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INTRODUCTION |
The ribosome is the central constituent of the protein synthesis
machinery (1). During translation of messenger RNA into protein,
the ribosome is helped by several soluble factors that operate in a
sequential manner to improve both efficiency and fidelity of this
process (2, 3). How this extraordinary coordination is performed is not
yet exactly understood. Still, because most of the translation factors
are GTPases, the driving of the factors by the ribosome is likely to be
controlled by GTP hydrolysis (4). A small portion of the 28 S rRNA
designated as the GTPase center is known to be involved in GTP
hydrolysis activation (5). The GTPase center is connected directly to the stalk (6), an elongated and very flexible protuberance interacting
with elongation factors (7-10). However, despite decades of research,
the organization and functions of the proteins constituting the stalk
remain unclear. The number and the nature of the proteins constituting
the stalk are different depending on the biological system, although
their general organization, made of five proteins, is likely to be
similar. In prokaryotes, four identical proteins (L7/L12) are linked to
the GTPase center by L10 connected itself with a sixth protein, L11
(11). In mammals, the equivalents of the four L7/L12s are two different
proteins, P1 and P2, each being present in two copies. These proteins
are bound to P0, the equivalent of L10, which is itself bound to L12,
the eukaryotic equivalent of L11, and to the GTPase center (6, 12). In
plants, an additional protein, P3, has been described (13). In yeast, there are two variants of both P1 (P1
and P1
) and P2 (P2
and P2
); the precise repartition and function of each variant remains unsettled (14). This structural heterogeneity seems to correspond to
functional differences, and data obtained in one system cannot be
extrapolated directly (10). Both L7/L12 and P1/P2 have in common their
size (around 110-120 residues), their acidity, and the fact that N-
and C-terminal domains are joined by an alanine-rich flexible
region (15). The C-terminal protruding region (16, 17) is identical in
P0, P1, and P2 and contains phosphorylation sites not found in
prokaryotes. In the rat, P2 phosphorylation has been shown to stimulate
the proteosynthetic activity of the ribosome (18) and the GTPase
activity of eEF-2 (19). The N-terminal domains of P1 and P2, although
of very different lengths in eukaryotes and prokaryotes, are involved
in P0 and L10 binding, respectively (20, 21). In eukaryotes, an
exchange between the ribosome-bound P1 and P2 (but not P0) and a
cytoplasmic pool of these proteins has been shown (22-24), a situation
not found with L7/L12 in prokaryotes. The functions and conditions of
this exchange remain unexplained. P0, contrary to L10, contains the
flexible alanine-rich region and the phosphorylable C-terminal
domain found in P1 and P2. Mobility complicates the study of the
components of the stalk that represent two of the last three proteins
not shown in the crystallographic structure of the 50 S ribosomal
subunit of the archaea, Haloarcula marismortui (25, 26).
Besides, structural studies of the isolated proteins are incomplete
(27). This mobility is biologically relevant (28), and the conformation
of the stalk was shown to be different depending on the step of the
ribosomal cycle (8, 9, 29, 30). Hence, it is a major goal to understand
how these dramatic conformational changes operate at a molecular level.
In previous works from our laboratory, rat recombinant P1 and P2
proteins were overproduced and studied when linked to the ribosome (18)
and as isolated proteins (19). The lack of P0 prevented us from going
further in the study of the functions of these proteins in a
ribosomal context. Here, recombinant P0 was successfully overproduced
and shown to be effective in reconstituting a functional stalk together
with P1 and phosphorylated P2. Several approaches were used to
determine the interactions between the stalk components and led us to
propose a new model for its functional architecture.
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EXPERIMENTAL PROCEDURES |
Materials
The RNAgents total RNA isolation system kit, the
oligo(dT)15 used as a primer for the reverse
transcription, the BamHI and SmaI
restriction enzymes, the T4 DNA ligase, and the Escherichia coli cells (JM109 strain) were from Promega. The primers used for
the DNA polymerase chain reactions were from Isoprim. Pwo DNA polymerase, RNase H, and
-octylglucoside were acquired from Roche Molecular Biochemicals. Superscript II RNase
H
reverse transcriptase was from Life
Technologies, Inc. The pQE-30 plasmid and the
Ni2+-nitrilotriacetic acid-agarose gel came from Qiagen.
Preparation and properties of the monoclonal antibody (4C3) used to
detect the P-proteins in the Western blot have been previously
described (31).
