Disulfide interaction in situ between two neighbouring proteins in mammalian 60-S ribosomal subunits

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Biochimica et Biophysica A cta, 579 (1979) 10--19

© Elsevier/North-Holland Biomedical Press

BBA 38243 DISULFIDE INTERACTION IN SITU BETWEEN TWO N E I G H B O U R I N G PROTEINS IN MAMMALIAN 60-S RIBOSOMAL SUBUNITS ISOLATION OF THE CONTACT REGION OF THE L A R G E R PROTEIN

HEINZ NIKA and TORE HULTIN Department of Cell Physiology, Wenner-Gren Institute, University of Stockholm, S-11345 Stockholm (Sweden)

(Received December 19th, 1978) Key words: Ribosomal protein; Disulfide interaction; Chymotryptic cleavage; (Rat)

Summary A disulfide complex is formed in situ under gentle conditions between two neighbouring proteins in the 60-S subunits of mammalian ribosomes. The proteins have been identified as L 4 and L 29. The complex is easily isolated from whole ribosomes, and can be utilized for preparing the two proteins in a very pure state for further characterization. Chymotryptic cleavage of the complex or the isolated larger protein (L 4) in the presence of SDS produces two unequal fragments of this protein in nearly quantitative yield. The smaller fragment (approx. 12 000 daltons) contains the contact sequence. Only this fragment of protein L 4 is labelled when rat liver ribosomes are incubated w i t h iodo[14C]acetate under conditions of complex formation. Protein L 29 is resistant to chymotrypsin in the presence of sodium dodecyl sulfate.

Introduction

The 60-S subunits of mammalian ribosomes contain two adjacent proteins, previously referred to as proteins 10 and IIIA, which interact reversibly in situ under gentle conditions by forming an intermolecular disulfide bridge [1,2,]. The interaction is greatly facilitated by incubating the ribosomes at increased ionic strength or in the presence of intercalating heteroaromatics [1--4]. This causes a reversible unmasking of the larger protein, which can be followed quantitatively by use of molecular probes [1--5]. For disulfide interaction the absence of soluble thiols is essential, and the reaction is prevented by thiol reagents [1 ].

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The disulfide-linked protein couple can be isolated by preparative polyacrylamide gel electrophoresis [1]. As will be shown below, this provides a convenient m e t h o d of preparing the two proteins in a very pure state for further characterization. A fragment of the larger protein, containing the contact region, can be isolated after c h y m o t r y p t i c cleavage of the isolated complex. Materials and methods

Ribosomes Rat liver ribosomes and 60-S ribosomal subunits were prepared as described [6,7]. Disulfide interaction between the two investigated ribosomal proteins was induced as described previously [1]. In brief, ribosomes were dialyzed overnight (0°C) against a thiol-free medium containing 75 mM KC1, 5 mM MgC12 and 20 mM Tris-HCl, pH 7.7, incubated for 10 min (35°C) with 0.9 M KC1 or 0.6 mM atebrin, diluted with plain medium and repelleted b y centrifuging for 60 min at 165 000 X gay. For the labelling of exposed SH groups ribosomes (50--60 A260 units/ml) were incubated for 10 min (35°C) with 0.01 mM iodo[14C]acetate in the presence of 0.9 M KC1 or 0.6 mM atebrin. Unlabelled iodoacetate (1 mM) was added, and incubation was continued for 5 min. The reaction was stopped with 50 mM mercaptoethanol. Proteins were extracted from whole ribosomes with 0.2 M HC1 [8], and from 60-S subunits with acetic acid [9].

Polyacrylamide gel electrophoresis Preparative electrophoresis in 7.5% polyacrylamide gel slabs (100 X 200 X 4.5 mm) was run at pH 4.3 essentially as described [1]. Urea concentration was 8 M. The gels were prerun overnight in gel buffer [10] containing 1 mM mercaptoethylamine. During separation the electrode buffer (70 mM ~-alanine in 27.5 mM acetic acid) completely surrounded the cells. It was maintained at 0--4°C and was circulated continously between the electrode compartments. Preparative SDS gradient gel electrophoresis was run in 7--15% polyacrylamide gels (16°C) utilizing the system of Laemmli [11]. When disulfide bonds were to be preserved, mercaptoethanol was omitted, and glycerol was replaced with 2 M urea. The thickness of the gels was 3 mm. Diagonal SDS gel electrophoresis was run in 10--20% gradient gels using the same system without mercaptoethanol in the first separation. After rapid staining and destaining [12], 5 mm gel strips were equilibrated for 15 min (35°C) with the mercaptoethanolcontaining sample buffer [11 ] and submitted to a second electrophoresis in the same system. The thickness of the gels was 1.0 and 1.5 mm in the first and second separations, respectively. Two-dimensional gradient gel electrophoresis for analytical separation was based on the system of Reisfeld et al. [10] in the first dimension. The 10--15% polyacrylamide gradient gels (pH 4.3) contained 8 M urea, and were 1.0 mm thick. The second separation in 10--20% SDS gradient gels was as described above for diagonal electrophoresis. Two-dimensional electrophoresis for the cross-identification of ribosomal proteins was essentially as described b y Welfle et al. [13]. The first separation was in 4.5% polyacrylamide gel, pH 8.9, the second separation in 10% gel, pH 7.2, containing 0.1% SDS [14,15].

