Comparative analysis of ATPase of yeast elongation factor 3 and ATPase associated with Tetrahymena ribosomes

Biochimie (1995) 77, 7 i 3-718 © Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris

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Comparative analysis of ATPase of yeast e|ongation factor 3 and ATPase associated with Tetrahymena ribosomes O Kovalchuke, J Ziehler, K Chakraburtty* Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wi 53226, USA (Received 3 October 1994; accepted 24 February 1995)

Summary m Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translationa! apparatus. The biochemical function of EF-3 has recently been defined. The protein removes deacylated tRNA from the ribosomal exit site (E-site) thus facilitating occupation of the ribosomal A-site by aa-tRNA. A functional homolog of yeast EF-3 has not been identified in non-fungal species. Yeast EF-3 is a ribosome-depend~ra ATPase that can also accept GTP and ITP as substrates. The function of EF-3 in ribosomal reactions requires ATP hydrolysis. An ATPase activity associated with higher eukaryotic ribosomes has been claimed to be a direct functional homolog of yeast EF-3. Comparative analysis of biochemical, immunological and functional properties of ATPase activity associated with the ribosomes isolated from the ciliated protozoan Tetrahymena pyriformis with that of yeast EF-3 ATPase indicates that these two activities are significantly different. Results reported in this communication strongly suggest that the ribosome associated ATP hydrolytic activity of Tetrahymena pyriformis is not a functional homolog of yeast EF-3. elongation of translation / ribosomal ATPases / evolution of eukaryotes / Saccharomyces cerevisiae / Tetrahymena pyriformis

Introduction

Two soluble protein factors are essential for polypeptide chain elongation reactions in all organisms [1]. Eukaryotic elongation factor 1 (EF-lot) and factor 2 (EF-2) are functional homologs of bacterial translational factors EF-Tu and EF-G [2]. In addition to EF-1 ~ and EF-2, fungal ribosomes require a third protein, elongation factor 3 (EF-3) for polypeptide chain synthesis [3--6]. The function of EF-3 in ribosomal reactions has been defined recently [6, 7, 24]. EF-3 induces conformational change of the ribosome from the post to the pre-translocational state thus facilitating removal of deacylated tRNA from the E-site and binding of aa-tRNA to the A-site. The function of EF-3 depends on ATP (GTP) hydrolysis [6, 8-11]. The hydrolytic activity of EF-3 is enhanced by two orders of magnitude by the yeast ribosome due to an increase in the turnover rate of EF-3 ATPase [ 1 1, 12]. A direct correlation has been established between activation of EF-3 ATPase and the functional state of the ribosome [ 121.

*Correspondence and reprints

Plants (wheat, chlorella), protists (Tetrahymena), and higher eukaryotes (mammals) do not have a soluble fonn of EF-3 [9, 13-15]. However, it has been postulated that ribosome-associated ATPase may function as an analog of EF-3 in the Tetrahymena translational system [15]. In this communication, results on comparative analysis of biochemical and immunological properties of yeas! EF-3 with those cf riboson'le-associated ATPase from Tetrahymena pyriJbrmis are presented to evaluate the function of this protein in translation.

Materials and methods

All biochemicals and radiochemicals were obtained from standard sources as described previously [11, 121. tRNA ehe was pdrchased from Boehringer Mannheim. Aminoacylated tRNA was prepared according to a protocol published before [121.

Isolation of yeast elongation factors and the ribosome Yeast ribosomes EF-lot, and EF-2 were isolated from a low protease strain and purified to homogeneity according to previously published methods [11]. A homogeneously purified preparation of EF-3 was obtained from a strain over-expressing EF-3 by a modified protocol of Kamath and Chakraburtty Ill, 161.

