(Received for publication, March 26, 1997, and in revised form, May 30, 1997)
From the Laboratoire de Biochimie Médicale, Institut de Biologie et Chimie des Protéines, Centre National de la Recherche Scientifique, 7, passage du Vercors, 69367 Lyon Cedex 07, France
The acidic ribosomal proteins P1-P2 from rat liver were overproduced for the first time by expression of their cDNA in Escherichia coli. They were tested for their ability to reactivate inactive P1-P2-deficient core particles derived from 60 S ribosomal subunits treated with dimethylmaleic anhydride, in poly(U)-directed poly(Phe) synthesis. The recombinant P1-P2 were unable to reactivate these core particles although they could bind to them. When recombinant P1-P2 had been phosphorylated first with casein kinase II, they were as efficient in the reactivation process as P1-P2 extracted with ethanol/KCl from the 60 S subunits. Reconstitution experiments were carried out using all possible combinations of the two recombinant proteins phosphorylated or not. Reactivation of the core particles required the presence of both P1 and P2 with the latter in its phosphorylated form. These experiments reveal a distinct role for P1 and P2 in protein synthesis. Phosphorylated P2 produced a partial quenching of the intrinsic fluorescence of eukaryotic elongation factor 2, which was not observed with the unphosphorylated protein. This result demonstrates the existence of an interaction between phosphorylated P2 and eukaryotic elongation factor 2. P2 also quenched part of the intrinsic fluorescence of P1, due to the interaction between the two proteins.
The large subunit of eukaryotic ribosomes contains 12-kDa acidic P proteins, which seem more numerous in lower eukaryotes than in higher organisms (1). In these, two types of P proteins are found, designated as P1 and P2 (2, 3). They share similar properties with prokaryotic proteins L7-L12, which form a pentamer with protein L10, (L7/L12)4-L10, constituting the lateral stalk of the 50 S ribosomal subunits (4). The eukaryotic P proteins, as their prokaryotic counterparts, seem to play an essential role in the interaction with elongation factors and in factor-dependent GTPase activity (5). However, some specific properties are observed with eukaryotic P proteins. First, these proteins exist on the ribosome as phosphorylated derivatives. They can be phosphorylated in vitro by either casein kinase II or by an endogenous ribosome-bound enzyme (6). Second, proteins P1-P2 present on the ribosome can exchange with a cytoplasmic pool of these unphosphorylated proteins (7). Phosphorylation of P1-P2, which appears to be necessary for ribosome activity, was originally suggested to be a requirement for the binding of these proteins to ribosomes (5), but recently this hypothesis has been challenged by new results obtained in yeast (8).
We have shown previously that active rat liver 60 S ribosomal subunits could be reconstituted from inactive core particles prepared with 2,3-dimethylmaleic anhydride (DMMA)1 (9), by adapting a method previously used with yeast ribosomes (10). Reactivation of the rat liver core particles was obtained not only with DMMA-split proteins containing several proteins including P1-P2 but also with a 50% ethanol, 0.08 M KCl extract containing P1-P2 exclusively. Dephosphorylation of P1-P2 with alkaline phosphatase completely inhibited the reactivation process (11). The poor ability of the ethanol/KCl core particles to be reactivated with this extract was shown to be related to a conformational alteration which destabilizes the 5 S RNA·protein complex (12).
Using this relatively simple in vitro reconstitution system, we here report results obtained by studying separately the role of P1 and P2 overproduced by cloning their cDNA in Escherichia coli. We have focused our work on the effects of phosphorylation of each of the two proteins, their mutual interaction, and the interaction of P2 with elongation factor eEF-2.
The primers used for the polymerase chain
reaction were from Life Technologies, Inc. AmpliTaq DNA polymerase was
from Perkin-Elmer. The pQE-30 plasmid and the
Ni2+-nitrilotriacetic acid-agarose gel came from Qiagen.
Restriction enzymes, T4 DNA ligase, and E. coli cells were
from Promega. Ampicillin came from Boehringer Mannheim, BenzonaseTM
from Merck, and isopropyl--D-thiogalactopyranoside from
GERBU Biotechnik. ATP and glutathione-Sepharose 4B were from Pharmacia
Biotech Inc. Bovine thrombin came from ICN. DMMA and N-acetyltyrosinamide were from Sigma.
[
-32P]ATP (specific activity, 11 Bq/pmol) and
L-[14C]phenylalanine (specific activity, 20 Bq/pmol) were purchased from NEN Life Science Products. Preparation of
rat liver eEF-2 (95% pure) and of 60 S ribosomal subunits by zonal
centrifugation has already been described (13, 14). A monoclonal
antibody prepared against recombinant P1 and reacting with P0-P1-P2 was a gift from Dr. Monier, Immunology Department, University Lyon 1, Lyon,
France. Protein concentration was determined with the Coomassie Blue
plus protein assay reagent kit from Pierce.
