J. Mol. Biol. (1976) 108, 781-787

Close Proximity of Escherichia coli 50 S Subunit Proteins L7/L12 and LIO and L l l

Dhnethyl adipimidate has been used to study the proximity relations between Escherich~ coli 50 S ribosomal proteins. The composition of two cross-linked protein complexes has been determined. Proteins L7/L12 involved in GTP hydrolysis dependent on elongation factor G, elongation factor Tu or initiation factor 2 are found to be neighbours of protein L10 and of protein Lll , which probably forms part of the peptidyl transferase centre.

Bifunctional reagents have proved useful in the analysis of neighbourhood relations between ribosomal proteins (Bickle eta/. , 1972; Chang & Flaks, 1972; Lutter ~ ~., 1972; Slobin, 1972; Shih & Craven, 1973; Traut et al., 1973; Barritault ~ al., 1975). However, very few applications of these reagents to the study of protein neighbour- hoods in the 50 S ribosome have been reported (Clegg & Hayes, 1972,1974; Expert- Bezan?on et al., 1975). Here we briefly describe results obtained in experiments in which two of the protein complexes produced by treatment of Escherichia coli 50 S ribosomal subunits with dimethyl adipimidate have been purified and their constituent proteins identified. One of the complexes contains proteins L7/L12, which are known to be involved in GTP hydrolysis dependent on elongation factor G, elongation factor Tu or initiation factor 2 (Hamel et al., 1972; Sander et a~., 1972; Fakunding ~ al., 1973 ; Terhorst ~ al., 1973) and protein L l l , which probably forms part of the peptidyl transferase centre (Nierhaus & l~ontejo, 1973), and the other proteins L7/L12 and L10.

Autoradiography of dried polyacrylamide gel slabs upon which the protein com- plement of dimethyl adipimidate-treated, 35S-labelled 50 S ribosomes has been separated by two-dimensional electrophoresis (Kaltschmidt & Wittmann, 1970; Barritault et al., 1975) reveals the presence of many new radioactive products which do not exist in the protein complement of untreated 50 S ribosomes. Several of these new products possess electrophoretic mobilities lower than that of any normal 50 S protein, and almost certainly correspond to complexes of two or more proteins joined by intermolecular cross-links. One of them situated in the left half of the two-dimen- sional gel slab above the spot containing ribosomal protein L9 (position occupied by spot a/b in Fig. 2(b)) was selected for analysis. The region of the gel slab containing this product was cut out and its content of 35S-labelled material, which will be referred to as complex a/b, was eluted. Figure 1 shows the results of autoradiography of a polyacrylamide gel slab upon which a sample of the eluted material was subjected to electrophoresis in the presence of sodium dodecyl sulphate. I t can be seen that most (>80~ of the radioactivity migrated in a single band containing material whose molecular weight, estimated by comparison with those of ribosomal proteins $4 and L2 and haemoglobin dimer (Hb2), is 30,0004-2000. Unlabelled carrier 50 S proteins were added to the remainder of the eluate of the 35S-labelled cross-llnKed material, the mixture was subjected to ammonolysis to cleave cross-links (Barritault d al., 1975), cleavage products were separated by two-dimensional gel electrophoresis and the gel slab was stained, dried and autoradiographed. Of the four spots visible

781

782 A. E X P E R T - B E Z A N ( ~ O N E q ' A L .

FIG. 1. Molecular weight and homogeneity of dimethyl adipimidate cross-linked complex a/b. Samples of 25S-labelled complex a/b and of radioactive and non-radioactive control proteins

were subjected to electrophoresis in a 15~o (w/v) polyaerylamide gel in the presence of sodium dodecyl sulphate (Laemmli, 1970). The gel slab was then stained and dried (Barritault st al., 1975) and an autoradiograph prepared after exposure for 6 days using Kodak RPS-X-Omat X-ray film.

a, 7000 cts/min of 3BS-labelled complex a/b; b, 1000 cts/min of 35S-labelled complex a/b; e, 2000 ors/rain of 35S-labelled protein L2.