Methods
Construction of the P0-pQE-30 Expression Vector--
The
cDNA of P0 was obtained by reverse transcription-polymerase chain
reaction of total RNA prepared from 1 g of rat liver (Wistar
strain) using the general guanidinium thiocyanate plus 2-mercaptoethanol method (32). The reverse transcription step was
performed under Superscript II standard conditions. Polymerase chain
reaction (30 cycles) was performed using 0.2 µl of the reverse transcription product and 10 µM 5' (TAT GGA TCC ATG CCC
AGG GAA GAC AGG GCG ACC) and 3' (TAT CCC CGG TTA GTC GAA GAG ACC GAA
TCC CAT) primers. The annealing temperature was 59 °C. P0 cDNA
(960 base pairs) was cloned (18), and its sequence was in full
agreement with the Swiss-Prot P0 sequence of Rattus
norvegicus (entry name: RLA0 RAT; primary accession number:
P19945) corrected for three conflicts compared with the previously
published sequence (33, 34).
Overproduction and Purification of P0--
P0, overproduced as
described for P1 and P2 (18), was mostly insoluble and purified from
inclusion bodies. The pellet of the bacterial lysate was submitted to a
sequential extraction with 12 volumes of 1.5-3 M and 6 M guanidine. P0, extracted in the 3 and 6 M
guanidine fractions, was eluted via Ni2+-nitrilotriacetic
acid-agarose gel chromatography with 190 mM imidazole in a
buffer containing 4 M guanidine, 50 mM ammonium phosphate, pH 7.5, 300 mM KCl, 0.1 mM EDTA,
10% (v/v) glycerol. After dialysis against this buffer, the
preparation, divided into aliquots, was frozen at
80 °C. The final
yield from a 1-liter culture was 15 mg.
Cloning, Overproduction, and Purification of the Mutants of P1
and P2--
Truncated mutants of P1 and P2 were used in this work. N1
and N2 comprise the amino acids 1-63 and 1-65 from P1 and P2,
respectively. These proteins were overproduced after the cloning and
sequencing of their cDNAs as described previously (18). cDNAs
were obtained by polymerase chain reaction using P1-pQE-30 and
P2-pQE-30 vectors as templates (18), the 5' primers previously
described (18), and the following 3' primers: ATT AAG CTT TTA TAC ATT
GCA GAT GAG GCT TCC for N1 and ATT AAG CTT TTA CAC ACT GGC CAG CTT GCC AAC for N2. N2 was overproduced in the supernatant only and purified as
described for P1 and P2 (18). N1, found only in inclusion bodies, was
purified following the procedure used for P0, except that 8 M guanidine was necessary to solubilize and purify it.
Solubilization of P0 and N1--
Renaturation was carried out by
removing the guanidine with an overnight dialysis at 4 °C. 10 µM P0 (or 20 µM N1) and 20 µM ligand(s) (P1 alone or N1 plus either P2 or N2, in the case of P0, or
N2 or P2, in the case of N1) in 2 M guanidine, 40 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM 2-mercaptoethanol, 1 µM EDTA, and 20% (v/v) glycerol were dialyzed against the same buffer but without guanidine. A similar procedure was used for N1 using P2 or N2 as
ligands. The yields of solubilization of either P0 or N1 by increasing
ligand concentrations were determined according to the following
procedure. Mixtures containing a fixed concentration of either P0 or N1
were dialyzed with increasing ligand concentrations and then
centrifuged at 17,000 × g for 30 min. In each test,
the concentration of the proteins in the supernatant (solubilized P0 or
N1 plus the ligands) was determined using the Coomassie Blue plus
protein assay reagent kit from Pierce. Then, the soluble P0 or N1
concentration was obtained by subtracting the ligand concentration
from the measured concentration. This calculation is well
founded, because ligands behaved as soluble proteins that were not
found in the pellets in a significant amount after the renaturation process. These measures were checked by analyzing aliquots
of both the supernatants and the pellets via
SDS-PAGE1 and by quantifying
the bands corresponding to either P0 or N1 using a Personal
Densitometer SI (Molecular Dynamics) equipped with the Image QuanNT
software as previously described (19).
Analysis of the Complexes between the P-proteins by
Two-dimensional Electrophoresis--
The supernatant of the dialyzed
solutions of the P-proteins was loaded onto a non-denaturing,
1.5-mm-thick, 6% polyacrylamide electrophoresis gel (13 cm × 13 cm) cooled at 4 °C (35) that contained 50 mM Tris/HCl,
pH 8.5, 50 mM KCl, 5 mM
-octylglucoside, 5 mM MgCl2, and 15% (v/v) glycerol. A
premigration was carried out during 1 h at 80 V before loading the
samples. The sample buffer contained 150 mM Tris/HCl, pH
8.5, 300 mM KCl, 5 mM MgCl2, 20 mM
-octylglucoside, 20 mM dithiothreitol,
20% (v/v) glycerol, and 0.02% bromthymol blue. The overlay buffer
poured onto samples was identical, except that it contained only 10%
(v/v) glycerol and 50 mM KCl. Migration was performed at
100 V for 1 h, 200 V for 2 h, and then 250 V until the
tracking dye reached the end of the gel. Electrophoresis buffer (150 mM Tris/HCl, pH 8.5, 50 mM KCl, 5 mM MgCl2 and 5 mM dithiothreitol)
was changed every hour. The gel was Coomassie Blue-stained, and the
slab corresponding to the first electrophoresis was cut and included in
the stacking layer of a 2-mm-thick 17% SDS-polyacrylamide gel
(36).
