©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Highly Conserved Protein P0 Carboxyl End Is Essential for Ribosome Activity Only in the Absence of Proteins P1 and P2 (*)

(Received for publication, March 1, 1995; and in revised form, May 15, 1995)

Cruz Santos Juan P. G. Ballesta (§)

From the Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cient&ıacute;ficas and Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein P0 together with proteins P1 and P2 form the stalk in eukaryotic ribosomes. P0 has a carboxyl-terminal domain about 100 amino acids long that has high sequence similar to the ribosomal proteins P1 and P2. By sequential deletion of this region, a series of Saccharomyces cerevisiae truncated P0 genes have been constructed that encode proteins lacking 21, 87, and 132 amino acids from the carboxyl terminus, respectively. These constructions have been used to transform yeast P0 conditional null mutants to test their capacity to restore cell growth. Removal of only the last 21 amino acids causes a small effect on cell growth in wild-type strains; however, this deletion is lethal in strains having P protein-deficient ribosomes. A P0 lacking 87 amino acids allows cell growth at a low rate, and ribosomes bind P proteins with much less affinity. Lastly, removal of 132 amino acids totally inactivates P0; this deleted protein is unable to bind to the particles, causing a deficiency in active 60 S subunits and making the cell nonviable. These results indicate that at least one out of the five protein P-like carboxyl termini present in the ribosome has to be firmly bound to the particle for protein synthesis and cell viability, and this structure can be provided by protein P0. The part of P0 from around positions 230-290 is important for the interaction of proteins P1/P2 with the ribosome, but it is not essential for protein synthesis. Finally, the region including from residues 185 to 230 is required for the interaction of P0 with the rRNA.


INTRODUCTION

Protein P0 is a component of the eukaryotic ribosome ``stalk.'' The stalk is a highly flexible lateral protuberance of the large ribosomal subunit that plays a central role in the interaction of the soluble factors with the ribosome during protein synthesis (Möller and Maassen, 1986). This structure has been well characterized in the bacterial ribosome (Strycharz et al., 1978; Marquis et al., 1981) and seems to be universally present in all ribosomal particles (Lake, 1985). In eubacterial particles, the stalk is formed by a protein pentameric complex made of one copy of protein L10 and two dimers of the acidic protein L7/12 (for review, see Liljas(1991)). The acidic protein dimers interact with the C-terminal region of L10 through their amino ends, exposing the carboxyl end to the exterior. The whole complex binds directly to the 23 S rRNA through the amino-terminal side of L10 (Gudkov et al., 1980). The L10bullet(L7/12)(4) complex binding site is located in a highly conserved region of 23 S rRNA overlapping the binding site of protein L11 (Dijk et al., 1979; Schmidt et al., 1981). This region of the ribosome structure has been defined as the GTPase center of the ribosome where the supernatant factors and some protein synthesis inhibitors, like thiostrepton, interact (Beauclerk et al., 1984; Egebjerg et al., 1989, 1990).

The protein L7/12 equivalents have been characterized in a number of eukaryotic and archaebacterial species, and their genes have been cloned. They are generically called P proteins and show a low sequence homology with the bacterial polypeptides, although the overall structure seems to be not very different (Shimmin et al., 1989). Functional data on systems other than eubacterial are not very abundant (Sanchez-Madrid et al., 1979; MacConnell and Kaplan, 1980; Köpke et al., 1992), but it is accepted that these proteins play a similar role in all cases. The eukaryotic acidic P proteins have, however, some peculiarities that differentiate them from the bacterial polypeptides. Probably the most interesting one is their capacity to be phosphorylated (Zinker and Warner, 1976; Horak and Schiffmann, 1977a, 1977b; Issinger, 1977; Leader and Coia, 1977; Kruiswijk et al., 1978; Tsurugi et al., 1978; Sanchez-Madrid et al., 1981). This modification strongly affects the activity of the proteins regulating their affinity for the ribosome (MacConnell and Kaplan, 1980; Sanchez-Madrid et al., 1981; Juan-Vidales et al., 1984), which supports their involvement in some regulatory mechanism of the ribosomal activity (Ballesta et al., 1993).

