Eur. J. Biochem. 98, 409-416 (1979)

Acidic Ribosomal Proteins from Eukaryotic Cells Effect on Ribosomal Functions

Francisco SANCHEZ-MADRID, Rambn REYES, Pedro CONDE, and Juan P. G. BALLESTA

Instituto de Bioquimica de Macromoleculas. Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas y Universidad Autonoma de Madrid

(Received February 5, 1979)

Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribo- somes are released. At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be sustituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.

The existence in eukaryotic ribosomes of acidic proteins with electrophoretic mobility and molecular weight similar to that of proteins L7 and L12 from Escherichia coli ribosomes has been reported [l-41. In some cases antibodies against the bacterial proteins were reported to cross-react with the eukaryotic pro- teins [ I ] .

Functional similarities between acidic proteins from bacterial and higher cells have been reported [2,6] using particles deprived of these proteins by washing the ribosomes with 1 M NH4Cl and 50% ethanol as described for E. coli [7]. It was later found that this treatment induces the release of two proteins in addition to the acidic ones [3]. Therefore, the con- clusions drawn from this type of experiment must be re-examined.

We have found experimental conditions that per- mit the selective release of most acidic proteins from rat liver and yeast ribosomes. The roles of the acidic proteins in both types of ribosomes have been in- vestigated using these particles. The results presented in this report indicate differences in the requirements of acidic proteins for the function of eukaryotic and prokaryotic ribosomes.

Ahhreviatiun. EF-1, 2, etc, elongation factors 1, 2, etc

MATERIALS AND METHODS

Preparation of Ribosomes

Saccharornyces cerevisiae, strain Y 166 was grown in yeast extract/peptone/glucose medium [S] in a Lab- Line/S.M.S. Hi-density fermentor, up, to late expo- nential phase. Cells were broken by sea-sand grinding and the ribosomes were washed in high-salt buffers [9] and finally resuspended in 80 mM KCI, 10 mM MgCL and 20 mM Tris-HCI, pH 7.4 (buffer 1). The particles were stored in liquid nitrogen. Rat liver ribosomes were prepared as described elsewhere [lo].

Preparation of Core Particles and Split Protein Fractions

The ribosomes were dissolved at 4 mg/ml in 10 mM imidazol-HC1 buffer pH 7.4, containing 20 mM MgC12, 1 mM 2-mercaptoethanol and either 0.4 M or 1 M NH4Cl. Ethanol was added up to 50% (v/v) final concentration in two steps and the treatment was prolonged for 15 min. During the whole procedure the preparation was kept at 0 "C. After treatment the particles were centrifuged at 12000 rev./min for 15 min and resuspended in buffer 1. Treatment was not re-

410 Acid Proteins from Eukaryotic Ribosomes

peated. The treatment was usually carried out on ribosomes freshly prepared and the activity tests were performed immediately after the treatment. It is not recommended to freeze the core particles.

The split protein fractions SPo.4 and SP1.o were dialyzed against 2 o/, acetic acid and then lyophilized. Precipitation with acetone is not advised since loss of activity has been observed.

When radioactive ribosomes were needed the cells were grown either in 32P02- using a low-phosphate medium [ l l ] with about 1 mCi radioactive phos- phate/l (Junta de Energia Nuclear, Spain) or in 2 % glucose and 0.7 %, Difco yeast nitrogen base supple- mented with histidine, tryptophan and methionine in the presence of 1 mCi 14C or 3H-labelled mixture of amino acids/l (The Radiochemical Center, Amersham, England). In some cases, as indicated, the ribosomes were labelled after isolation by reductive methyla- tion [12].