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Preparation of Tetrahymena ribosome Ribosomes from the ciliated protozoa were prepared according to a modified protocol published by Miyazaki and Kagiyama [151. Tetrahymena (strain CU4al) were grown in a medium containing 2% proteose peptone, 1% yeast extract, and 0.6% dextrose at 29°C to a density of approximately 5 x 105 cells/ml. Cells were harvested by centrifugation at 1500 g for 10 rain, washed once with a solution containing 60 mM Tris-HCl (pH 7.0), 5 mM MgCI2, 50 mM NH4CI, 2 mM spermidine (buffer A) and stored at -80°C. Cells were disrupted by grinding with four volumes (wt/wt) of autoclaved sea s ~ d and one volume of buffer A containing 1 mM sediam az;de. l h e cells were vortexed for 6 rain with intermittent coolh~g ,~d c~:r~trifuged for 30 rain in a Beckman JA20 rotor at 16000 ~m. The supematant fraction ($30) was centrifuged for 3 h at 45000 rpm in a Beckman Ti 70 rotor. Ribosomal pellets were resuspended in a buffer containing 10% glycerol and 500 mM KCI, clarified by centrifugation for 10 min at 10000 rpm (JA20 rotor), layered over a 25% glycerol cushion of buffer A containing 500 mM KCI, centrifuged for 3.25 h at 45000 rpm in a Ti 70 rotor. Pelleted ribosomes (once washed ribosomes) were rinsed ~,ith buffer A, resuspended, clarified and centrifuged through a second glycerol cushion containing 0.75 M KCI. This final pellet (twice washed ribosomes) was resuspended in buffer A containing 25% glycerol and stored at -80°C.

Assay conditions Details of the reaction conditions for poly(U)-dependent polyphenylalanine synthesis and ribosome-dependent ATP hydrolytic activity have been described [ 12, 16, 171. For immunoblot analysis, equivalent amounts of ribosomes (10 pmol) or S 100 fractions (10 lag) were electrophoresed on SDS-PAGE along with standard EF-3 (10 pmol) and l l4C]-Iabeled mol wt markers 1181. Separated proteins were transferred onto a presoaked PVDF membrane [191. A 5000-fold dilution of purified EF-3 anti-sera was used as primary antibody. Radioiodinated protein A was used to identify the cross-reacting protein bands by autoradiography 1201.

Results

activity of unwashed Tetrahymena ribosomes is inhibited by an unidentified component (possibly a loosely associated nucleotide hydrolase activity that depletes nucleotide pool) [15]. Tetrahymena ribosomes purified through a glycerol-salt cushion (once washed ribosomes) demonstrate ATP hydrolysis. A significant amount of this ATP hydrolytic activity remains associated with the ribosomal fraction after extraction with 0.75 M KCI (fig 1A, z~v). The EdieHofstee plot presented in figure I B shows that once washed ribosomal fraction (A--A) iS composed of at least three ATPases; one of these ATPases with the lowest Km for ATP is lacking in the twice washed ribosomes (fig 1B, c~Q). In any case, the kinetic parameters of the ATPase that eluted off the ribosome and the fraction that remained associated with the ribosome after high salt extraction (twice washed ribosomes) are very similar (fig I B, Edie-Hofstee plot). From these results, we conclude that the same group of proteins contributes to the hydrolytic activity associated with once washed and twice washed ribosomes. In order to evaluate the role of ribosome-associated ATPases in translation, the nucleolytic activity and poly(U)-dependent polyphenylalanine synthesis by the yeast and the Ten'ahymena ribosomes were analyzed along with the effect of adenine nucleotides on these reactions. Results of these analyses are presented in figure 2. In the yeast system, under limiting concentration of GTP pool (20 laM), poly(U)-dependent polyphenylalanine synthesis is significantly stimulated by ATP (fig 2A, ~-t-~ and [8]) and inhibited by ADP (A-A). GTP hydrolysis by the yeast ribosomes is similarly affected by ATP and ADP (fig 2D).

