(Received for publication, March 1, 1995; and in revised form, May 15, 1995)
From the
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.
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 L10(L7/12)
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, YP1-YP1
and
YP2
-YP2
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. (
)
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)P0
(P2)
.
In spite of
the clear functional and structural similarities, some conspicuous
characteristics differentiate the bacterial and eukaryotic
P0P1/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 P0
P1/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.
Escherichia coli JM83,
TG1, and DH5 were used as the hosts for the routine maintenance
and preparation of plasmids used in these studies and were grown in LB
medium.
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.
Figure 2: Construction of plasmid series pFLhisD. See details under ``Materials and Methods.'' Xm, XmaI. Remaining restriction sites as in Fig. 1.
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.
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).
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.
In S. cerevisiae D67, the genes encoding
the two members of the acidic proteins YP1 group, YP1 and
YP1
, 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.
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.
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, YP1, YP1
,
YP2
, or YP2
, 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.
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)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 P0P1
P2 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 P0P1/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
P1
P2 complex that remains bound to P0 only through the amino end.
The deletion of 132 amino acids seems to block the binding of the
P1
P2 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 (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.
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).