Estimation of Ribosomal Proteins

Proteins from the particles were extracted by acetic acid [13] and precipitated by acetone [14]. The pro- tein pellets were exhaustively washed with acetone, in order to remove traces of acetic acid, dissolved in 20 mM NaHC03 and 8 M urea and separated by two-dimensional electrophoresis according to the standard methods [15] or in the presence of sodium dodecyl sulphate in the second dimension [16]. When radioactive proteins were used, the spots were cut out and extracted with acetic acid, dried on glass- fiber filters [17] and then radioactivity was estimated. For colorimetric determinations the absorbance at 605 nm of the acetic acid extract was measured. Kruiswijk and Planta [18,19] and international [20] protein nomenclature for yeast and rat liver respec- tively have been used. When required, protein was estimated according to Lowry et al. [21].

Activity Tests

Synthesis of polyphenylalanine was carried out using pH 5 fraction as source of supernatant factors and rat liver ribosomes as previously described [lo]. In some experiments the pH 5 fraction was substituted by 0.014 mg/ml of purified elongation factor 1 (EF-1) and 0.05 mg/ml of purified EF-2. EF-1 was obtained from rabbits reticulocites [22] and EF-2 from rat liver [23]. Both factors were kindly provided by Dr Fernan- dez Puentes. In the case of yeast ribosomes the ionic conditions were similar except for the MgC12 concen- tration that was increased to 12.5 mM. As a source of elongation factors the fraction 100000 x g super- natant precipitated between 30 % and 70 "/, saturation of (NH4)2S04 was used. ATP and GTP at 1 mM and 50 mM concentrations respectively were also included

in the reaction mixture of 50 p1 that was supplemented with the amounts of ribosomes indicated in the text. [14C]Phenylalanine with 830 counts min-' pmo1-l was used in the experiments. Incubation was carried out at 30°C for 20 min and the reaction stopped by the addition of 2 ml 5% trichloroacetic acid. The precipitated samples, after incubation at 90 "C for 10 min were filtered through glass-fiber filters, washed with 5 % trichloroacetic acid and ethanol, dried and radioactivity estimated on 5 %, Permablend (Packard Instruments Co.) in toluene.

Elongation-Factor-2(EF-2)-dependent GTP Hydrolysis

The reaction was carried out in 5O-pl mixtures containing 40 pg ribosomes in 50 mM Tris-HC1 buffer pH 7.4,60 mM KCI, 8 mM MgC12 and 10 mM 2-mer- captoethanol. The concentrations of GTP ranged from 7 pM to 10 pM depending on the experiment. A par- tially purified preparation of EF-2 from reticulocytes, prepared according to previously reported procedures [4] and kindly provided by Dr Carmen Fernandez- Puentes, was used at a final concentration of0.5 mg/ml. The mixture was incubated at 37 "C for 10 min and the reaction stopped by addition of 150 pl of 4% active charcoal in 0.7 M perchloric acid and 2.5 mM KzHP04. After centrifugation at 5000 rev./min for 15 min aliquots of 100 pl were taken and radioactivity estimated in a mixture with 3 ml of Bray's solution.

Initiation Complex Formation

The reaction was carried out in two steps. First, the formation of ternary complex takes place in 180-4 volume reaction mixtures containing 0.2 mM GTP, 2 mg/ml crude initiation factors of rabbit reticulo- cytes and 0.055 mM [35S]Met-tRNAf (specific activity 50000 counts min-' pmol-') in 20 mM Tris-HCI pH 7.4, 100 mM KCI and 8 mM 2-mercaptoethanol. The mixture was incubated at 37°C for 10 min. Secondly 20 pl of a mixture having 200 pg ribosomes, 8 pg poly(A,U,G) and 30 mM MgC12 were added. After ten additional minutes at 37°C the samples were applied to 15 - 30 % sucrose gradients made in 20mM Tris-HCI pH 7.4, 60mM KCI and 10mM MgCI2 that were centrifuged at 40000 rev./min in an SW 50.1 Spinco rotor for 2 h 10 min.

When the reactivity of the bound Met-tRNA towards the puromycin had to be tested, 50 p1 of a solution of 0.01 M puromycin was added after the last incubation and the mixture was kept at 37 "C for 10 additional rnin before applying it to the gradients.