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Ribosome-associated ATPase activity has been claimed to substitute the function of fungal EF-3 in the Tetrahymena translational system [151. T h i s conclusion is based on the observation that the postribosomal supematant fraction prepared from the ciliated protozoa lacks an EF-3 equivalent but that the translation in this system is stimulated by ATE a function attributed to EF-3 [I l] and that a polyclonal antiserum to yeast EF-3 inhibits translation in the protozoan system [15]. We have tested these criteria and additional parameters in order to evaluate the role of ribosome-associated ATPase in translation. Data presented in figure I demonstrate the ATP hydrolytic activity stringently associated with the Tetrahymena ribosomes. All experiments presented in this communication were carried out with well washed ribosomes (once or twice washed) isolated from Tetrahymena or from yeast. This is because the

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A small but consistent stimulatory effect of ATP in GTP hydrolysis by the yeast ribosome is, very likely, due to the sparing effect of ATP on the limited GTP pool (20 laM) used in these experiments. In the Tetrahymena system, once washed ribosomes are less active in translation (fig 2B) compared to their twice washed counterparts (fig 2C). Repeated extraction of the ribosomes with buffer containing high salt removes a significant amount of the nucleoiytic activity (fig I A, B). This point is re-emphasized in the differential rate of GTP hydrolysis by the once and twice washed Tetrahymena ribosomes (fig 2E, F). It is of interest to note that extraction with high salt containing buffer reduces the nucleolytic activity and increases the translational activity of the Tetrahymena ribosomes (fig 2B, C). The relatively poor translational activity of once washed Tetrahymena ribosome (Fig 2B) compared to its twice washed counterpart (Fig 2C) is attributed to the depletion of limited amount of GTP (20 {aM) used in these experiments. The situation with the yeast ribosomes is just the reverse. High salt washed yeast ribosomes demonstrate significantly less translational activity compared to the unwashed yeast ribosomes. The salt extracted yeast ribosomes regain translational activity upon addition of purified EF-3 [ 12]. Addition of EF-3 has no effect on the translation by Ten'ahymena ribosomes (data not shown).

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Fig 2. Effect of adenine nucleotides on poly(U)-dependent polyphenylalanine synthesis (A-C) and GTP hydrolysis (D-F) on yeast (A, D) and Tetrahymena(B, C, E, F) translational systems. Experiments in the yeast system were with twice washed yeast ribosome. For the Teo'ahymenasystem, both once washed (B, E) and twice washed (C, F) ribosomes were analyzed. Assays were performed under standard conditions described elsewhere [12]. The concentration of GTP was kept at 20 laM in all assays. Poly(U)-dependent polyphenylalanine syntheses were performed with 1 pmol of either yeast or Tetrahymena ribosomes, 5 pmol of yeast EF-lct, 1 pmol of yeast EF-2, 10 pmol of |3H]PhetRNA phe and 1 lag of poly(U) in a reaction volume of 15 lal. EF-3 (1 pmol) was added in the assays with yeast ribosome (A, D). Circles (o-c~) represent experiments with GTP alone, triangles (A-A) represent GTP plus ADP (1 mM) and squares (~J-!l) represent GTP and ATP (300 laM).

Results presented in figure 3 demonstrate the effect of polyclonal antibody on poly(U)-dependent polyphenylalanine synthesis by yeast and Tetrahymena ribosomes. The translation assays consist of either yeast or Tetrahymena ribosomes purified though glycerol-salt cushions, purified yeast elongation factors EF-let and EF-2. The yeast EF-lcx and EF-2 are fully active in heterologous system~ [9, 21]. Factor 3 was included in all reactions with yeast ribosomes as indicated. As expected, translation in the yeast system is completely inhibited by anti-EF-3 antibody. The antibody effect is reversed by EF-3 (fig 3A, 13, o - e ) . A 10-15% inhibition of Tetrahymena ribosomes is observed either with control sera or with EF:'-3 specific antisera (fig 3A, A--A, A--A). Unlike the yeast system, the low level of inhibition of the Tetrahysnena translation by EF-3 antisera is not reversed by EF-3 (data not shown). Therefore, we conclude that a 10-15% inhibition of translation in the Tetrahymena system in the presence of excessive amount of either control sera or anti-EF-3 antisera is a non-specific effect. Immunological cross-reactivity of EF-3 specific antisera with Tetrahymena proteins was analyzed by Western blots. Three independent preparations of EF-3 specific polycional amisera were used for these analyses. Two of these antisera were raised against native EF-3 and one was raised against the ,denatured protein. Results from one of these Western blots are