Reconstitution of Active Dimethylmaleic Anhydride (DMMA)
Particles--
Extraction of P0 from 60 S subunits and reconstitution
of the biological activity after addition of either the split or
recombinant proteins were adapted from the method described by Nieto
et al. (37) with the following modifications. 1) 60 S
subunits and DMMA concentrations were 0.8 µM and 21.6 mM, respectively; 2) all steps were performed on ice; 3)
split proteins were separated from the DMMA particles by
ultracentrifugation through a 15% sucrose layer; 4) core particles and
split proteins were dialyzed against a buffer containing BisTris, pH
6.0 in place of sodium cacodylate; 5) reconstitution was performed in
the same buffer at pH 7.0; and 6) proteins not bound to DMMA particles
were separated by a second ultracentrifugation through a 15% sucrose
layer. The proteosynthetic activity of the subunits was measured by the
polyphenylalanine synthesis test (38).
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RESULTS |
P0 Is Overproduced as an Insoluble Protein--
P0 overproduced in
an E. coli system fused with an N-terminal poly(His) tag was
found mostly in inclusion bodies, contrary to what had been found
previously with P1 and P2 overproduced under similar conditions (18).
Coproduction of P0 with the chaperonines GroES/GroEL or with
thioredoxin did not improve the amount of soluble P0 in the bacterial
supernatant. Hence, P0 was purified from inclusion bodies using 4 M guanidine. Several methods to obtain non-denatured
soluble P0 from this solution were unsuccessful. Dilution or dialysis
of the guanidine under different conditions resulted in P0
precipitation, as did an attempt to refold it immobilized on the
Ni2+-nitrilotriacetic acid-agarose gel affinity
column used for its purification. This led us to try to renature P0 in
the presence of its available potential ligands in the stalk, the
soluble proteins P1 and P2.
P1 but Not P2 Can Solubilize P0--
P0 was mixed with recombinant
proteins P1 and P2 in 2 M guanidine to keep it soluble.
When guanidine was removed by dialysis (Fig.
1), nearly all P0 remained soluble in the
presence of P1 alone and (P1 + P2) but surprisingly not in the presence
of P2 alone (compare the insoluble proportion of P0 in lanes
2, 4, and 3, respectively, with that in
lane 1, corresponding to P0 alone). The small amounts of P1
and/or P2 that were found in the pellets in lanes 2-4 were
also found when P1 and/or P2 were submitted to the solubilization
process in the absence of P0 (data not shown). This precipitation
should originate from a very limited denaturation of P1 and P2 under
the conditions of the solubilization process. This experiment suggested
that P1 but not P2 could interact with P0 to make a soluble complex.
Quantitative data were obtained, and the effect of P1 concentration on
P0 solubilization led to the determination of the stoichiometry of this
complex (see Fig. 4, filled circles).

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Fig. 1.
Differential effects of P1 and P2 on P0
solubilization. Solubilization of 28 µg (0.8 nmol) of P0 (35.2 kDa) was performed by an overnight dialysis of the guanidine in the
absence of any other protein (lane 1) or in the presence of
1.6 nmol (21 µg) of either P1 alone (12.9 kDa) (lane 2) or
P2 alone (13.1 kDa) (lane 3) or in the presence of 1.6 nmol
of (P1 + P2) (lane 4). After centrifugation, the proteins
contained in the pellets were analyzed via 15% SDS-PAGE (36). P1 and
P2 are known to migrate at a higher molecular mass than predicted from
their sequence. MW, molecular mass; kD,
kilodalton.
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Rationale for the Design of Shortened Mutants of P1 and
P2--
Because it had been suggested that N-terminal domains in P1
and P2 were involved in the binding to P0 in yeast (21), we prepared
truncated mutants of rat P1 and P2. N1 and N2, the N-terminal domains
of P1 and P2, respectively, contained the first 63 and 65 residues of
P1 and P2 preceded by a poly(His) tag. N1 and N2 sequences are
highlighted in gray in Fig. 2,
in which hydrophobic regions are shown under the sequences. N2,
predicted to be a very hydrophilic protein, was purified from the
bacterial supernatant. In contrast, N1, mostly made of hydrophobic
regions, was overproduced and purified only from inclusion bodies in 8 M guanidine.