Crude initiation factors were prepared as previ- ously described [5] and were generously provided by Dr M. Fresno. [35S]Met-tRNAf was prepared ac- cording to previously reported procedures [5].

F. Sinchez-Madrid, R. Reyes, P. Conde, and J . P. G. Ballesta 41 1

Preparation qf Specific Antisera

Split proteins (SP0.4) prepared as described above, were dissolved in 10 mM Tris-HC1 pH 7.4 and 2 mM 2-mercaptoethanol and injected (0.2 mg/rabbit) intra- muscularly as a water-in-oil emulsion with 3 vol. of Freund complete adjuvant (Difco).

After 15 days the animals were again injected sub- cutaneously with 0.3 mg protein/rabbit in the same conditions but using Freund incomplete adjuvant (Difco). Starting from the 15th day after the last injection the rabbits were bled from the ear and the serum titre checked by double immunodiffusion.

Inzmunodiflision Tests

in 10 mM Tris-HC1, 150 mM NaCl, pH 7.4. The tests were carried out using 1 % agar plates

RESULTS

Proteins Released from 80-S Ribosomes by NH4CIIEthanol Treatment

As in the case of rat liver ribosomes [3], two- dimensional gel electrophoresis of proteins from yeast ribosomes treated with 1 M NH4Cl and 50% ethanol (Pl.ocores) revealed the absence of at least four proteins, one of them (spot S31) corresponding to the 40-S sub- unit, the most acidic spots L44/45, and spot L15, corre- sponding to the 60-S subunit (using Kruiswijk and Planta nomenclature [18,19]. When the concentra- tion of ammonium salt in the washing buffers was lowered to 0.4 M (P0.4 cores) only proteins L44/45 were absent from the ribosomes. Particles with char- acteristics similar to P0.4 cores were obtained when the treatment was performed with 1 M NH4C1 in the presence of only 35 % ethanol.

The conclusions obtained by comparison of the intensity of spots in the gels were confirmed by quanti- tative estimation of the proteins present in the par-

ticles. Estimation was carried out either colorimetri- cally by eluting the Coomassie blue from the stained spots or by measuring the radioactivity of the spots when labelled ribosomes were used. Proteins L44/45 were reduced to about 20 % and 10 % of controls in Po.4 and PI.^ cores respectively, while proteins L15 and S31 appear drastically diminished only in the second type of particles (Table 1).

It is important to note that ethanol precipitation of 80-S ribosomes in the presence of 60 mM NH4C1 (80-S pp particles) leaves practically unaltered pro- teins L15 and S31 and only slightly affects the con- tents of protein L44/45.

Polymerizing Activity of' Treated Particles

The polymerizing activity of yeast ribosomes precipitated with ethanol at different ammonium con- centrations was tested using a polyphenylalanine syn- thesizing system. It was observed that precipitation at low ammonium ion concentration partially in- activates the particles obtained (80-S pp particles). Apparently, the -older the preparation the higher the inactivation observed. The extent of inactivation ranges from 0 to 60 depending on the preparation of ribosomes. This effect does not seem to be due to a partial release of ribosomal proteins, as they are not detected in the supernatant from the treatment by two-dimensional electrophoresis. Moreover, there is no correlation between the amount of acidic proteins present in the particles (Table 1) and the loss of activity. Nevertheless, the content of total protein per ,4260 unit of 80-S pp particles, measured in the 670/;, acetic acid extract [21], was about 25% lower than in untreated particles, indicating that a release of proteins does actually take place, probably conta- minating non-ribosomal proteins (i.e. initiation, elon- gation and termination factors) that might affect the activity of the ribosomes.

