced alterations in the conformation of ribosomes is restricted by the difficulties involved in quantitative protein separation. The resolving power of polyacrylamide-gel electrophoresis alone is clearly unsatisfactory even with I8o-mm columns, and it has not yet been possible to combine this method with other fractionation procedures without seriously reducing the possibilities of quantification. The experiments described deal with a protein in the larger ribosomal subunit (Protein IO), which has a particularly favorable location in the electrophoretic pattern. Several pieces of evidence suggest that this fraction is essentially uniform: (I) Its marked resistance to solubilization from the particles by displacement with protamine in the presence of MgC12 (ref. 4). (2) Its characteristic pattern of tryptic degradation in situ, notably the formation of a well-defined fragment (T) which shows the same resistance to protamine extraction as the parent protein 9. (3) The various specific conditions, described in the present paper, which lead to the complete abolishment of its normal resistance to chymotrypsin. The difficulty with which Protein IO is solubilized from the ribosomes with protamine in Mg~+-containing media 4, in combination with its relatively low electrophoretic mobility in 12.5 % polyacrylamide gel, suggests that the molecule is of appreciable size, and under normal conditions deeply anchored in the ribosomal structure. The trypsin-sensitive loop may be located near one end of the polypeptide chain, since the tryptic fragment T is only moderately displaced towards the more rapidly migrating part of the disc-electrophoretic pattern. The sensitization of the protein Biochim. Biophys. Acta, 182 (1969) 147-157

CONFORMATIONAL ALTERATION IN RIBOSOMES

157

to chymotrypsin may involve the uncovering of less terminal and more deeply located sequences, since the chymotryptic fragments (C) have a higher electrophoretic mobilit y in the gel than the T-fragments, and are less intensely stained. Although the C-fragments seem to be somewhat less well defined than the T fragments, it is notable that they have approximately the same position in the disc-electrophoretic pattern independently of the procedure used to induce the chymotrypsin sensitivity. No evidence of a reversible detachment of Protein IO from the particles has been obtained under the conditions described here. Both the uncovering of chymotrypsin-sensitive sites of Protein IO and its reversal are mediated through a temperature-dependent reaction. The ability of acridine reagents to induce this reaction suggests that the shielding depends on specific features in the RNA structure 12,15. The (moderate) sensitivity of the shielding pattern to ribonuclease points in the same direction. No correlation was observed between the uncovering of Protein IO and ribosomal dissociation. The exposed, trypsin-sensitive part of Protein IO was not of functional importance for the reversible, conformational reactions for which this protein served as a marker. The T-fragment was sensitized to chymotrypsin under the same exacting conditions as the intact protein was, and formed the same kind of chymotryptic fragment. The reversibility of the unshielding could also be demonstrated with the trypsin-treated ribosomes. The structural flexibility of the larger ribosomal subunit around Protein IO would obviously be compatible with a translocation function of this region in peptide synthesis. However, the experimental data clearly indicate that neither the part of Protein IO which is normally trypsin sensitive nor that which becomes chymotrypsin sensitive by incubation of the particles in the presence of proflavine or high ionic strength are necessary for endogenous or poly U-dependent amino acid incorporation. ACKNOWLEDGMENTS

This investigation was supported by research grants from the Swedish Cancer Society (67:113) and the Swedish Natural Science Research Council (307-9). We thank Mr. L. Ericson, Miss Kristina Irvall and Mrs. Birgit Lundberg for valuable technical assistance. REFERENCES I T. HULTIN AND U. 0STNER, Biochim. Biophys. Acta, 147 (1967) 178. 2 U. 0STNER AND T. HULTIN, Biochim. Biophys. Acta, 154 (1968) 376. 3 T. HULTIN AND U. 0STNER, Biochim. Biophys. Acta, 16o (1968) 229. 4 J- S. ROTH AND T. HULTIN, F E B S Letters, I (1968) 16. 5 M. BERGMANN AND J. S. FRUTON, Adv. Enzymol., I (1941) 63. 6 R. RENDI AND T. HULTIN, Exptl. Cell Res., 19 (196o) 253. 7 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. EARR AND R. J. RANDALL, dr. Biol. Chem., 193 (1951) 265. 8 R. A. REISFELD, O. J. L E w i s AND D. E. WILLIAMS, Nature, 195 (1962) 281. 9 T. HULTIN, Abhandl. Deut. Ahad. Wiss. Berlin, in the press. IO Y. TASI.IIRO AND T. MORIMOTO, Biochim. Biophys. Acta, 123 (1966) 523I I J. STAHL, G, R. LAWFORD, B. WILLIAMS AND P. N. CAMPBELL, Biochem. J., lO9 (1968) 155. 12 A. ~v~. FURANO, D. F. BRADLEY AND L. G. CHILDERS, Biochemistry, 5 (1966) 3044 . 13 T. HULTIN AND K. A. ABRAHAM, Biochim. Biophys. Acta, 87 (1964) 232. 14 I. B. WEINSTRIN AND I. H. FINKELSTEIN, J. Biol. Chem., 242 (1967) 3757. 15 R. I. COTTER, P. MCPHIE AND W. B. GRATZER, Nature, 216 (1967) 864.

Biochim. Biophys. Acta, 182 (1969) 147-157