The plasmids pP1-13 and
pP2-11 encoding, respectively, rat liver ribosomal proteins P1 and P2
were a gift from Dr. I. G. Wool (University of Chicago) and had
been prepared as described (3). P1 cDNA was polymerase chain
reaction-amplified from pP1-13 plasmid using a 5 primer (5
TAT GGA
TCC ATG GCT TCT GTC TCT GAG CTT GCC 3
) and 3
primer (5
TAT AAG CTT
TTA GTC AAA AAG ACC AAA GCC CAT 3
). P2 cDNA was polymerase chain
reaction-amplified from pP2-11 plasmid using a 5
primer (5
TAT GGA
TCC ATG CGC TAC GTT GCC TCT TAT CTG 3
) and a 3
primer (5
TAT AAG CTT
TTA ATC AAA CAG GCC AAA TCC CAT 3
). The primers introduced
BamHI and HindIII restriction sites, and the
amplified cDNAs were digested by the endonucleases after gel
purification and ligated into the corresponding sites of linearized
pQE-30 (Qiagen) or pGEX-KT plasmids.
The pQE-30 plasmid allowed the production of a protein carrying a
6-histidine tag at the N terminus. The pGEX-KT plasmid (a gift from Dr.
D. Nègre and Dr. J. C. Cortay, CNRS, Lyon, France), constructed according to Hakes and Dixon (15) is designed to generate
an in-frame fusion protein composed of glutathione
S-transferase (GST) and P1 or P2. pGEX-KT includes a
thrombin cleavage site and an upstream glycine linker engineered
between the GST and ribosomal protein sequences. The four recombinant
plasmids were named pQE30-P1, pQE30-P2, pGEX-P1, and pGEX-P2. E. coli JM 109 cells (endA1, recA1,
gyrA96, thi, hsdR17
(rk, mk+), relA1,
supE44,
(lac-proAB), (F
, traD36,
proAB, lacIqZ
M15)) were
transformed with the ligation products and grown on agar plates
supplemented with ampicillin (50 µg/ml). Correct recombinants were
identified by multiple restriction digests.
E. coli cells transformed
with the recombinant plasmid were grown at 37 °C in LB medium (1%
(w/v) bacto-tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl) at pH
7.5 containing 100 µg of ampicillin/ml, until the absorbance at 600 nm reached 0.7 unit. Expression of recombinant proteins was induced
with 2 mM isopropyl--D-thiogalactopyranoside for 3 h at 37 °C. Cells were harvested by centrifugation at
5,500 × g for 10 min at 4 °C and resuspended in 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA, 6 mM MgCl2, 1% (v/v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 100 units of
Benzonase/ml. The cells were lysed using a SLM-Aminco French pressure
cell press at 1,200 p.s.i. with a 40-ml capacity cell and centrifuged
at 30,000 × g for 30 min.
The acidic
proteins expressed with pQE-30 plasmid were purified through a
Ni2+-agarose affinity chromatography, according to the
Qiagen instruction manual. The fusion proteins expressed with pGEX-KT
plasmid were purified through glutathione-Sepharose 4B affinity
chromatography, cleaved by thrombin (enzyme/substrate ratio, 1:160, by
mass), and the released ribosomal proteins were purified after a
subsequent glutathione-Sepharose 4B chromatography, according to the
Pharmacia instruction manual. Recombinant proteins, purified in both
systems, were then dialyzed against 50 mM Tris-HCl, pH 7.4, 20 mM KCl, 10% (w/v) glycerol, 4 mM
2-mercaptoethanol and frozen at 80 °C.
DMMA core particles were prepared from 60 S ribosomal subunits as described previously (9). The selective extraction of proteins P1-P2 from 60 S subunits by treatment with ethanol and 0.08 M KCl has been also described (11). The reconstitution process was carried out using the DMMA core particles and either the ethanol extract (11, 12) or the recombinant proteins (phosphorylated or not). The amount of split proteins was three times in excess in the case of ethanol extract and two times in the case of the recombinant proteins.
Phosphorylation of Recombinant Proteins by Casein Kinase IIThe casein kinase II was a gift from Dr. Chambaz (University
of Grenoble, Grenoble, France). Recombinant proteins were
phosphorylated by casein kinase II (enzyme/substrate, 1:800, by mass)
at 37 °C for 4 h in 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 20 mM MgCl2, 1 mM dithiothreitol, 2% (w/v) glycerol containing 2 mM ATP. The same procedure was used to phosphorylate the
DMMA core particles and the reconstituted 60 S ribosomal subunits with
50 µM [-32P]ATP (specific activity, 5 Bq/pmol). After phosphorylation, these materials were submitted to an
electrophoresis in a 15% SDS-polyacrylamide gel and
autoradiographed.