Four acidic P protein genes have been characterized and cloned in yeast (Mitsui and Tsurugi, 1988; Remacha et al., 1988; Newton et al., 1990). The four proteins can be grouped in two pairs, YP1alpha-YP1beta and YP2alpha-YP2beta based on amino acid sequence comparisons to their mammalian counterparts (Wool et al., 1991). Gene disruption studies have shown that the absence of each individual protein is not very deleterious (Remacha et al., 1990, 1992), and, in fact, the four P proteins can be eliminated without a lethal effect on the cells. (^1)

The protein L10 equivalent is probably the stalk component that shows more dissimilarities between eubacterial and other systems. The eukaryotic protein was called P0, and although it was identified originally as an anti-acidic P protein sera cross-reacting polypeptide (Towbin et al., 1982), it was only when the gene was cloned (Rich and Steitz, 1987; Mitsui and Tsurugi, 1988; Newton et al., 1990) and its amino acid sequence analyzed that it was proposed to be the protein L10 corresponding polypeptide (Shimmin et al., 1989). The amino acid sequence provided an explanation for the anti-P protein sera cross-reactivity of P0, since the carboxyl end of both protein types was found to be highly homologous (Shimmin et al., 1989).

Protein P0, like the eukaryotic acidic P proteins, is phosphorylated in the cells (Zinker and Warner, 1976; Elkon et al., 1986; Mitsui et al., 1987), but contrary to the P proteins, it is not found free in the yeast cytoplasm (Mitsui et al., 1988; Santos and Ballesta, 1994), although a cytoplasmic pool of P0 seems to exist in mammalian organisms (Elkon et al., 1986). Moreover, opposite to the P proteins, disruption of the P0 gene is a lethal event in yeast (Santos and Ballesta, 1994), resembling the typical ribosomal proteins, which are generally required for cell viability.

As expected from the proposed role of P0 in the stalk structure, the formation of a complex between P0 and both acidic P protein-types, P1 and P2, has been reported (Elkon et al., 1986; Rich and Steitz, 1987). In agreement with the dimeric state of the acidic proteins (van Agthoven et al., 1978; Juan-Vidales et al., 1984), P1 and P2 are found as dimers in this complex (Uchiumi et al., 1987). Gene disruption experiments have also shown that acidic protein binding to ribosomes requires the presence of both P1 and P2 proteins (Remacha et al., 1992), confirming, therefore, that the ribosome stalk structure is formed by at least one molecule of P0, one dimer of P1, and one dimer of P2. In fact, quantitative estimation of acidic proteins in exponentially growing ribosomes using antibodies cross-reacting with the carboxyl end of P0 and acidic P proteins gave a value close to five copies of antigen/ribosome (Saenz-Robles et al., 1990) indicating that the stoichiometry of the complex is, indeed (P1)(2)bulletP0bullet(P2)(2).

In spite of the clear functional and structural similarities, some conspicuous characteristics differentiate the bacterial and eukaryotic P0bulletP1/P2 protein complex. Thus, the bacterial structure is extraordinarily stable, resisting the presence of very high urea concentrations to a point that it was initially considered as one individual ribosomal protein called protein L8 (Pettersson et al., 1976). Moreover, the whole complex can be released from the bacterial ribosome, together with protein L11, by washing with ethanol and high salt concentration (Highland and Howard, 1975). In both aspects the eukaryotic protein complex is different. In eukaryotic cells, no protein L8 equivalent has been reported, and ethanol-ammonium wash releases only the acidic P proteins but not the P0 that remains bound to the ribosome (Sanchez-Madrid et al., 1979; Towbin et al., 1982; Rich and Steitz, 1987; Santos and Ballesta, 1994). The relative lability of the P0bulletP1/P2 complex is not unexpected, since an exchange between the P proteins in the cytoplasm and in the ribosome has been reported to take place in the cell during protein synthesis (Zinker and Warner, 1976; Tsurugi and Ogata, 1985).

The main structural difference between eubacterial L10 and P0 is, obviously, the carboxyl-terminal addition corresponding to the P protein-like sequence. In order to analyze the role of this extension in P0 function, a deletion analysis was performed.


MATERIALS AND METHODS

Yeast and Bacterial Strains

Saccharomyces cerevisiae W303-1b (MATalpha, leu2-3, 112, trp1-1, ura3-1, his 3-11, 15, ade2-1, can1-100). S. cerevisiae W303GP0 (MATalpha, leu2-3, 112, trp1-1, his 3-11, 15, ade2-1, can1-100, rpP0::URA3-GAL1-rpP0) was derived from S. cerevisiae W303-1b by integrating through homologous recombination in the rpP0 locus a construction carrying the P0 coding region fused to the GAL1 promoter (Santos and Ballesta, 1994). S. cerevisiae D67 (alpha; ura3-1, his 3-11, 15, ade2-1, can1-100, rpYP1alpha::LEU2, rpYP1beta::TRP1; rpYP2alpha, rpYP2beta) has been described previously (Remacha etal., 1992). The yeasts were grown in either rich YEP medium (1% yeast extract, 2% peptone) or minimal SD medium (Sherman et al., 1983) supplemented with the necessary nutritional requirements. In both cases, either 2% glucose or 2% galactose were used as carbon sources as indicated.