Taking as controls the 80-S pp particles, the ac- tivity of the cores and P1.0 cores prepared from

Table 1 Ribosomal protein released from the ribosomes by NH4Cl/ethanol Estimation of protcins L15 and S31 was carried out using ribosomal proteins labelled by reductive methylation The values given correspond to the maximum, minimum and average obtained using as internal standards five ribosomal proteins not affected by the ethanol treatment For the determination of proteins L44/45, ribosomes labelled in vivo with 32P04 and ''C-labelled ammo acids were also used and the second dimension of the gel electrophoresis was run in sodium dodecyl sulphate The values given are the average of seven experiments using different core preparations

Particles Protein L15 Protein S31 Acidic proteins L44145 ~ _ _ ~ - -~ .- _ _ - - ~

max min av max min d V max min av

of controls - ~ ____ - - ~~ ~ ~ ____ - ___ - - -_- - - -

80-s pp 99 90 94 122 14 98 104 14 82 Po 4 cores 94 70 81 149 71 86 29 14 21 PI cores 40 32 31 56 35 43 19 7 12

412 Acid Proteins from EukdryOtiC Ribosomes

n A B n

Y

0 0 20 0 10 20 Amount of particles (pmol)

Fig. 1. Polyphenylalanine synthesis by yeast ( A ) and rat liver ( B ) ribosomes. (A) Untreated 80-S ribosomes; (0) 80-S pp; ( x ) (m) PI 0 cores. Conditions of the test as indicated in Materials and Methods

cores;

D

T

I

Fig. 2. Initiation conzpk.u fijrmation by yeast ribosomes. The reaction mixtures in the conditions described in Materials and Methods were run in 10-30”/, sucrose gradients and then scanned at 260 nm; at the same time fractions were collected and precipitated with 5 % trichloroacetic acid, filtered on glass filters and counted. (Al 80-S ribosomes; (B) P0.4 cores; (C) Pl.0 cores and (D) 80-S ribosomes minus initiation factors. (+O) A260; (0---0) radioactivity

the same batch of ribosomes differed substantially. Fig.1 shows the results of a typical preparation in- dicating a drastic inactivation of Pl.0 cores while the Po.4 cores displayed more than 70% of the activity in the controls. Similar results were obtained when particles prepared from rat liver ribosomes were used (Fig. 1 B).

The polymerizing activity of the P0.4 particles could be due to the existence of acidic proteins in the prep- arations of factors used for the activity tests. Two- dimensional gel electrophoresis and double-diffusion tests carried out with sera from rabbits immunized with sP0.4 from yeast did not reveal the presence of acidic proteins in the 100000 x g supernatant fraction (not shown). Nevertheless, the absence of any con-

taminant protein in the polymerization tests was secured by using purified elongation factors. The Po.4 cores displayed in this case similar activity with respect to the 80-S pp controls than in less purified systems (not shown).

Initiation Complex Formation by Core Particles

The capacity of the particles lacking acidic protein to form the initiation complex was also tested. Fig.2 shows that, in the presence of crude initiation factors, Met-tRNAf and poly(A,U,G) all the particles, even Pl.0 cores, were able to bind the initiator tRNAf and form an 80-S complex. The extent of the reaction of the bound Met-tRNA with puromycin was similar

F. Sinchez-Madrid, R. Reyes, P. Conde, and J. P. G. Ballesta

to controls in the case of Po.4 cores and something lower for Pi .o cores (Table 2). The significance of these results is, however, dependent on the absence of acidic ribosomal proteins from the crude initiation factor preparation from reticulocytes, as these ribosomal proteins are functionally exchangable with the equiv- alent proteins of yeast (unpublished results).

Electrofocusing in acrylamide gels (Fig. 3) shows that bands in the pH range of the acidic ribosomal proteins are undetected in the initiation factor sample. The sensitivity of the technique (0.5-1 pg protein) indicates that ribosomal acidic proteins, if present in the initiation factor preparations, are at concentra- tions of less than 1 - 2 %.

Therefore, considering the optimal concentration of acidic proteins required for reconstituting the GTPase activity of the core particles (Fig.5), it is highly improbable that the results shown in Fig.2 are due to contamination of the initiation factor preparations.