12 Preparative gels were stained for 15 min in a solution of 0.25% Coomassie brilliant blue, 7% acetic acid and 50% methanol. Analytical gels were stained overnight. Destaining was in 7% acetic acid containing 50% methanol.

Peptide analysis Proteins, dissolved in 10 mM HC1, were incubated for 16 h at 37°C with pepsin (enzyme to substrate ratio 1 : 50). Incubation was continued in 50 mM ammonium bicarbonate with trypsin (enzyme to substrate ratio 1 : 50). At 4 h a second portion of trypsin was added. After 16 h the digest was acidified with acetic acid, lyophilized, and dissolved in 0.3 M pyridine acetate, pH 2.5. The peptides were analyzed by high-pressure liquid chromatography using a 2 X 150 mm column of DC-4A cation exchange resin. The peptides were eluted at a rate of 16 ml/h with a gradient of pyridine acetate buffer as indicated. The eluate was reacted with ninhydrin for 4 min (100°C) and passed through the 10 pl flow cell (10 mm light path) of a Labotron UDC p h o t o m e t e r provided with a 570 nm interference filter.

Limited proteolysis of ribosomal proteins in SDS Incubation with chymotrypsin (enzyme to substrate ratio 1 : 10) was as described by Cleveland et al. [12] with the following modifications: glycerol was replaced by 2 M urea. Heating at 100°C was omitted in the case of the disulfide-containing protein complex to avoid dissociation. Protein concentration was 0.5--0.8 mg/ml. Split products were analyzed by SDS gradient gel electrophoresis and diagonal electrophoresis as described above.

Chemicals ~-Chymotrypsin (EC 3.4.21.1, 3 times crystallized) was obtained from Sigma Chemical, St. Louis, Mo, cation exchange resin DC-4A from Durrum Chemical Corp., Palo Alto, Ca, and iodo[2-14C]acetic acid (57 Ci/mol) from the Radiochemical Centre, Amersham, England. Results

Identification of the interacting proteins The two proteins in the 60-S subunits of mammalian ribosomes which are capable of disulfide interaction in situ were identified previously by two-dimensional polyacrylamide gel electrophoresis, and preliminarily designated as proteins 10 and I I I A [1,2]. The proteins are indicated by arrows in the twodimensional gradient gel electrophoretic pattern shown in Fig. 1A, which represents proteins from partially purified rat liver 60-S ribosomal subunits. The recent nomenclature of rat liver ribosomal proteins has been developed on the basis of partially different electrophoretic principles [13,16,17]. The separations shown in Fig. 1B,C were run in the two-dimensional system used by Welfle et al. [13] to facilitate the cross-identification of ribosomal proteins separated by different electrophoretic methods. In Fig. 1C the proteins from 60-S ribosomal subunits had been supplemented with the dissociation products of the isolated disulfide complex. The products were superimposed on t w o spots corresponding to proteins L 4 and L 29. Like protein " I I I A " [1,2], pro-

13

B

1

2

l

i

Fig. 1. P r o t e i n s f r o m p a r t i a l l y p u r i f i e d 6 0 - S r i b o s o m a l s u b u n i t s a n a l y z 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 . ( A ) T h e p o s i t i o n s ( a r r o w s ) o f t h e t w o i n t e r a c t i n g p r o t e i n s (1) as d e t e r m i n e d b y g r a d i e n t gel e l e c t r o p h o r e s i s in (1) a c i d / u r e a [ 1 0 ] , (2) S D S / T r i s [ 1 1 ] o f a 5 0 ~ g p r o t e i n s a m p l e . (B,C) C r o s s - i d e n t i f i c a t i o n o f t h e s a m e p r o t e i n s u s i n g t h e e l e c t r o p h o r e t i c s y s t e m o f Welfle e t al. [ 1 3 ] . ( 1 ) T r i s / b o r a t e , p H 8 . 9 , (2) S D S / p h o s p h a t e . In (B) 1 5 0 ~zg o f r i b o s o m a l p r o t e i n s w e r e u s e d . In (C) 3 0 /~g o f isol a t e d p r o t e i n c o m p l e x h a d b e e n a d d e d t o 1 2 0 ~zg o f r i b o s o m a l p r o t e i n s b e f o r e e l e c t r o p h o r e s i s . T h e dissociated components comigrated with proteins corresponding to L 4 and L 29 (arrows). Encircled area, weakly staining acid protein [15].