Escherichia coli JM83, TG1, and DH5alpha were used as the hosts for the routine maintenance and preparation of plasmids used in these studies and were grown in LB medium.

Enzymes and Reagents

Restriction endonucleases were purchased from Boehringer Mannheim GmbH, New England BioLabs, Inc., and Amersham Corp. and were used as recommended by the suppliers. T4 DNA ligase, calf intestinal alkaline phosphatase, and the DNA polymerase I Klenow fragment were from Boehringer Mannheim, and DNA polymerase I and T4 DNA polymerase were from New England BioLabs. [alpha-P]dCTP (3000 Ci/mmol) was obtained from Amersham Corp.

Cell Transformations

Bacterial transformations were performed according to the procedure of Hanahan (Hanahan, 1985). Yeasts were transformed using either spheroplasts (Burges and Percival, 1987) or intact cells. Either the LiCl method (Ito et al., 1983) or electroporation in a Gene Pulser (Bio-Rad) following the manufacturer's instructions were used in the last case.

Plasmids

The pFL series was derived from pUC19 by the addition of auxotrophic markers and replication origins and were obtained from Dr. P. Slonimski (Centre de Genetique Moleculaire, CNRS, Gif-sur-Yvette, France); pFL-C does not have any genetic marker, and pFL39 carries a HIS gene. pSceL10e was obtained by cloning a 3.7-kb (^2)EcoRI-SalI fragment from S. cerevisiae, containing the P0 gene and a portion of the pUC13 cloning site, into the EcoRI site of pEMBL8- (provided by Dr. P. P. Dennis, University of British Columbia, Vancouver, Canada). The YDp-H plasmid series was provided by Dr. F. Hilger (Berben et al., 1991). pSU-P0 and BSP0 were obtained by inserting a 3.7-kb EcoRI-BamHI fragment and 2.8-kb EcoRI-AvaII fragment from pSceL10e, both containing the complete P0 gene, in the multiple cloning site of plasmids pSU2719 (Martinez et al., 1988) and Bluescript, respectively.

Construction of Plasmids Carrying C-terminal Deleted Forms of Protein P0

pFLhisP0-C

Construction was performed as indicated in Fig. 1. Site-specific mutagenesis by heteroduplex hybridization of double-stranded DNA (Morinaga et al., 1984), using the oligonucleotide TGCTCCAGCTTAATAACTGCAGCTGAAGAA, was used to introduce two consecutive termination codons and a new PstI restriction site at the 3`-end of the P0 gene coding region in plasmid pSU-P0. The mutated gene, which expressed a P0 protein lacking the last 21 amino acids, was transferred as a 2.5-kb PstI-PstI fragment to the PstI site in Bluescript, and from this construction, the P0 gene was finally introduced into the multiple cloning site of pFL39 as a 2.5-kb EcoRI-SmaI insert.


Figure 1: Construction of plasmid pFLhisP0-C. See details under ``Materials and Methods.'' A, AvaII; B, BamHI; Cl, ClaI; E, EcoRI; Ec, EcoRV; H, HindIII; Hc, HincII; K, KpnI; N, NcoI; P, PstI; Pv, PvuII; Sc, SacI; Sl, SalI; Sm, SmaI; Sp, SphI; Ss, SspI; Xb, XbaI; Xh, XhoI; Xn, XmnI.



pFLhisP0D Series

As summarized in Fig. 2, progressive deletions at the 3`-region of the P0 gene in a 2.5-kb EcoRI-PstI fragment from plasmid BS-P0 were made by treating for increasing periods of time with exonuclease 3 (Sambrook et al., 1989). The treated fragments were religated to the same plasmid, and the size of the truncated insert in different clones was estimated by agarose electrophoresis. XhoI-BamHI inserts ranging from 1.9 to 1.5 kb were subcloned into the SalI-BamHI sites of plasmid pFL-C. pFL-C was derived from pFL39 by removing the HIS3 and URA3 markers. Finally, a 1.1-kb BamHI fragment from plasmid YDp-H (Berben et al. 1991), containing a HIS3 marker flanked by a sequence having termination codons in the three reading frames was linked to the 3`-region of the truncated P0 gene. The DNA inserts from a number of plasmids of different size were sequenced to determine precisely the deleted portion of the P0 gene. Two plasmids, pFLhisP0D2 and pFLhisP0D7, were selected for further studies. pFLhisP0D2 carries a 1.7-kb DNA insert coding for a polypeptide ending at leucine 226 in protein P0 plus five additional amino acids corresponding to the polylinker region in the insert from plasmid YDp-H previous to the termination codon. The polypeptide encoded by the1.55-kb insert in pFLhis-P0D7 ends at proline 181 and contains six additional amino acids from the polylinker.