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Fig. 2.
Location of the hydrophobic regions
in P0, P1, and P2. Hydrophobicity of the P-proteins was studied
following the method of Kyte and Doolittle (48) using MPSA
software (49, 50). Hydrophobic (Hy) regions
are displayed with a + under the protein sequence. The
common C-terminal sequence is written in bold black
characters, and the alanine/proline-rich region (the hinge) is
shown in gray bold letters. Sequences of the (1-63) and
(1-65) N-terminal domains of P1 and P2, respectively, are highlighted
in gray.
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N1 Interacts with Both N2 and P0--
Dialyses of N1 in the
presence of P2 or N2, which were potential ligands for N1, were carried
out to determine whether it was possible to solubilize N1 in the
absence of guanidine (Fig. 3A). N1, a completely
insoluble protein (Fig. 3A, lane 1), was solubilized by both N2 (lane 2) and P2 (lane 3).
The effect of increasing concentrations of either N2 or P2 on N1
solubilization (20 µM) was studied (Fig. 3B).
For that, N1 solubilization was carried out in the presence of
increasing concentrations of either N2 or P2, and the concentrations of
N1 remaining soluble after dialysis of the guanidine were determined
using the procedure described under "Experimental Procedures" and
in the legend to Fig. 3. Using either P2 or N2, we observed that the
soluble N1 concentration was directly proportional to that of P2 or N2
up to 20 µM. The shapes of these curves revealed
equimolar complexes between N1 and P2 or N1 and N2, because the slopes
were equal to one. Furthermore, the level of the plateau indicated that
the solubility of the complex N1-P2 (Fig. 3B, open
squares) was 20 µM at least, this
concentration corresponding to the solubilization of all the N1
available in the test. The complex N1-N2 (Fig. 3B, filled diamonds) was slightly less soluble (about 18 µM), because a small fraction of N1 remained insoluble
for any concentration of N2 tested. This result indicated that P1 and
P2 could bind each other and that their N-terminal domains were
involved in the process of heterodimerization. This suggested also that
the common C-terminal domain might play a main part in the P-protein solubility, which might be due to its high hydrophilicity (see Fig. 2).
On the two-dimensional electrophoreses displayed in Fig. 5, P1-P2 and
N1-P2 complexes were visualized as complexes C1 in panel A and C2 in panel B,
respectively (see below for more details).

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Fig. 3.
Effects of P2 or N2, the (1-65) domain of
P2, on the solubilization of N1, the (1-63) domain of P1.
A, solubilization of 16 µg (2 nmol) of N1 (8.0 kDa) in 100 µl was performed by an overnight dialysis of the guanidine in the
absence of any protein (lane 1) and in the presence of an
equimolar amount of N2 (8.0 kDa) (lane 2) or P2 (lane
3). After centrifugation, insoluble proteins were loaded onto an
18% SDS-polyacrylamide gel (36). N2 (as P2) was a fully
soluble protein under these conditions and does not contribute to the
band shown in lane 2. N1 alone (lane 1) is a
fully insoluble protein under these conditions. MW,
molecular mass; kD, kilodaltons. B,
solubilization in 100 µl of 2 nmol of N1 (16 µg) was performed by
an overnight dialysis of the guanidine using increasing concentrations
of N2 ( ) or P2 ( ). After centrifugation, the concentration of
soluble N1 was measured by subtracting the N2 or P2 concentration
from that of the total soluble protein concentration, because
both N2 and P2 were fully soluble. In the case of P2, the accuracy of
this method was verified by quantifying the bands corresponding to N1
in the pellet and in the supernatant after separation via SDS-PAGE, as
described under "Experimental Procedures" and in Ref. 19. Using N2,
this method could not be applied to quantify N1 in the supernatants,
because N1 and N2 had the same molecular weight.
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To determine the stoichiometry of the complexes between P0 and its
ligands, an approach similar to that used for N1 in Fig. 3B
was applied to P0 (Fig. 4). Using P2 as a
potential ligand, no enhancement of P0 solubility was shown, regardless
of the P2 concentration (Fig. 4, filled triangles).
Using P1 as a ligand (Fig. 4, filled circles), the
concentration of soluble P0 increased linearly with P1 concentration,
with a slope of about 0.5 (P0 (µM)/P1 (µM))
up to about 20 µM P1. This slope suggested that a complex
containing two P1 molecules for one P0 was formed (P0 concentration in
the test was 10 µM). Above 20 µM P1, a
plateau corresponding to the solubilization of all the P0 available was observed. A similar result was obtained using (P1 + P2) in place of P1
alone. These results showed that two P1 molecules (or two heterodimers,
P1/P2) had to associate with P0 to solubilize it.

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Fig. 4.