Table 2. Iiiitiation-factor-drpcndendent binding of’ Met-t RNA Experiments were carried out as described in Fig. 1 and in Materials and Methods. The data correspond to radioactivity under the 80-S ribosome peak in the gradients of samples incubated in the absence and in the presence of puromycin

Particles Met-tRNAf bound ~ ~ - ~~

- puromycin + puromycin puromycin reactive

counts/min control ~- ~ ~ _ _ _ ~

8 0 3 18 823 1 4 049 25.4 Po 4 cores 19 674 15021 23.7

80-S 15 395 10221 33.6 PI cores 12357 10164 17.8

A

f,-----

413

EF-2-Dependent GTP Hydrolysis

The acidic proteins L7 and L12 from E. coli play an important role in the interaction of elongation factors with ribosomes [26]. We have tested this inter- action in eukaryotic ribosomes by measuring the EF-2- dependent GTPase activity of yeast and rat liver particles using partially purified EF-2 from reticulo- cytes in the assays.

As in the case of the polyphenylalanine synthesis, the GTPase activity of the 80-S pp particles is also affected.

1 2 3 4

Fig. 3. Electrof’ocusing of’initiutiunf’actors and acidic protein prepura- tions. (1 and 2) 200 pg and 300 pg respectively, of reticulocyte initiation factors. (3) 10 pg reticulocyte acidic proteins (SPo.4 frac- tion). (4) 10 pg yeast acidic proteins (SPo.4) The arrows indicate the position of the bands corresponding to the acidic proteins. The electrofocusing was carried out from pH 3.5 to 10 under conditions previously reported (2).

0

0 5 0 0 10 2 0 Amount of part icles (pmol)

Fig.4. EF-Z-dqendent GTP hydrolysis by yeast ( A ) and rat liver ( B ) ribosomes. EF-2 from reticulocytes was used to test the activity of 80-S riboSomes (0), 80-S pp (O), P0.4 cores ( x ), Pl,0 cores (A)

414 Acid Proteins from Eukaryotic Ribosomes

Concerning the activity of particles lacking pro- teins, the GTPase of P0.4 cores is affected more than their polymerizing activity (Fig. 4). On average the activity of GTPase is reduced to less than 50% of that of the controls and, contrary to the polyphenyl- alanine synthesis, GTPase activity is stimulated by the addition of the split proteins fraction (SP0.4) (Fig. 5).

Proteins L7 and L12 from E. coli (SPl.0 fraction) that strongly stimulate the EF-G-dependent activity of their own core particles do not affected the activity of the P0.4 cores from yeast and conversely, yeast acidic proteins are inactive on E. coli particles (Fig. 6).

Immunological Similarities among Acidic Proteins from Dijferent Origins

The lack of stimulatory activity of bacterial pro- teins L7 and L12 in the EF-2-dependent GTP hydro- lysis of Po.4 cores induced us to test the structural

0 10 20 sp0.4 ( p g )

Fig. 5. EjJecr ofSPo.4,frornyeusr on polyphenylalanine sjnthesis ( x ) and EF-2-dependent GTPusr (0 ) of’ yeust Po cores. 100 ”/, values correspond to 7500 counts/min [‘4CJphenylalanine polymerized and 164 pmol GTP hydrolyzed

similarity of acidic proteins from ribosomes of differ- ent origins. Using antisera raised against SPo.4 from yeast and SPl,o from E. coli ribosomes, cross-reactivity is only detected in the homologous systems, specifically between L7/L12 and anti-SP1 .O from E. coli and SP0.4 from yeast and its own antiserum (Fig.7). Proteins from Artemia salina and from rat liver show no cross- reaction with either antiserum.