Figure 2: Construction of plasmid series pFLhisD. See details under ``Materials and Methods.'' Xm, XmaI. Remaining restriction sites as in Fig. 1.



Electrophoretic Methods

Proteins were resolved by SDS-polyacrylamide gel electrophoresis. Proteins in the gel were electrophoretically transferred to Immobilon-P membrane (Millipore) using LKB Novablot buffer (39 mM glycine, 48 mM Tris-HCl, 0.0375% SDS, 20% methanol) as both anode and cathode electrode solutions. Electrophoresis was performed at 0.8 mA/cm^2 for 1 h. Treatment with antibodies was performed as described by Towbin et al.(1979).

RNA Blots (Northern)

Yeast RNA, extracted by phenol from intact cells was prepared as described by Warner and Gorenstein(1977). RNA was fractionated in 1.0% agarose gels and transferred to nylon membranes (Hybond-N, Amersham Corp.). Hybridization was performed using specific DNA probes indicated in the text, according to standard procedures (Sambrook et al., 1989).

Cell Fractionation

Cells in 20 mM Tris-HCl, pH 7.4, 80 mM KCl, 10 mM MgCl(2) were broken with glass beads in the presence of a mixture of protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatine A) and centrifuged in a Sorvall SS34 rotor for 15 min at 15,000 rpm obtaining an S-30 fraction. Preparation of ribosomes and S-100 fractions from S-30 has been described previously (Sanchez-Madrid et al., 1979). The acidic proteins were extracted from the ribosomes by ammonium-ethanol treatment and acetone precipitation (Sanchez-Madrid et al., 1979).

Polysome Analysis

Polysomes were obtained from exponentially growing cells treated with cycloheximide (100 µg/ml) 5 min before harvesting and broken with glass beads as previously indicated but this time including 100 µg/ml cycloheximide in all buffers. Extracts were centrifuged for 2.5 h in a TST 41,14 rotor through 7-45% sucrose gradients in 100 mM Tris-HCl, pH 7.4, 100 mM KCl, 30 mM MgCl(2), 5 mM 2-mercaptoethanol.

Immunological Methods

Antisera specific to P0 were obtained by injecting into rabbit truncated recombinant P0 protein lacking the last 21 amino acids, which correspond to the highly conserved carboxyl end, to avoid cross-reaction with the acidic P proteins (Santos and Ballesta, 1994). Monoclonal antibodies to the acidic P proteins were obtained as described previously (Vilella et al., 1991).


RESULTS

Construction and Characterization of S. cerevisiae W303 Strains Expressing Truncated P0 Proteins

Using either site-directed mutagenesis to introduce a new termination codon or exonuclease III treatment to produce nested deletions in the 3`-end of the P0 gene, three truncated forms of the gene were obtained, called P0-C, P0D2, and P0D7 (see ``Materials and Methods'' and Fig. 1and 2). These will express protein P0 derivatives lacking 21, 87, and 132 amino acids from the carboxyl end, respectively.

S. cerevisiae W303GP0, which carries a genomic P0 protein gene copy under the control of the inducible promoter GAL1 (Santos and Ballesta, 1994), was transformed in galactose medium with plasmids pFLhisP0-C, pFLhisP0D2, and pFLhisP0D7, which carry the truncated P0 genes. In each case, his colonies were selected in galactose media and characterized by Southern analysis of their DNA. The results confirmed that the his strains selected from each transformation, called S. cerevisiae W303GP0-pC, W303GP0-pD2, and W303GP0-pD7, respectively, have a genomic GAL1-controlled P0 gene as well as the corresponding plasmids carrying the truncated genes (not shown). Similarly, expression of the truncated genes was confirmed by Northern analysis of RNA extracted from the transformed strains grown in glucose.