These were performed using a SLM Aminco 8000C spectrofluorometer as described previously (17). The eEF-2 concentration used was 0.1 µM, and the measurements were performed in a cuvette of 1 ml in 50 mM Tris-HCl, pH 7.4, 20 mM KCl, 10% (w/v) glycerol, 4 mM 2-mercaptoethanol. The quenching of eEF-2 or P1 fluorescence was deduced from the decrease of the fluorescence intensity in the presence of increasing amounts of P2 phosphorylated or not. Fluorescence was corrected for dilution. Correction for inner filter effect of P2 was made using N-acetyltryptophanamide as a standard.
Fig. 1 shows that the ribosomal
protein P2 with the 6-histidine tag at the N terminus (A) as
well as the GST-P2 fusion protein of Mr 40,000 (B) were highly overexpressed upon
isopropyl--D-thiogalactopyranoside induction, as a
soluble form in the supernatant. Using the first system, affinity
chromatography on a Ni2+-agarose column was a particularly
efficient purification procedure in which the recombinant ribosomal
proteins were selectively bound and then subsequently eluted with 150 mM imidazole. In the second system of expression, an
efficient purification of the fusion protein was obtained by affinity
chromatography through a glutathione-Sepharose 4B column. This fusion
protein was very sensitive to thrombin cleavage, generating isolated P2
and GST without any apparent secondary cleavage. P2 was then purified
using a second glutathione-Sepharose 4B column, as the unbound
fraction, while GST and the small amount of uncleaved fusion protein
were retained. The yield was 100 mg of protein, approximately 99%
pure, per liter of E. coli culture using the first system
and 10 mg using the second one. Similar results were obtained for
ribosomal protein P1 overexpressed in both systems, but P1 released
after cleavage of the GST-P1 fusion protein was insoluble.
The Mr of the two recombinant proteins estimated by their migration in SDS-polyacrylamide gel electrophoresis was significantly higher than the theoretical values of 11,490 and 11,684 for P1 and P2, respectively. A similar difference between observed and expected values has been already observed using native P1 and P2 (2). In our case, it is also necessary to take into account the supplementary N-terminal residues. To verify the correct processing of the recombinant proteins, these were electroblotted onto a polyvinylidene difluoride membrane and submitted to N-terminal sequencing by automatic Edman degradation using a 473 A liquid sequencer (Applied Biosystem). The correct N-terminal residues of both proteins were found, preceded by GS in the case of the protein obtained from GST-P2 and by MRGS(H6)GS in the case of the recombinant proteins with the 6-histidine tag.
Reconstitution of Active 60 S Ribosomal Subunits Using Phosphorylated Recombinant P1-P2Core particles were prepared
from 60 S ribosomal subunits treated with DMMA as described previously
(9). The absence of P1-P2 proteins from these particles was confirmed
using a monoclonal antibody prepared against recombinant P1 and which
reacted with P0, P1, and P2 in the 60 S subunits (Fig.
2A). The core particles contained P0, but no P1 nor P2 (Fig. 2B), and exhibited only
5% of the original activity of the 60 S subunits in poly(U)-directed polyphenylalanine synthesis. After incubation with recombinant P1-P2,
the activity of the core particles was still very low, as compared with
that measured after addition of an ethanol extract of 60 S subunits
containing native P1-P2 (14% instead of 82%) (Table
I). Recombinant P1-P2 were then
phosphorylated with casein kinase II and ATP. As shown in Fig.
3, there was no remaining unphosphorylated form after incubation of P1 and P2 with the kinase under the phosphorylation conditions used. The extent of core particle
reactivation observed (85%), after addition of the two recombinant
proteins previously phosphorylated, was identical to that
observed with native P1-P2. Addition of phosphorylated P1 and
unphosphorylated P2 produced a very small reactivation (10%).
Interestingly, addition of phosphorylated P2 and unphosphorylated P1 gave a much better reactivation (63%). The same results were obtained using either P2 with the 6-histidine tag or P2 overexpressed with the pGEX-KT plasmid. Addition of either phosphorylated P1 or
phosphorylated P2 alone was ineffective (8%).
|
To see whether or not unphosphorylated P1-P2 were able to bind to the
core particles, reconstituted subunits were phosphorylated with casein
kinase II and [-32P]ATP and their protein content
analyzed by gel electrophoresis and autoradiographed (Fig.