Effects of increasing concentrations of
potential ligands on P0 solubilization. Solubilization of 1 nmol
of P0 (35 µg) in 100 µl was performed by removing the guanidine
with an overnight dialysis in the presence of increasing concentrations
of various ligands. After centrifugation, the concentration of soluble
P0 was measured by subtracting the ligand concentration from
that of the total soluble protein concentration. Furthermore, results
given by this calculation were assessed by quantifying the bands
corresponding to P0 in the pellets and in the supernatants after
separation via SDS-PAGE, as described under "Experimental
Procedures" and in Ref. 19. When two proteins were added together,
they were in equal molar concentration. The proteins tested for P0
solubilization were P1 ( ), P2 ( ), N1 ( ), (N1 + P2) ( ), (N1 + N2) ( ). (P1 + P2) gave a curve superposable with that given by P1
alone, and that of N2 was identical to that of P2.
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To test whether N1, the insoluble N-terminal domain of P1, interacted
with P0, a trial of (N1 + P0) cosolubilization was performed (Fig. 4,
filled squares). It resulted in no solubilization of P0 and
even in a reduction of its solubility. This suggested that a complex
between N1 and P0 was formed but was insoluble, which might originate
from the fact that the hydrophobic regions constituting mainly P0 and
N1 were not all buried in the complex (see Fig. 2). Then, the ability
of the complexes N1-P2 and N1-N2 to solubilize P0 was studied (Fig. 4).
Contrary to P2 or N2 alone (Fig. 4, filled triangles)
that did not modify the solubility of P0, the addition of increasing
concentrations of either (N1 + P2) (open circles) or (N1 + N2) (filled diamonds) resulted in a substantial increase in
P0 solubility. These observations indicated that P2 did not bind to P0
directly but through an interaction between its N-terminal domain and
that of P1. Still, the shapes of the curves suggested that these
complexes were less efficient than full-length P1 (or (P1 + P2)) in
achieving P0 solubilization. Indeed, the maximal effect was obtained
with a higher molecular ratio (~3 versus ~2), suggesting
that the P0 affinity for N1-P2 or N1-N2 was lower than that for P1 or
P1-P2. Furthermore, the maximal solubility of these complexes was lower
(around 6.5 µM) than that given by P1 (or (P1 + P2)) (at
least 10 µM; the highest solubility was not obtained). These data indicate that the hinge regions of P0 and P1 and perhaps also the C-terminal domains may be involved in the stabilization of the
complex P0-P1.
The existence of the complex resulting from the association of P0 with
P2 through the N-terminal domain of P1 was directly shown via
two-dimensional electrophoresis of a mixture of P0, N1, and P2 (complex
C3 in Fig. 5B). A
significant part of P0 precipitated in the well (Fig.
5B, L), probably because of the low salt
concentration in the first dimension electrophoresis buffer and
to a stabilization of the aggregates by oxidation of the P0 cysteine.
P2 migrated in excess compared with P0 and N1, because about 40% of P0
and N1 had precipitated during the dialysis (Fig. 4, open
circles). The free form of P2 was found to migrate as the broad
band labeled F in Fig. 5, which might originate from
an association/dissociation equilibrium of P2 dimers under these
conditions (see below and Ref. 17).

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Fig. 5.
Two-dimensional gel electrophoreses of
P-protein complexes. A, an equimolar mixture of P1 and
P2 was loaded onto a non-denaturing 6% polyacrylamide gel at
pH 8.5, and its migration is represented by the horizontal slab stained
with Coomassie Blue. This slab was cut and included in the staking
layer of an 18% SDS-polyacrylamide gel (36). The
two-dimensional electrophoresis reveals that P1 and P2 migrate
mainly as a complex (C1) and that almost no P2 is found in
the broad band F containing free P2 (compare with
panel B). B, after dialysis and centrifugation,
the soluble fraction of a P0, N1, and P2 mixture (molar ratio of 1:2:2)
was separated in bands L, C3, and C2
using a non-denaturing 6%-polyacrylamide gel at pH 8.5 stained
with Coomassie Blue. L, at the basement of the well,
contained aggregated proteins unable to enter into the gel. The
slab was cut and included in the staking layer of an 18%
SDS-polyacrylamide gel (36). After migration and staining with
silver nitrate, the gel showed that L contained only
P0, that C3 was a complex of P0 with N1 and P2, and that
C2 was a complex of N1 with P2. P0 and N1 that are insoluble
did not migrate in the absence of P2 under these non-denaturing
conditions. The broad band F represents the migration of
free (unbound) P2 (see "Results"). MW, molecular mass;
kD, kilodaltons.
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Incorporation of P1 and P2 into the Ribosomal Stalk Requires the
Prior Formation of P1-P2 Dimers--
The preceding results led us to
conclude that P1 and P2 associated as a heterodimer to P0. However, the
questions could be asked whether P1 and P2 could form homodimers and
whether these homodimers might participate in the stalk formation.