Detection of the Truncated Proteins

As in the previous experiment, S. cerevisiae W303GP0-pC, W303GP0-pD2, and W303GP0-pD7 were grown on glucose for 24 h, and total cell extracts (S-30 fractions) as well as ribosomes were obtained. Only a small amount of cells was recovered from strain W303GP0-pD7 (see below), and, moreover, the yield of ribosomes from these cells was extremely low as previously reported for the parental W303GP0 strain growing in the same conditions (Santos and Ballesta, 1994).

Total cell extracts were resolved by SDS gel electrophoresis, and the presence of P0 was tested using specific antibodies (Fig. 3). Extracts of cells grown in galactose were also included as a control. In galactose-grown cells, only wild-type protein P0 is detected in all the cases; in glucose, the expression of the intact protein is repressed, and the presence of the truncated polypeptides is then detected. In the case of W303GP0-pD7 extracts, only the presence of wild-type P0 is found, indicating that the P0-D7 protein is very unstable, and only the P0 protein remaining from the galactose-grown cells is present.


Figure 3: Immunoblot of P0 protein in total cell extracts (S-30 fractions) from S. cerevisiae W303GP0 (1), W303GP0-pC (2), W303GP0-pD2 (3), and W303GP0-pD7 (4) grown in either galactose or glucose media. 25 µg of total cell extracts from the different strains were resolved by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and detected with rabbit antibodies to protein P0. The position of the different P0 forms is indicated.



The proteins extracted from ribosomes of the different glucose grown cells were also resolved by SDS gel electrophoresis and the presence of P0 detected by immunoblotting. The corresponding truncated protein was found in the ribosomes from W303GP0-pC and W303GP0-pD2; on the contrary, the ribosomes from the cells recovered from the W303GP0-pD7 culture, as in the corresponding S-30 extracts (Fig. 3), did not show the presence of a truncated P0, and only a band in the position of the intact P0 protein was detected, as in the case of the S-30 extracts (Fig. 4A).


Figure 4: Immunoblot of proteins in the ribosomes from S. cerevisiae W303GP0 (1), W303GP0-pC (2), W303GP0-pD2 (4), and W303GP0-pD7 (3). Ribosomal proteins were resolved by SDS electrophoresis, and detected with either anti-P0 serum (A) or with antiserum to the acidic protein P1/P2 (B).



To test whether the P0 truncation has any affect on the formation of the ribosome bound complex with the acidic proteins, the presence of P1/P2 was tested in the ribosomal particles from the different strains using a specific antibody. Only in the case of the wild-type and the W303GP0-pC strains was a strong P1/P2 protein signal detected. A weak band of acidic proteins was present in the ribosomes from W303GP0-D7 and, surprisingly, no acidic proteins were found in the particles from W303GP0-pD2 (Fig. 4B). The absence of acidic P proteins from W303GP0-pD2 was confirmed by isoelectrofocusing of ribosome extracts (data not shown).

Effect of Truncated P0 Protein Expression on Cell Metabolism

As expected, the yeast strains carrying the different truncated P0 proteins grow in a rich medium containing galactose with a doubling time around 120 min, similar to that of the wild-type strain. When they were transferred to a glucose medium and allowed to grow for 24 h, the expression of the wild-type GAL1-controlled P0 gene copy is repressed, and only the truncated forms of the protein are expressed. In these conditions, strain W303GP0 does not grow, and the parental wild-type strain has a doubling time of 90 min; the presence of P0 lacking the last 21 and 87 amino acids from the carboxyl end reduces the growth rate of strains W303GP0-pC and W303GP0-pD2 to approximately 160 and 210 min, respectively. The expression of P0 lacking the last 132 amino acids has more severe consequences; when W303GP0-pD7 is transferred to a glucose medium, a progressive reduction of the growth rate is detected, and after about 24 h, the cell growth stops. Moreover, a very important decrease in cell viability is detected, and after 4 days in glucose, only about 2% of the cells are able to form colonies when plated in galactose agar plates.

When extracts from strain W303GP0-pD7 growing in glucose for 6 h are analyzed by sucrose gradient, the presence of relevant shoulders in the peaks is detected, along with a substantial reduction in the amount of polysomes (Fig. 5). These shoulders correspond to ``half-mers,'' polysomes that carry at the 5`-end only an initiating 40 S ribosomal subunit and denote a deficiency of active 60 S ribosomal subunits in the cell. This effect is not found in the strains W303GP0-pC and W303GP0-pD2 grown in similar conditions. In fact, the results obtained with strain W303GP0-pD7 are in this respect analogous to those previously reported for the parental W303GP0 strain (Santos and Ballesta, 1994), indicating that the truncated protein is totally inactive.