The positions of stained bands containing Hb, Hb2, Hb4 (monomer, dimer and tetramer of haemoglobin, respectively) and proteins L2, and $4 are indicated.

on the a u t o r a d i o g r a p h (Fig. 2), th ree comigra te wi th t he s t a ined 50 S pro te ins L10, L l l a n d L7]L12 and one (spot a /b in Fig . 2(b)) migra tes as t he or iginal mate r ia l . Spots cor responding to each of the 50 S r ibosomal p ro te ins and to t he p roduc t s vis ible on the a u t o r a d i o g r a p h were cut ou t and the i r r a d i o a c t i v i t y measured . The resul ts ob ta ined , p resen ted in Table 1 and in t he h i s tog ram of F igure 3, a re i n t e rp re t ed as follows.

P

(a)

7tl 2~: tuna!am P-lmWW |

j i v - -

2O

U

C:3

~ac

32 33+,t 9,

I j.. ~-.

(b) FIo. 2. Identif icat ion of the components of complex a/b.

(a) Autoradiograph of the two-dimensional eleetrophoretie separat ion of the products of ammonolysis of 200,000 cts]min of asS-labelled complex a/b. After ammonolysis and several precipitations with acetone in the presence of cold carrier 50 S proteins, 50~o of the input radio- ac t iv i ty was recovered. The recovered produets were then separated by two-dimensional electro- phoresis on a polyaerylamide gel. The autoradiograph of the gel slab was developed after exposure for 2 weeks.

(b) Drawing showing the positions of stained spots (carrier 50 S ribosomal proteins) in the gel slab whose autoradiograph is shown in (a). Prote in L31 is not seen. Proteins L33 and L34 migrate close to the buffer front and are not separated. Spot a/b is detected only on the autoradiograph (top).

784 A. E X P E R T - B E Z A N ~ O N E T A L .

I 000

c

u

> ,

0 500

i f )

I 00

L7 /L I2

_10

4 68101515171921 25 30

7/12 II 50S ribosomel proteins

54

FIG. 3. Histogram of the 3sS distribution in the 50 S ribosomal proteins shown in Fig. 2. All the spots corresponding to 50 S proteins were cut out, hydrolysed by incubation overnight

in 50% (v/v) H202 at 50~ and counted. After deduction of a background of 50 to 100 cts/min (see Fig.), spots a/b (unreacted cross-linked material) L7/L12, L10 and Lll were found to contain 2950 to 3000 (value not shown in Fig.), 970 to 1020, 650 to 700, and 700 to 750 cts/min of 35S, respectively. About 45 % of the total recovered radioactivity was therefore present in ammonolysis products L7/L12, LI0 and Lll. The very low overall recovery of radioactivity (100,000 cts/min of 35S applied to the first-dimension gel, approx. 5500 ets/min of 35S recovered in spots a/b, L7/L12, L10 and L 11) is due to experimental losses during the 2-dimensional electrophoretic separation of ammonolysis products and the hydrolysis and counting of gel samples, and not to loss of material during the ammonolysis reaction (this problem will be discussed in detail elsewhere).

Proteins L7/L12 contain three sulphur atoms (Terhorst etal. , 1973) and proteins L10 and L l l each contain six or seven sulphur atoms (Dzionara etal. , 1970; Kalt- schmidt et al., 1970; Bakardjieva & Crichton, 1974). By assuming uniform 35S- labelling of our 50 S particles (cells were labelled during 6 to 8 generations), the stoichiomctry of the cross-linked material can be evaluated by comparison of the radioactivity per equivalent sulphur atom in the ammonolysis products. The results presented in the Table show that the eluted cross-linked material contains proteins L7/L12, L10 and L l l in a mol ratio of approximately 3 : 1 : 1 . Considered together, this result and the estimated molecular weight of the eluted cross-linked material (30,000=}=2000, see above and Fig. 1) exclude the presence of complexes (LT/L12)3- L10-L l l , (L7/L12)2-L10-Lll and L7/L12-L10-Ll l (calculated molecular weights 76,000, 64,000 and 51,000, respectively) but do not distinguish between the ternary complex (L7/L12)3 and the binary complexes L7fL12-L10, L7 /L12-Ll l and L10- L11 (calculated molecular weights 38,000, 32,000, 33,000, and 40,000, respectively). However, the presence of complexes L 1 0 - L l l and (L7/L12)3 in the cross-linked material can be eliminated for the following reasons. (1) Complex L 1 0 - L l l has already been identified in the form of three closely neighbouring spots situated just above 50 S protein L1 in two-dimensional gels, i.e. in the upper part of the right half of the gel slab (spots 19 S, 19, 19i in Fig. 1 of Expert-Bezancon et al., 1975) at a considerable distance from spot a/b in Figure 2, and at a position corresponding