14 tein L 29 [13] is characterized by a remarkably low rate of migration in the SDS/phosphate system [ 14]. Isolation o f proteins L 4 and L 29 The specific disulfide interaction between proteins L 4 and L 29 was utilized for preparing these two proteins in a pure state from whole, preconditioned [1] ribosomes. A 40--50% complex formation was regularly obtained in these ribosomes. The extracted proteins were first separated by preparative gel electrophoresis, usually in the acid/urea system [1,10]. Provided that the distance of migration was sufficient, the L 4-L 29 complex was obtained in an almost pure state as analyzed by SDS gradient gel electrophoresis (Fig. 2A,B). After reduction, proteins L 4 and L 29 were isolated by preparative SDS gel electrophoresis (Fig. 2B). Remaining, non-complexed proteins L 4 and L 29 could be recovered from the first, preparative gel in a partially purified form (Fig. 2A). Since both proteins have a relatively low mobility in the SDS as compared with the acid urea system [1,2], effective purification was obtained by subsequent SDS gel electrophoresis (Fig. 2C,D).

A

B

C

D

,t

O

,t

Fig. 2. I s o l a t i o n of p r o t e i n s L 4 a n d L 29 f r o m w h o l e r a t liver r i b o s o m e s b y use of t h e specific disulfide c o m p l e x (A,B), a n d b y t h e d i r e c t e l e c t r o p h o r e t i c m e t h o d ( A , C , D ) . ( A ) P r e p a r a t i v e e l e c t r o p h o r e t i c separat i o n ( a c i d / u r e a s y s t e m ) o f p r o t e i n s f r o m p r e c o n d i t i o n e d r i b o s o m e s i n c u b a t e d f o r 10 rain ( 3 5 ° C ) w i t h 0.9 M KCl. T h e L 4-L 29 c o m p l e x a n d t h e sites of r e m a i n i n g , u n c o m p l e x e d p r o t e i n s L 4 a n d L 29 are indic a t e d b y a r r o w s . (B) P r o t e i n s f r o m t h e e x c i s e d L 4-L 29 b a n d ( A ) w e r e s e p a r a t e d b y p r e p a r i t i v e SDS g r a d i e n t gel e l e c t r o p h o r e s i s b e f o r e a n d a f t e r m e r c a p t o e t h a n o l t r e a t m e n t , (C) P r o t e i n s f r o m t h e L 4 a n d L 29 sites ( A ) w e r e s e p a r a t e d b y p r e p a r a t i v e SDS g r a d i e n t gel e l e c t r o p h o r e s i s . T h e d i s t i n c t b a n d s i n d i c a t e d b y a r r o w s c o n t a i n e d essentially p u r e p r o t e i n s L 4 a n d L 29, as s h o w n b y SDS g r a d i e n t gel e l e c t r o p h o r e s i s ( D ) a n d c o n f i r m e d b y a n a l y t i c a l t w o - d i m e n s i o n a l g r a d i e n t gel e l e c t r o p h o r e s i s ( n o t i l l u s t r a t e d ) .

15 The purified proteins L 4 and L 29 produced distinct and strikingly different peptide patterns after pepic-tryptic hydrolysis, indicating few sequence homologies (Fig. 3). As would be expected from the marked difference in molecular weights [ 1,13,18], protein L 4 was characterized by greater structural complexity. Contact region o f protein L 4 When the isolated L 4-L 29 complex was iacubated with chymotrypsin in the presence of SDS [12], the proteolytic attack was largely confined to one narrow site on the L 4 chain (Fig. 4). The cleavage was very rapid, and prolonged incubation (30 min) did not lead to further degradation. Protein L 29 was resistant under these conditions, and remained in the residual complex. In the electrophoretic pattern this complex migrated at a rate intermediate between proteins L 4 and L 29 (Fig. 4C,F). A large split product of protein L 4 migrated in the gel slightly ahead of the degraded complex. By mercaptoethanol treatment the complex was dissociated into intact protein L 29 and a minor fragment, which evidently contained the contact sequence of protein L 4 (Fig. 4D). In the electrophoretic system used in these experiments [11] the minor fragment migrated slightly ahead of the B chain of the concomitantly dissociated chymotrypsin (formular weight 13 927) [19]. In the experiment shown in Fig. 5 the chymotryptic digest of the L 4-L 29 complex was submitted to diagonal SDS gel electrophoresis using mercaptoethanol for disulfide reduction between the two electrophoretic separations. As