Figure 5: Sucrose gradient analysis of total cell extracts from S. cerevisiae W303GP0-pD7 grown in galactose (A) and after 6 h in glucose (B). Arrows indicate the position of the half-mers.



Preparation of a P-protein-deficient S. cerevisiae Strain Carrying a GAL1-controlled P0 Gene

Previous reports using disrupted yeast strains carrying inactivated acidic protein P1-P2 genes have shown that the presence of these proteins bound to ribosome is not required for cell viability and, therefore, for ribosome activity (Remacha et al., 1992). However, since those strains contain a protein P0 in the ribosome, its carboxyl terminus may provide a protein P-like structure able to carry out its function with enough efficiency to maintain protein synthesis and cell viability. To test this possibility, the construction of a yeast strain carrying acidic protein P-free ribosomes and conditionally expressing a truncated P0 protein was carried out.

In S. cerevisiae D67, the genes encoding the two members of the acidic proteins YP1 group, YP1alpha and YP1beta, have been disrupted, and in these conditions the remaining YP2 proteins in the cell are unable to bind to the ribosome (Remacha et al., 1992). In this strain, therefore, protein synthesis is carried out on acidic protein-free ribosomes.

The genomic P0 gene copy in strain D67 was substituted by a GAL1 controlled copy as previously reported for construction of strain W303GP0 from wild-type S. cerevisiae W303 (Santos and Ballesta, 1994). The gene substitution was confirmed by Southern analysis of the transformed strain (Fig. 6). As expected, all of the selected strains, called D67GP0, were unable to grow on glucose agar plates.


Figure 6: Southern analysis of DNA from S. cerevisiae D67 (1), D67GP0 (2), and D67GP0-pC (3). DNA treated with EcoRI was resolved in agarose gels, transferred to membranes and hybridized with a DNA fragment containing the coding sequence of the P0 gene. The fragment corresponding to each one of the different gene constructs is indicated.



Effect of Truncated P0 Gene in S. cerevisiae D67 Strain

Cells of S. cerevisiae D67GP0 growing in galactose were transformed with plasmid pFLhisP0-C selecting for his transformants in the same medium. The presence of the plasmid in the transformed D67GP0-pC cells was confirmed by the presence of the corresponding restriction band in the Southern blot of the transformant's DNA (Fig. 6).

Strain D67 and its derivatives, D67GP0 and D67GP0-pC, grow in galactose with similar doubling times due to the expression of an intact P0 protein. When transferred to glucose, D67 grows with a doubling time of 225 min, but D67GP0 cannot grow due to the P0 repression. On the other hand, the expression of the truncated P0-C, that rescues the growth of W303GP0 in these conditions, is unable to support the growth of D67GP0.


DISCUSSION

Protein P0 interacts with the acidic proteins P1/P2 in the eukaryotic ribosome forming a complex that, by analogy with the bacterial particles, is presumed to form the stalk of the large ribosomal subunit (Uchiumi and Kominami, 1992). In this respect, P0 plays the same role played by protein L10 in the eubacterial ribosome. A comparison of P0 and L10 amino acid sequences shows a low but significant homology (Shimmin et al., 1989) that supports the functional equivalence of the two polypeptides. There are, however, important structural differences between the proteins. The most conspicuous difference is the presence in P0 of a carboxyl end domain of approximately 100 amino acids that resembles the sequence of the acidic P proteins, including the terminal decapeptide DDDMGFGLFD (Fig. 7) universally conserved from yeast to human ribosomes (Rich and Steitz, 1987; Newton et al., 1990).


Figure 7: Sequence comparison of the C-terminal half of protein P0 and acidic ribosomal proteins using the Gap program from the Wisconsin University Biotechnology Center GGC software. The deletions in the different truncated P0 protein forms are indicated.



The construction of yeast conditional null mutants unable to grow at the restrictive conditions has previously shown that the presence of P0 is required for ribosome activity and, therefore, for cell viability (Santos and Ballesta, 1994). Using these mutants, the activity of different truncated forms of P0 has been tested. The results show that S. cerevisiae cells are able to grow with a doubling time of 160 min, only 70% higher than the parental strain, when ribosomes carry a P0 lacking the last 23 amino acids. The data indicate, therefore, that the presence of the highly conserved carboxyl end is not absolutely required for protein P0 function, probably due to the existence of a similar structure in the acidic proteins P1/P2.