DISCUSSION The reason for the partial inactivation of yeast ribo-

somes upon ethanol precipitation at low ammonium concentration (60 mM NH4CI) is not well under- stood. Apparently there is no direct relationship be- tween that inactivation and the release of ribosomal proteins (Table 1). Protein is actually released by the precipitation and in the ammonia/ethanol wash there is some material distinct from the acidic proteins but immunologically similar to some components of the 100000 x g supernatant fraction (not shown). This might indicate that some factors contaminating the ribosomal preparations are washed off by the treat- ment, causing a diminution of the polymerizing effi- ciency of the particles. Alternatively ethanol, by af- fecting the conformation of the ribosomes, might cause an inactivation that would be irreversible in those particles having structural damage i.e. rRNA breaks. This hypothesis is compatible with the ob- servation that in general the inactivation is higher as the preparation becomes older.

The results obtained using particles treated with 0.4 M NH4Cl (P0.4 cores) suggest that, in the case of ribosomes from S. cerevisiae and rat liver, the most acidic proteins from the large subunit are not an absolute requirement for their polymerizing and initi- ation complex formation activities in the conditions used in these experiments. The core particles deprived of at least 80% of these proteins, lose no more that 30 of their polyphenylalanine synthesis capacity.

I I I ‘0 5 10 0 5 10

Acidic proteins (pg)

Fig. 6. Reconstitutior? o/ G7‘f’tr.w trc./ii.it,v of Feust und l ? u ( ~ ~ ( v / u / ('arcs /?J, trc.idic,/,,.ort,rri.s. Yeast Prl.l cores (A) and E. coli P, (I cores (B) were tested in the presence of increasing concentrations of SPu.4 from yeast (0) and SP, from E. co/i (0). In each casc the factor homologous to the particles was used. 100 activity correspond to 140 pmol hydrolyzed by yeast ribosomes and 1800 pmol by E. coli particles. The split proteins were dissolved in 10 m M Tris-HCI. pH 7.8, and 2 mM 2-mercaptoethanol

F. Shnchez-Madrid, R. Reyes, P. Conde, and J . P. G. Ballesta 41 5

Fig. 7. L)ouhk.-~j~~~.sion f ~ s t . (A) Central well: antiserum to yeast SP,,,,. (B) Ccntral well: antiserum to E. coli SPl.0. (1) SPI:.~ from yeast (10 pg); (2) SP0.4 from rat liver (10 pg); ( 3 ) L7 and L12 from E. coli (6 pg); (4) SPo.4 from A . salina (8 pg)

The discrepancies between our results and those reported previously [2,6], suggesting a fundamental role for the acidic proteins on the polymerizing activity of yeast ribosomes, can be explained in terms of the core particles used in both studies. Indeed, particles treated with 1 M NH4Cl (PI,(> cores) either from yeast ([6] and this report Fig. 1) or rat liver [2,3] lack poly- merizing activity, although it can be restored upon addition of the split protein fraction [3,6]. However, since cores lack about 60% of proteins L15 and S31, the use of these cores does not permit conclusions to be drawn on the role of the acidic proteins. How- ever, the results showing that acidic proteins from E. coli can reconstitute the activity of Pl.0 cores from yeast [ 6 ] , suggests the possibility that the preparation of bacterial proteins used in those experiments [6] contains a component or components that can replace the non-acidic proteins lacking in the yeast cores.

Discrepancies can also be attributed to the activity of the particles used as controls. It is clear from the

results shown here that precipitation of the 80-S ribo- somes may affect their activity without loss of ribo- somal proteins. The inactivation may be substantial depending on the preparation of ribosomes. It is, therefore, important to use appropriate controls, that is, ribosomes precipitated without loss of proteins, in order to study the effect of the release of acidic pro- teins on their activity.