To answer these questions, N1, the insoluble N-terminal domain of P1,
was mixed with either full-length P1 (Fig.
6A, lane 1) or P2
(lane 2); the amount of solubilized N1 was approximately equal in both lanes. From this experiment, one may conclude that P1
associated significantly to N1 and hence that two P1s (or more) could
bind by their N-terminal domains. The ability of P2 to dimerize was
deduced from its behavior when passed through a gel filtration column;
it was eluted as a single peak, the elution volume of which
corresponded to the mass of a P2 dimer (data not shown).

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Fig. 6.
Compared effects of P1 and P2 on the
solubilization of N1, the N-terminal domain of P1, either alone
(A) or in the presence of P0
(B). A, solubilization of 19 µg (2.4 nmol) of N1 was performed by an overnight dialysis of the guanidine in
the presence of an equivalent molar amount (31 µg) of P1 (lane
1) or P2 (lane 2) in 100 µl. After centrifugation,
soluble proteins were loaded onto an 18% SDS-polyacrylamide
gel (36). N1 alone is a fully insoluble protein. B,
solubilization of 0.6 nmol of P0 (21 µg) and 1.2 nmol of N1 (9.6 µg) was performed under the same conditions in the presence of 1.2 nmol (15.5 µg) of either P1 (lane 1) or P2 (lane
2) in 50 µl. After centrifugation, soluble proteins were loaded
onto an 18% SDS-polyacrylamide gel (36).
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However, by the two-dimensional electrophoresis of the equimolar
mixture of P1-P2 (Fig. 5A), it was shown that all the
proteins were involved in the P1-P2 complex. Indeed, almost no P2 was
found in the broad band F, corresponding to unbound P2
(compare with Fig. 5B). This indicated that in the absence
of P0, purified P1 and P2 bound one another to form heterodimers and
that there was a disruption of the homodimers.
To elucidate whether P1 and P2 could bind P0 as homodimers, we mixed
the two insoluble proteins, P0 and N1 (molar ratio 1:2), in the
presence of either P1 or P2 (molar ratio to P0 of 1:2) (Fig.
6B). We observed that in both cases, a part of P0 and N1 was
solubilized but in different proportions. In the presence of P1 (Fig.
6B, lane 1), a large amount of P0 was
solubilized, whereas the majority of N1 had precipitated (compare the
ratio of P1 to N1 in panel A with that found in panel
B). In the presence of P2, the situation was the opposite (compare
lane 2 with lane 1 in Fig. 6B); the
amount of solubilized P0 was lower, and that of N1 was higher. From
these results, one may deduce that P1 bound poorly to N1 in the
presence of P0. Consequently, this experiment suggested that the
N-terminal domain of P1 had a common binding site for both P0 and P1
and therefore that it could bind simultaneously only P0 and the
N-terminal domain of P2 but not a second P1. Therefore, it was likely
that the P1 homodimers observed in purified solution had to dissociate
to allow the binding to P0 in the stalk. The binding of the N-terminal
domain of P2 to that of P1 explained the situation illustrated in Fig.
6B, lane 2; P2 had to associate first with
N1, and the N1-P2 complex has already been shown to be less effective
in solubilizing P0 than P1 alone in the experiments reported in Fig. 4
(open circles).
60 S Subunits Reconstituted Using the Three Recombinant Proteins
Are Active--
Because both P0 and N1 were solubilized recombinant
proteins, it was required to test whether they were functional. This
was accomplished by measuring the proteosynthetic activity of 60 S subunits in which native P0, P1, and P2 had been removed and replaced by the recombinant proteins. Extraction of the native P-proteins was
performed by adapting a long established method utilizing DMMA (37).