1D

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LU 0 Z


r.I,

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MINUTES F i g . 3. P e p t i d e p a t t e r n s o f p r o t e i n s L 4 ( A ) a n d L 2 9 ( B ) a f t e r p e p t i c - t r y p t i c h y d r o l y s i s . T h e h y d r o l y s a t e s (12--15 nmol) were analyzed on a 2 X 150 mm cation exchange column (DC-4A) maintained at 52°C. A gradient w i t h a starting b u f f e r o f 0 . 1 M p y r i d i n e a c e t a t e ( p H 3 . 5 ) a n d a l i m i t i n g b u f f e r o f 2 . 0 M p y r i d i n e a c e t a t e ( p H 5 . 0 ) w a s u s e d for e l u t i o n at a rate o f 1 6 m l / h . ( A ) 6 0 0 ~ g o f p r o t e i n L 4. ( B ) 3 9 0 / ~ g o f p r o t e i n L 29. , Absorbance; ...... , transmittance (100---0%).

t t

*a

-25760 -23426

Fig. 4. Isolation of a chymotryptic fragment of the L 4-L 29 complex, containing the contact region of protein L 4. The L 4-L 29 complex, isolated as in Fig. 2. was incubated with chymotrypsin in the presence of 0.5% SDS and analyzed by SDS gradient gel electrophoresis [ill. (A) Complex incubated without enzyme. (B) Chymotrypsin alone (without and with incubation). (C) Complete chymotryptic digest. Residual complex and large split product of protein L 4 indicated by arrows. (D) Same as (C). but mercaptoethanol added before electrophoresis to release contact fragment of protein L 4 (arrow). (E) Same as (B), but mercaptoethanol added to dissociate chymotrypsin into subunits B and C (13 927 and 10 157 daltons). (F) Same as (A), but mercaptoethanol added to dissociate the intact disulfide complex into proteins L 4 and L 29. Additional molecular weight designations refer to the following protein markers: ovalbumin (43 OOO), chymotrypsinogen A (25 760). papain (23 426). and myoglobin (17 200).

in the previous experiment (Fig. 4), very little of the complex remained intact after proteolysis (Fig. 5, encircled spots). The large fragment of protein L 4 was unaffected by mercaptoethanol together with small amounts of unspecific split products. The modified complex was dissociated by mercaptoethanol into protein L 29 and the small contact fragment of protein L 4. The latter was not quite homogeneous, but included traces of chains with slightly different mobility. Chymotrypsin was dissociated by mercaptoethanol to subunits (B,C) which were also displaced from the diagonal pattern. The main component of the separated contact fragment of protein L 4 was excised from the gel and resubmitted to SDS gel electrophoresis [ 111. Only one component was observed (data not shown). Although essential for the identification of the contact fragment, the intermolecular disulfide bridge of the L 4-L 29 complex was without importance for the selectivity in the chymotryptic cleavage. In the experiment shown in Fig. 6

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Fig. 5. D i a g o n a l S D S g r a d i e n t gel e l e c t r o p h o r e s i s o f c h y m o t r y p t i c split p r o d u c t s o f t h e L 4 - L 2 9 c o m p l e x . I n c u b a t i o n a n d first e l e c t r o p h o r e s i s w e r e as in Fig. 4 C . T h e first d i m e n s i o n gel w a s t r e a t e d w i t h m e r c a p t o ethanol before the second separation. The large fragment of protein L 4 (not displaced by mercaptoethan o D , t h e r e l e a s e d p r o t e i n L 2 9 , a n d t h e s m a l l c o n t a c t f r a g m e n t o f p r o t e i n L 4 are i n d i c a t e d b y a r r o w s . E n c i r c l e d w e a k s p o t s i n d i c a t e d i s s o c i a t i o n p r o d u c t s (L 4, L 2 9 ) o f u n d e r g r a d e d c o m p l e x . T h e d i s s o c i a t e d B a n d C c h a i n s o f c h y m o t r y p s i n serve as i n t e r n a l m a r k e r s (cf. Fig. 4).