A not very different reduction of the growth rate was previously detected when any one of the four yeast acidic proteins, YP1alpha, YP1beta, YP2alpha, or YP2beta, is suppressed by gene disruption (Remacha et al., 1990). Since the P proteins are present as dimers (Juan-Vidales et al., 1983; Uchiumi et al., 1987) and the presence of one protein from each type, P1 and P2, is required for ribosome binding of the protein P complex (Remacha et al., 1992), at least five identical C-terminal peptides must be found in a standard active ribosome, including the one in P0, a figure that is close to experimental determinations (Saenz-Robles et al., 1990). Our results indicate that one of these C-terminal structures, or even two of them, if they correspond to P proteins of different type (Remacha et al., 1992), can be removed causing a relatively small effect on the protein synthesis capacity of the ribosomes.

The suppression of two acidic proteins from the same group, as in strain D67, caused a more dramatic effect; in these conditions, a protein P-free ribosome was found in the cell, and a 3-4-fold increase of the doubling time is detected. These defective ribosomes were, nevertheless, still able to maintain cell viability and showed a surprisingly high in vitro activity (Remacha et al., 1992). The disruption of three and even the four acidic protein genes has similar effects.^1

In this report it is clearly shown that the truncated P0 protein is unable to maintain the viability of strain D67GP0 when the expression of the wild-type P0 protein is suppressed by growing in glucose. These results indicate that the activity of the protein P-free ribosomes is dependent on the presence of the acidic protein-like carboxyl end in protein P0 and that this presence seems to be sufficient to allow ribosome activity and protein synthesis. This conclusion confirms the relevance of this so highly conserved structure for ribosome function, probably related to supernatant factor interaction, and at the same time indicates that this function can be performed by only one copy strongly bound to the ribosome. The presence of multiple copies of acidic proteins increases the efficiency of translation notably, but it does not seem to affect the basic polymerization process, since the accuracy of the protein P-free ribosomes is not appreciably affected. Therefore, the reported simultaneous presence of the acidic protein C-terminal in two different locations in the ribosome, at the tip of the stalk and in the body of the particle (Olson et al., 1986), is probably not essential for ribosome activity although confirms the high flexibility of this structure (Cowgill et al., 1984).

The deletion of about 70 more amino acids from the P0 carboxyl domain, removing almost 80% of the P-protein like-addition (Fig. 7), causes an additional negative effect in the wild-type W303 strain growth, increasing the doubling time to 210 min. The binding of the P1/P2 proteins to the ribosomes in these cells is very weak, and they are removed easily from the particles, indicating that the segment of the P0 sequence between residues 225 and 290 is required for a stable binding of the P1/P2 protein complex. On the other hand, the 21-amino-acid C-terminal peptide does not seem to have an important role in the P proteins binding to the ribosome since the washed ribosomes from the W303DP0-pC have a normal amount of P proteins.

Interestingly, the formation of the P0/P1-P2 complex has been suggested to be based on the existence of bilateral hydrophobic zippers in the structure of both types of proteins (Tsurugi and Mitsui, 1991). In agreement with this hypothesis, the region where these hydrophobic zippers have been proposed in P0 corresponds precisely to the sequence between residues 205 and 282, mostly missing in the truncated P0-D2 (Fig. 7).

The absence of 138 amino acids from the C terminus completely abolishes the activity of the protein P0. In this case, corresponding to S. cerevisiae W303GP0-pD7, the growth rate and viability of the cells declines when the cultures are shifted to glucose media, inducing the expression of the truncated protein; after about 24 h, the growth has completely stopped, and the number of viable cells decreases dramatically. Similarly, a decrease in the amount of polysomes and the presence of half-mers is detected in cells growing in glucose. All of these results are very similar to those obtained when the expression of the intact P0 is repressed in the parental W303dGP0 strain growing in glucose (Santos and Ballesta, 1994) and indicates that the P0-D7 protein is totally unable to functionally substitute for the wild-type polypeptide. In these conditions, a deficiency in large subunits occurs and finally ribosome synthesis seems to stop, causing an alteration of cell metabolism, which results in cell death. Nevertheless, the time required for cell growth to stop after shifting to the restrictive medium is unexpectedly long. In fact, the effect on the growth rate is rather slight during the first few hours in spite of the disappearance of the P0 mRNA from the cells after only 60 min in glucose medium (Santos and Ballesta, 1994). It seems that exponentially growing cells contain an excess of ribosomes that is able to buffer the effect of an halt in the ribosome synthesis for a substantial period. A similar effect has been reported as yeast passes through a normal growth cycle; rRNA and r-protein synthesis stop at an early stage, but cells continue to grow at normal log rate for some time (Ju and Warner, 1994).