Under classical conditions (molar ratio of DMMA to 60 S subunits
equal to 15.000), P1 and P2 were entirely extracted contrary to P0, and
a molar ratio above this value resulted in an inability to restore the
DMMA particle proteosynthetic activity (38). Here, we used a 27.000 molar ratio but under milder experimental conditions (See
"Experimental Procedures" for details). As shown in the immunoblot
revealing P0, P1, and P2 (Fig. 7), there
was no P0 left in DMMA particles obtained under these new conditions (lane 2). The residual amount of P1 in this lane in the
absence of P0 might be due to the high salt concentration of the
extracting buffer that reinforced unspecific hydrophobic interactions
and could make P1 stick to the DMMA particles. A comparison of the ratio of P0 to (P1 + P2) in native subunits (Fig. 7, lane
1) with that found in the split proteins before dialysis
(lane 3) indicated that some P0 had precipitated during the
extraction process. A comparison of these ratios before (Fig. 7,
lane 3) and after dialysis (lane 4) showed that
soluble remaining P0 precipitated during the dialysis needed to
regenerate the amino groups of the proteins removed by the DMMA
extraction. This precipitation was probably due to the disruption of
the complex P0-P1 by the DMMA treatment and explained why the use of a
molar ratio of DMMA to 60 S subunits above 15.000 (classical
conditions) resulted in the inability to reactivate the DMMA particles
with the split proteins. Indeed, results in Table
I showed that the DMMA particles lacking
P0 were poorly reactivated when reconstituted with either the split proteins (27%) or a mixture of recombinant P1 and phosphorylated P2
(26%). Addition of P0 to the last mixture restored most of the
activity (83%). Interestingly, a mixture containing P0, phosphorylated P2, and N1 instead of P1 was shown to reactivate the DMMA particle (80%) to the same extent the mixture containing full-length
P1. From these results, one may conclude that both recombinant
P0 and N1 were functional after solubilization and that the
intermediary and C-terminal domains of P1 were dispensable for the
proteosynthetic activity of the ribosome.

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Fig. 7.
Immunodetection of the P-proteins in the DMMA
particles and in the split proteins. DMMA particles were prepared
as described under "Experimental Procedures." After protein
separation via 15% SDS-PAGE (36) and transfer on a nitrocellulose
membrane, the content of P-proteins was determined using a monoclonal
antibody probing the common C-terminal epitope (31). Lane 1,
native 60 S subunits; lane 2, DMMA particles;
lane 3, split proteins before the dialysis required for the
regeneration of amino groups; lane 4, split proteins after
this dialysis. The amounts of DMMA particles and split proteins
corresponded to the amount of 60 S subunits that they derived
from. MW, molecular mass; kD, kilodaltons.
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Table I
Proteosynthetic activity of 60 S subunits reconstituted from
recombinant P-proteins and DMMA particles
Proteosynthetic activities were measured under conditions in which
subunits were limiting (38) and expressed in pmol of polymerized
phenylalanine per pmol of either reconstituted particles or control 60 S after subtraction of the residual DMMA particle activities (4.8 ± 0.8 pmol/pmol). Control 60 S subunits were treated the same way as
DMMA particles but without DMMA. Split proteins corresponded to the
proteins displayed in lane 4 of Fig. 7. Values were
calculated from six different experiments.
L-[14C]phenylalanine specific activity was 20 Bq/pmol. In these experiments, P2 previously phosphorylated was used,
because such phosphorylation increased the activity of reconstituted
particles (18). P2p, phosphorylated P2.
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DISCUSSION |
The experiments presented here were designed to elucidate how the
acidic ribosomal proteins P0, P1, and P2 associate into the stalk of
the mammalian ribosome and the role of each component. To fulfill this
aim, we overproduced recombinant P0 in addition to P1 and P2, which had
already been obtained and studied as isolated proteins (18, 19).
Getting P0 has made possible the study of the association of the
P-proteins as they are in the stalk. Truncated mutants of the latter
proteins were designed and prepared to locate the binding domains and
to study their functions.
Biological Activity of Recombinant P0--
The fact that
recombinant P0 was overproduced in inclusion bodies, contrary to P1 and
P2, the other ribosomal stalk components, raised the question whether
it was functional after being refolded. Therefore, a method to assess
the biological activity of recombinant refolded P0 was developed and
showed that P0, in addition to binding P1, was able to efficiently
reconstitute the proteosynthetic activity of ribosomes deprived from
native P0 (Fig. 7 and Table I). It is noteworthy that native P0 has
been reported previously to be insoluble (39), in agreement with our
observation (Fig. 7) and the fact that no P0 is found in the
cytoplasmic pool of mammalian cells (24). Therefore, the observed
insolubility of P0 is probably an intrinsic property of the protein and
not a consequence of its misfolding.
P0 Interacts with P1 but Not with P2--
Our results indicate
that only P1 forms a stable complex with P0. The stoichiometry of the
complex is two P1 molecules for one P0, which strongly suggests that P0
solubilization by P1 does not involve an unspecific interaction (Fig.
4). No complex between P0 and P2 is shown in our experiments, either in
the solubilization experiments (Figs. 1 and 4) or in the
two-dimensional electrophoresis (Fig. 5B). Hence, it can be
concluded that P1 and P2 play a different part in the formation of the
stalk. Such a conclusion is in agreement with previous results obtained
with rat liver P1 and P2 (40) and with recent results showing that
P2
is unable to bind to P0 contrary to P1
in Saccharomyces
cerevisiae (41).