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shown in figure 4. Data presented in figure 4A indicate the Coomassie blue staining of the protein bands of an identical SDS-PAGE electroblotted on a PVDF membrane. Preparations of ribosomes and post ribosomal supematant (SI00) fractions from Tetrahymena pyriformis and S cerevisiae wel~ analyzed in these experiments. Panel B in figure 4 represents l-h exposure and panel C 100-h exposure of the same PVDF membrane. The shorter exposure (B) shows the position of the standard yeast EF-3 as a 116000 Da protein. No detectable cross-reacting materials are visible either in the pig liver or in the Tetrahymena S-100 or in the ribosomal fractions (B, lanes 6-10). After prolonged exposure of the autoradiogram (as indicated by the dark patch in the lanes with standard EF-3 and the yeast ribosomal fractions), several bands appeared in all lanes. None of these bands correspond to yeast EF-3 in molecular mass. The two taint (low mol wt) bands present in once washed Tetrahymena ribosome fraction (fig 4C, lane 7) are absent in the twice washed fraction (lane 8). These two ribosome preparations correspond to the materials analyzed for ATP hydrolytic activity in figure I A, B and in translational assays in figure 2B, C. These low tool wt anti EF-3 cro~s-reacting bands, in all probability, represent non-specific signals. Interestingly, Tetrahymena ribosomes that are free of anti-EF-3 cross-reacting bands are fully active in translation (fig 2B, C) and in ATP hydrolysis (fig 1).

Discussion An absolute dependence of fungal ribosomes on EF-3 remains an enigma. The question remains as to how ribosomes from prokaryotes and eukaryotes other than fungi carry out translational reactions in the absence of an EF-3 like protein. A search for a functional

20

Fig 3. Effect of EF-3 antisera on polyphenylalanine synthesis by Tetrahymena and yeast ribosomes. Closed symbols represent experiments in the presence of EF-3 specific antisera and open symbols are with control sera. Triangles represent experiments with Teo'ahymena ribosomes and circles are experiments with yeast ribosome. B. Reversal effect of purified EF-3 on antisera inhibited yeast system. Open circles represent polyphenylalanine synthesis with yeast ribosomes in the presence of control sera. For details of the experimental conditions, see [ 12].

homolog of EF-3 in these systems has consistently given negative results. Yeast EF-3 is an ATPase. The hydrolytic activity of EF-3 is stimulated by two orders of magnitude by the yeast ribosomes [11, 12]. A nucleotytic activity stringently associated with highly purified ribosomes and ribosomal subunits of nonfungal erigin has been implicated as a functional homolog of yeast EF-3 [15]. Results presented in this communication and elsewhere [12] strongly suggest the contrary. Translation by yeast ribosomes is stimulated by ATP and inhibited by ADP (fig 2A). In the Tetrahymena system, neither ATP nor ADP showed any significant effect on poly(U)-dependent polyphenylalanine synthesis (fig 2B, C). A small but consistent stimulatory effect of adenine nucleotides on the Tetrah3'mena system is attributed to the sparing effect of these nucleotides on the limited GTP pool (20 }aM) used in these analyses. This is evident when one compares the data presented in figure 2 (B, C, E, F). The experiments in figure 2 were carefully designed keeping the concentration of GTP below its K,, for EF-3 and ribosomal NTPases. The Km of GTP for yeast EF3 is - 1 5 0 laM and for Tetrahymena ribosome-associated NTPases is 40-200 laM. The kinetic parameters for the Tetrahymena 6bosomes were derived from the data presented in figure lB. Functionally, there are major differences between yeast and Tetrahymena ribosomes. Well washed yeast ribosomes that are fully competent in tRNA binding, are poorly active in translation with E F - I a and EF-2 in the absence of EF-3 [1]. Contrary to the yeast ribosomes, Tetrahymena ribosomes demonstrate poor translational activity prior to salt extraction (fig 2B and [15]). Upon removal of extraneous proteins and nucleolytic activities (fig I B), ribosomes efficiently translate poly(U) message (fig 2C). Addition of yeast EF-3 has no effect on these ribosomes (data not shown).