On the other hand, EF-2-dependent activities of the ribosomes are more sensitive to the removal of proteins L44/45. Although still having substantial activity, the P0.4 cores are more affected when tested in an EF-2-dependent GTPase assay than in a poly- merizing system. Moreover GTP hydrolysis and not polyphenylalanine synthesis is stimulated by the ad- dition of the acidic proteins. A dependence on the acidic proteins has been also reported for EF-1- dependent functions in Arternia salina and yeast ribo- somes [2]. The acidic proteins, therefore, seem to be dispensable for initiation and polymerization but partially required at least in the elongation-factor- dependent resolved assays. This apparent contradic- tion is not well understood. The presence in the super- natant factors preparations of unidentified compo- nent(s) that might compensate for the loss of the acidic proteins in the ribosome cannot be excluded when crude preparations of elongation factors were used. The presence of unkown factor(s) in the supernatants of E. coli has been suggested to explain the stimulation of EF-G . ribosome interaction when I00000 x g super- natant in present [27]. However, the use of pure elon- gation factors, clearly indicates that this is not the explanation for the polymerizing activity of the cores from yeast. Alternatively, the partial reactions might not reflect the actual polymerizing capacity of the particles. In this sense, recent data showing stimulation of EF-T-dependent GTPase by kyrro- mycin [28] and the acidic proteins L7 and L12 alone [29] raise questions about the physiological significance of uncoupled GTP hydrolysis by elongation factors.

In agreement with the lack of an absolute require- ment for the acidic proteins in the polymerizing activity of the ribosomes are the recent reports showing that particles lacking L7 and L12 derived from E. coli ribosomes are able to polymerize phenylalanine at a rate very similar to that of control ribosomes [30,31]. In fact the possibility of interaction of the ribosomes and elongation factors in the absence of acidic pro- teins has been previously shown [32]. All these data seem to suggest that these proteins, although physi- cally very close to the elongation factor binding site or even forming part of it, are probably not the only components involved in the interaction with the ribo- some. The important role that these proteins un- doubtedly play in the ribosome function could be related to the modulation of its activity. The existence of different forms of these proteins in bacterial as

416 F. Sanchez-Madrid, R. Reyes, P. Conde, and J . P. G. Ballesta: Acid Proteins from Eukaryotic Ribosomes

well as in higher cells is compatible with this hypo- thesis.

Establishing a correspondance between the ribo- somal proteins of the different species used in the field will be a very important step towards relating the findings of different investigators. In this sense our results suggest the structural and functional equivalence of proteins L15 and S31 from yeast and L12 and S25 from rat liver. It is also worth noting the similarities between protein L15 from yeast and protein L11 from E. coli. Both are heavily methylated proteins of the large subunit [33 - 361, easily removable by ammonia/ethanol washing ([37,38] and this report) and with similar mobility in the standard two-dimen- sional gel electrophoresis system.

However, the results on the structural resemblance of prokaryotic and eukaryotic ribosomal acidic pro- teins are conflicting. While in rat liver and chicken liver cross-reaction between the acidic proteins and anti-L7/L12 from E. coli has been clearly shown [l, 391; proteins from Artemia salina [40] and, in our study, from yeast and rat liver do not present such cross- reaction. These discrepancies might lie either in the different responses of the animals to raise the anti- bodies or in the different determinants exposed in the injected antigens, depending perhaps on the extent of denaturation of the protein used. Since stretches of the primary sequence of amino acids of acidic pro- teins from different origins seem to be similar [41], common antigenic determinants can be expected ; however, the secondary and tertiary structure of the proteins can differ substantially resulting in quite different immunogenic determinants. Thus, depending on the state of the sample injected the responses of the animals can be different.

This work was supported by an institutional grant to the Centro de Biologia Molecular from Comisidn del Descuento Complementario (Istituto Nacional de Previsibn) and personal grants from Essex Laboratories and Lilly Indiana of Spain. We thank Dr C. Fernandez Puentes and D r M. Fresno for the gift of elongation factors and [35S]Met-tRNA respectively, and Dr D. Vazquez for reading and commenting the manuscript.

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Bioquimica de Macromolttculas, Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas y Universidad Autonoma de Madrid, Facultad de Ciencias, Canto Blanco, Madrid-34, Spain