L E T T E R S TO T H E E D I T O R

TABLE 1

Stoichiometry measurements

785

S: Number of G: Molecular Protein weight sulphur atoms cts]10 r, ln G]S

( X 10 -3) per protein -- molecule background

L7]L12 12.5 3 9 7 0 0 - 1 0 2 0 0 3200-3400 LI0 19 6-7 6500-7000 930-1170 Ll l 19.6 6-7 7 0 0 0 - 7 5 0 0 1000-1250

Values for molecular weight and sulphur content are calculated from published data (Terhorst e~ al., 1973; Laemmli, 1970; Dzionara et al., 1970; Kaltschmidt et al., 1970). The latter are con- sidered to be accurate to within 4-1 sulphur atom.

to a pI value intermediate between those of the unmodified parent proteins. A similar relationship between the pI values of ribosomal proteins and their related cross-linked complexes has been observed in all cases so far examined in this labora- tory. Furthermore, the different forms of complex L 1 0 - L l l all migrate significantly more slowly than Hb2 and 50 S protein L2 in sodium dodecyl sulphate/polyacrylamide gels (Expert-Bezancon et al., 1975), whereas (see Fig. 1) the complexes present in the spot analysed in this report migrate faster than L2 or Hb2. (2) We have invariably observed that individual (i.e. non-cross-linked) ribosomal proteins containing imidoester substituents introduced by reaction with dimethyl adipimidate or dimethyl suberimidate display p I values slightly but significantly higher than those of the unmodified proteins (small rightward displacement in two-dimensional gels). A ternary complex containing L7/L12 would be expected to occupy a position above and a little to the right of tha t of unsubstituted L7]L12. The position of spot a]b is inconsistent with the presence of such a complex. (3) The presence of proteins L10 and L l l in cross-linked material with the electrophoretic mobility of spot a[b implies tha t they must be present as complexes with one or more acidic proteins such as L7/L12.

We conclude that the dimethyl adipimidate cross-linked material, whose analysis we have described, consists of a mixture of two binary complexes, L7]L12-L10 and L7]L12-Ll l . Since we have previously shown tha t a cross-link can be introduced between proteins L10 and L l l (Expert-Bezanc, on et al., 1975), the polypeptide chains of the protein pairs L7]L12-L10, L7]L12-Ll l , and L 1 0 - L l l must approach each other sufficiently closely at one or more points to bring reactive amino groups in the different chains to within at least 6-3 A of each other (the maximum distance between the two functional groups of dimethyl adipimidate is 6.3 A). Proteins L7/ L12, L10 and L l l are therefore neighbours in the 50 S subunit. Independent evidence showing tha t proteins L10 and L l l are involved in elongation factor G-dependent GTP hydrolysis (l~aassen & MSller, 1974,1975; Schrier & MSller, 1975; Van Agthoven el al., 1975) supports this conclusion. In addition, it has been shown tha t the 50 S component known as protein L8 is in fact a stable complex of proteins L7]L12 and L10 (Pettersson et al., 1976). The very strong interaction between these two proteins in the free state suggests tha t they probably also interact, and are therefore neighbours in the 50 S ribosome. The relative positions of spots L8 and a]b in two-dimensional

786 A. EXPERT-BEZANgON ET AL.

gel slabs (see Fig. 2) (tile latter displaced to tile right and upwards with respect to the former) are consistent with the presence of a complex containing unmodified L7/LI2 and IA0 in spot 8, aud of a dimethyl adipimidate-substituted complex of these two proteins in spot a/b.

Finally, if the proposal (Nierhaus & Montejo, 1973) that protein L l l contains or forms part of the peptidyl transferase site in the 50 S ribosomal subunit is correct, the results we present here show that this site may be situated close to the site for hydrolysis of GTP involving proteins LT/L12. However, some doubt still exists as to whether or not protein L l l forms part of the peptidyl transferase site (BaIlesta & Vazquez, 1974; Howard & Gordon, 1974), although recent experiments reinforce the conclusion that this is the case (Nierhaus et al., 1975).

Laboratoire de Chimie Celhflaire Institut de Biologic Physico-Chemique Fondation Edmond de Rothschild 13, Rue Pierre et Marie Curie 75005, Paris

i . EXPEBT-BEzANgON D. BARRITAULT M. MILET D. H. HAYES

Received 31 December 1975, and in revised form 2 August 1976

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