717 An inhibitory effect of anti-EF-3 antisera on translation by Tetrahymena ribosome has been used as an evidence by the group of M~yazaki and Kagiyama to argue for the presence of an EF-3 homolog in this system [15]. This group demonstrated inhibition of the Tetrahymena ATPase and polyphenylalanine synthesis by EF-3 antisera with an ICs0 of 4 mg/ml. The same preparation of EF-3 antiserum inhibits yeast ribosomes with an IC50 of 4 l.tg/ml. The significance of antibody inhibition in the Tetrahymena system is questionable as the effect is seen at such unusually high concentrations of the antibody. The Japanese group neglected to report the effect of an equivalent amount of control serum on translation. In our analysis, the inhibitory effect of a control serum on the Tetrahymena system is almost identical to the inhibition observed with the EF-3 specific antisera (fig 3A, ._~-A, A--A). Therefore, we conclude that a 10-15% effect of EF-3 antisera on the Tetrahymena system is non-specific. It is however understood that different antisera preparations against the same protein may recognize different epitopes. In any case, negative results do not provide any conclusive evidence for the presence or absence of an EF-3 like protein in Tetra-

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hymena. EF-3 is essential for translational elongation in yeast, therefore translation of poly(U) message was chosen as a test system to evaluate the significance of EF-3 cross-reacting proteins in elongation of translation. Yeast ribosomes cannot translate poly(U) without EF-3 [4]. Contrary to the yeast system, Tetrahymena ribosomes translate poly(U) without EF-3 (fig 2B) 1151. Removal of EF-3 cross-reacting proteins does not seem to influence poly(U) translation (fig 2B). Thus, immunological cross-reaction which may represent a low degree of structural similarity, or the presence of epitopes common to both EF-3 and Tetrahymena proteins, does not substantiate the conclusion of functional similarity between yeast EF-3 and ribosomal ATPase. EF-3-dependent translation by yeast ribosomes is inhibited by ADP (fig 2A). This, we believe, is due to competition with GTP for the nucleotide binding sites of EF-3 which requires ATP or GTP for its function [1 1]. In contrast to the yeast translational system, ADP stimulates translation by Tetrahyrnena ribosomes (fig 2B). The reason for this stimulation is essentially the same as for inhibition of EF-3 activity: competition between ADP and GTP for the nucleotidebinding sites of ribosomal NTPases. If ribosomal NTPases were essential for translation, one would expect to see inhibition of translation by ADP. ADP protects GTP from hydrolysis and thus preserves the GTP pool for elongation factors EF-ltx and EF-2 which are stringently dependent on GTP [ 1]. Therefore, the stimulatory effect of ATP on Tetrahymena

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Fig 4. Western blot analysis of ribosomal proteins. 10 pmol of either once or twice washed ribosome or l0 lag of (S-I 5) fractions were loaded per lane along with l0 pmoi of standard EF-3 (lane 2) and [14C]-labeled moiwt markers (lane5). Coomassie stained g~,l is shown in the upper panel. Autoradiograms of the Western blot after i h and 100 h exposure are shown in the two lower panels (B, C respectively). translation system reported by Miyazaki and Kagiyama [15] is, most likely, due to the sparing effect of adenine nucleotides on the GTP pool. Thus, results of immunological cross-reaction, functional and biochemical analysis presented in this communication strongly suggest that the ATPase activity associated with the Tetrahymena ribosome is not a functional analog of yeast EF-3. Similar studies with pig liver ribosomes indicate the absence of an EF-3 homolog in mammalian systems [12]. Animals are

718 considered to be closely related to fungi in the evolutionary scale [22, 23]. Absence of EF-3 may be one of the distinguishing features between higher and lower eukaryotes. Alternatively, fungi may have acquired the third elongation factor during evolution. Biochemical and immunological data presented in this communication and elsewhere [12] strongly suggest the absence of a direct homolog of EF-3 in eukaryotes other than fungi. Genetic complementation experiments are needed to resolve this issue. Such analyses are currently ongoing in our laboratory. Attempts to isolate a protein functionally analogous to yeast EF-3 from non-fungal sources have consistently given negative results. At this time, EF-3 remains an extremely attractive protein for both antifungal drug research and for studies on the molecular mechanism of protein synthesis. Acknowledgments This work was supported by a grant from National Institutes of Health (GM29795).

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