ribosomes were incubated with iodo[14C]acetate under conditions otherwise leading to complex formation. The alkylated proteins L 4 and L 29 were isolated as described. Aliquots were incubated with chymotrysin in the presence of SDS, and analysed by SDS gel electrophoresis together with undigested controls (Fig. 6A). Protein L 4 was completely split into two fragments of unequal size. As in the experiments with the isolated L 4-L 29 complex (Figs. 4,5) the small fragment migrated slightly ahead of the B chain of chymotrypsin, indicating identity with the contact fragment described above. The autoradiograph of the same gel showed that only this fragment had been labelled in situ by the iodo[~4C]acetate that prevented disulfide interaction (Fig. 6B). A small amount of labelled material migrated closely behind the main component (cf. Fig. 5). The isolated protein L 29 was not degraded under these conditions even at an enzyme to substrate ratio of 1 : 5.

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A

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Fig. 6. I s o l a t e d p r o t e i n s L 4 a n d L 2 9 p r e l a b e l l e d in s i t u w i t h i o d o [ 1 4 C ] a c e t a t e a n d i n c u b a t e d s e p a r a t e l y w i t h c h y m o t r y p s i n in t h e p r e s e n c e o f SDS. T h e l a b e l l e d p r o t e i n s w e r e i s o l a t e d as i l l u s t r a t e d in Fig. 2 C,D, b u t in t h e p r e s e n c e o f m e r c a p t o e t h a n o l . C h y m o t r y p s i n t r e a t m e n t w a s as f o r t h e L 4 - L 29 c o m p l e x (Figs. 4 , 5 ) . ( A ) S t a i n e d g r a d i e n t gel. (B) A u t o r a d i o g r a p h . ( a , b ) P r o t e i n L 4 i n c u b a t e d w i t h o u t a n d w i t h c h y m o t r y p s i n . C h y m o t r y p t i c f r a g m e n t s i n d i c a t e d b y a r r o w s . ( c , d ) P r o t e i n L 29 i n c u b a t e d w i t h o u t a n d w i t h c h y m o t r y p s i n . T h e l a r g e f r a g m e n t o f p r o t e i n L 4 (b) w a s u n l a b e l l e d .

Discussion

This study deals with two neighbouring proteins, L 4 and L 29 [13], in mammalian 60-S ribosomal subunits, characterized by high conformational and phylogenetic flexibility [2--4]. Protein L 29 is probably located near 'the peptidyltransferase center [20]. Under conditions of reversibly induced conformational alteration proteins L 4 and L 29 are capable of disulfide interaction in situ. We have found that a fragment of the larger protein (L 4) containing the contact sequence can be prepared in good yield from the L 4-L 29 complex or from the isolated protein L 4 after chymotryptic cleavage. The fragment is used at present in the further characterization of the contact region. The apparent molecular weights of ribosomal proteins, as determined by SDS gel electrophoresis, vary with the electrophoretic system, probably because of the high positive charge [21]. Using the system of Weber and Osborn [14], our early measurements [ 1 ] indicated minimum molecular weights for the two interacting proteins of 45 000 and 30 000, in close agreement with recent data on proteins L 4 and L 29 by Welfle et al. [13]. Using the electrophoretic sys-

19 tem of Laemmli [ 11], the corresponding values are appreciably smaller [18]. In the gradient gels described above the apparent molecular weights of the same proteins were 37 000 and 22 000, respectively. In the same gradient gels the two chymotryptic fragments of protein L 4 showed apparent molecular weights of approximately 12 000 (contact fragment) and 26 000 (Fig. 4). The latter fragment is probably identical with a fragment of protein L 4 obtained in situ by incubating comformationally altered ribosomes with chymotrypsin [3,4]. Although the chymotryptic attack on protein L 4 in the presence of SDS [12] was restricted to a narrow region at approximately 1/3 of the length of the protein chain, a small proportion of the .contact fragments had a slightly deviating electrophoretic mobility (Figs. 5, 6B). The internal region open to chymotryptic attack may contain several adjacent sites of which one is greatly preferred. Alternatively, some degradation may also occur at terminal sites of the molecule. Acknowledgment The work was supported by a grant from the Swedish Natural Science Research Council. We thank Mr. T. Hult and Miss Solveig Sundberg for valuable technical assistance. References 1 2 3 4 5 6 7 S 9 10 11 12 13 14 15 16 17 18 19 20 21

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