The N-terminal Domain of P1 (N1) Interacts with Both P0 and the
N-terminal Domain of P2 (N2)--
Experiments made with N1 indicate
that this domain binds both P0 and P2 (Figs. 3-6). N1 is a small
protein (63 residues), which suggests that the N-terminal part of P1
has evolved to promote specific interactions with both P0 and P2. This
might explain why P1 proteins from different species do not replace
each other despite important sequence homology (42). The fact that
P1-P2 association involves mainly their N-terminal domains (Fig.
3B) is intriguing, because N1 is mainly hydrophobic, whereas
N2 is mainly hydrophilic (see Fig. 2). Moreover, P1 and P2 interaction differs from that found in L7/L12, in which both the intermediate and
C-terminal domains participate in the dimerization by burying hydrophobic groups at the dimer interface (27). Because sequence identities are too low (15%) between L7/L12 and P1 or P2, no reliable model of the N1/N2 interaction using the available L7/L12 coordinate file (Protein Data Bank code 1DD4) can be built (27). Here, we
do not exclude the possibility that the hinge and the
C-terminal domains are also involved in the dimerization process, but
our data indicate that they do not have a prominent function (Table I).
In contrast, concerning the binding of P1 to P0, P1 intermediary (and
perhaps also C-terminal) domains might be involved more significantly in the binding, because the affinity and solubility of N1-containing complexes might be lower than those of the complexes involving full-length P1 (Fig. 4). However, DMMA particles reconstituted with
either N1 or P1 and both P0 and phosphorylated P2 have similar proteosynthetic activities. Therefore, the deleted hinge and C-terminal charged domains of P1 should not play a prominent function in protein
synthesis and in the interaction of the ribosome with eEF-1
and
eEF-2, the two elongation factors required in the in vitro
poly(U)-directed poly(Phe) synthesis test.
New Models of Association of the P-proteins in the Stalk--
P1
has been shown to interact by its N-terminal (1-63) domain both with
P0 and the N-terminal (1-65) domain of P2. In addition, no direct
interaction was shown between P0 and P2, albeit it should have been
revealed by the different methods used. Thus, between the three
possible models of the stalk that can be drawn (Fig. 8), model A (38) is inconsistent with our
experimental data because it is based on the assumption that both P1
and P2 bind to P0 and are present as homodimers. Here we present
several data indicating that there is a disruption of the homodimers of
both P1 and P2 (observed only in purified solutions in our experiments) to constitute P1/P2 heterodimers (Figs. 3, 5, and 6). That heterodimers of human P1/P2 can be formed more easily than homodimers of P1 and P2
has been recently reported (44). P1 preferentially binds P0
rather than N1 and is seemingly unable to bind them simultaneously (Fig. 6B). Therefore, only model C would be in agreement
with our results. Model C would also be in agreement with results
obtained for the prokaryotic model in which L10 is shown to have
distinct binding sites for each dimer of L7/L12 (43). The association of P1 and P2 into heterodimers, either free (as they are in the cytoplasm) or bound to P0 (as found in the stalk), suggests that P1 and
P2 bind directly to the ribosome as heterodimers and not sequentially.
However, in previous work, the presence of P1 and P2 homodimers in the
stalk has been suggested (40). Large modifications in the conformation
of the stalk in response to factor binding or changes in the A-site or
the P-site of the ribosome have been observed (8, 9, 29, 30). Then,
model B, which simulates a conformation of the stalk in agreement with
the presence of both homodimers and heterodimers of P1 and P2, and
model C, in agreement with our own results, might represent two
sequentially existing structures of the stalk.

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Fig. 8.
Models of interaction of the ribosomal
proteins of the stalk. P0, P1, and P2 are represented, in
spotted white, white, and gray,
respectively. The C-terminal domain and the hinge are structural
features common to the three proteins. Model A, built according to a
previously proposed model (40), is not in agreement with our results,
because we saw no direct interaction between P2 and P0. Model B differs
from model C by the presence of additional interactions between P1
molecules. Only model C would be in agreement with our experimental
data, suggesting strongly that a P1 molecule cannot simultaneously bind
another P1 molecule and P0 (see Fig. 6).
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Production of Functional Recombinant Proteins from Inclusion
Bodies--
In addition to providing precise and relevant details on
the organization of the lateral stalk of the mammalian ribosome, this
study emphasizes the interest of using protein ligands to promote
functional refolding of recombinant insoluble proteins. Indeed, a
similar procedure applied to refold both N1, the insoluble N-terminal
domain of P1, and P0 gave functional proteins (Table I). That
disordered fragments of the same protein reconstitute the native
structure upon association has already been shown (45) and would arise
from molecular recognition between disordered polypeptide chains in a
process coupling association with folding (46, 47). Here, we have
adapted this general principle to the folding of different interacting
proteins. This approach might be of general interest to promote the
functional refolding of recombinant insoluble proteins.