Previous data suggest that protein P0 is probably assembled into the ribosome in the cytoplasm and that, at least in yeast, the protein not bound to the particle is degraded (Santos and Ballesta, 1994). The fact that, in spite of the presence of the corresponding mRNA, the P0-D7 protein is not detected in the cells, indicates that the defective polypeptide is unable to bind to the 60 S subunit and, consequently, degraded. Therefore, the 44-amino-acid fragment present in P0-D2 and absent in P0-D7 (Fig. 7) must play an important role in the interaction of the protein with the ribosome, either directly, by being implicated in the interaction with the rRNA, or indirectly, by conferring the right conformation for binding to the polypeptide. The second alternative is probably correct since it has been shown that the N-terminal domain of bacterial protein L10 is the part of the polypeptide implicated in the interaction of the (L7/L12)(4)L10 with the ribosome (Gudkov et al., 1980).

It has to be noted, however, that although only the last 44-amino-acid fragment is essential for interaction with the ribosome; the absence of the other two P0 fragments is not innocuous and affects the cell growth rate. Moreover, the fact that none of the truncated proteins is detected when cells are growing in galactose indicates that the defective polypeptides cannot compete for binding with the wild-type P0 and are quickly degraded.

Summarizing these results a model can be proposed for the P0bulletP1bulletP2 complex (Fig. 8) in which the carboxyl-terminal extension in the P0 protrudes from the bulk of the protein, in an acidic protein-like conformation, forming a minimal ``stalk'' structure. At the same time, it acts as an anchor for the P1 and P2 complex that complete the stalk, making it fully efficient. In this structure, the central part of the P0 extension plays a key role in the interaction with the acidic protein complex, probably through hydrophobic zippers (Tsurugi and Mitsui, 1991), while the amino-terminal part of the extension has a role in the interaction of the complex with the ribosome.


Figure 8: Model of the P0bulletP1/P2 complex in the S. cerevisiae ribosome. The proposed complexes for the different truncated P0 proteins are shown. Two dimers of phosphorylated proteins P1 and P2 interact with the P1/P2-like C-terminal of P0. The absence of the last 21 amino acids from P0 (P0-C) seems not to affect the interaction of P1/P2 but the removal of 66 additional amino acids, including the leucine-zipper forming region (P0-D2), weakens the interaction of the P1bulletP2 complex that remains bound to P0 only through the amino end. The deletion of 132 amino acids seems to block the binding of the P1bulletP2 complex, and either directly or indirectly the interaction of P0 with the rRNA.



Ribosomes containing only the minimal ``P0 stalk'' are functional and can participate in a protein synthesis showing a correct decoding accuracy but low efficiency^1 (Remacha et al., 1992). Moreover, they seem to preferentially translate a distinct set of mRNAs, since a different protein expression pattern is detected in cells containing only P0 and lacking the P1/P2 acidic proteins.^1 Experimental evidence indicates that the amount of acidic P proteins found in the ribosome depends on the metabolic state of the cells (Saenz-Robles et al., 1990). Therefore, P protein-free ribosomes may not be just an artificial genetic construction; they could be present in the cell at some stage of the cell cycle. It is possible that these apparently deficient particles have a role in the control of protein synthesis by preferentially translating some specific mRNAs (Ballesta et al., 1993).


FOOTNOTES

*
This work was supported by Grant PB91-0011 from the Dirección General de Pol&ıacute;tica Cient&ıacute;fica (Spain) and by an institutional grant to the Centro de Biolog&ıacute;a Molecular from the Fundación Ramón Areces (Madrid). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Centro de Biologia Molecular, Universidad Autonoma, Canto Blanco, 28019 Madrid, Spain. Tel.: 34 1 3975076; Fax: 34 1 3974799.

(^1)
Remacha, M., Jimenez-Diaz, A., Bermejo, B., Rodriguez-Gabriel, M. A., Guarinos, E., and Ballesta, J. P. G.(1995) Mol. Cell. Biol., in press.

(^2)
The abbreviation used is: kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank M. C. Fernandez Moyano for expert technical assistance and Dr. P. P. Dennis (University of British Columbia, Vancouver, Canada) for providing plasmid pSceL10e. We also thank Dr. W. Strong and Dr. M. Remacha for reading and commenting on the manuscript.


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