4). As expected from the preceding results, no residual P1-P2 was labeled in the core particles treated in
this way (lane 1). On the other hand, subunits reconstituted from these core particles and unphosphorylated P1-P2 contained the two
labeled proteins (lane 2). Only P1 was labeled in subunits reconstituted using unphosphorylated P1 with P2 previously
phosphorylated with cold ATP (lane 3). In the reverse
situation (unphosphorylated P2 with P1 phosphorylated with cold ATP),
only P2 was labeled (lane 4).
Effect of Ribosomal Protein P2 on eEF-2 and P1 Intrinsic Fluorescence
The addition of phosphorylated P2 to eEF-2 induced a
partial quenching of the intrinsic fluorescence of eEF-2 (Fig.
5, open circles). eEF-2
contains 7 Trp residues and P2 none. A maximal quenching of 13% of
eEF-2 intrinsic fluorescence was observed with a slight blue shift
(from 332 to 328 nm). The concentration of phosphorylated P2, which
produced 50% of the maximal eEF-2 fluorescence quenching, was 100 nM. On the other hand, addition of the unphosphorylated P2
had no effect at the same concentrations (closed circles).
These results demonstrate the existence of an interaction between eEF-2
and P2, but only when the latter protein is phosphorylated. Using the
same technique to study the interaction between P1 and eEF-2 was
complicated by the fact that the presence of a Trp residue in P1
required the use of correction factors. In another experiment, we found
that P2 quenched 20% of the intrinsic fluorescence of P1 when both
proteins were added in an equimolar ratio (result non shown).
By expressing the cDNA of mammalian acidic ribosomal proteins
P1 and P2 in an E. coli system, it was possible to obtain
these proteins pure in large amounts and therefore to study the
function and the role of the phosphorylation of each protein. Active 60 S subunits could be reconstituted by incubation of inactive DMMA core
particles with the recombinant proteins previously phosphorylated. The
requirement for phosphorylation to obtain active material was not
surprising, since this result has also been obtained in reconstitution
experiments using P1 and P2 extracted from the ribosomes (5) or the 60 S subunits (11). More unexpected was the finding of a dissymmetry
between the effects of phosphorylated P1 and P2: phosphorylated P2 with
unphosphorylated P1 could reactivate the core particles, whereas
phosphorylated P1 with unphosphorylated P2 were almost ineffective. The
phosphorylation reaction was carried out with casein kinase II. It has
been shown that the two residues, which are phosphorylated by this
enzyme as well as by an endogenous ribosome-bound kinase, are the two
serine located in the common C-terminal peptide near the C terminus
(6). From the results of our experiments, it is clear that the
phosphorylation of P1-P2 is not required for their binding to the core
particles, since the unphosphorylated proteins can be incorporated and
then phosphorylated with [-32P]ATP. The presence in
the core particles of P0, which is assumed to be required for the
binding of P1-P2 (18), could be demonstrated by using a monoclonal
antibody. This antibody had been obtained using recombinant P1 as
antigen, and since it recognized the three proteins P0-P1-P2, it was
directed most likely against an epitope located in the C-terminal
peptide common to the three proteins. The major part of P0 was probably
phosphorylated, since this protein was not visible in the autoradiogram
of the core particles incubated with the kinase and
[
-32P]ATP (Fig. 4). In fact, it appeared weakly
labeled when the exposure time was increased (not shown). If the role
of P1-P2 phosphorylation is not to allow the binding of these proteins
to the core particles, the questions of why it is required for the
reactivation process and why P2 phosphorylation appears to have a
specific role remain. The experiment illustrated by Fig. 5 could give
at least part of the answer. It had been shown previously that
elongation factor eEF-2 could be cross-linked with protein P2, among
other ribosomal proteins (19). Our results obtained from intrinsic
fluorescence measurements demonstrate the existence of an interaction
of eEF-2 with phosphorylated P2. On the other hand, no interaction
between eEF-2 and unphosphorylated P2 was observed using this
technique. It will be interesting now to identify the amino acid
residues of P2, which are involved in this interaction by site-directed mutagenesis and to precise the exact role of P2 phosphorylation in the
eEF-2-dependent step of elongation. Interaction of P2 with P1 was also detected using the same technique, and the amino acid residues involved should also be identified.
We are grateful to Dr. I. G. Wool for the gift of the plasmids pP1-13 and pP2-11, to Dr. E. Chambaz for the gift of casein kinase II, to Dr. J. C. Monier for the gift of the monoclonal antibody reacting with P0-P1-P2, and to Dr. D. Hulmes for critical reading of our manuscript.