From the Departament de Bioquímica, Facultat
de Veterinària, Universitat Autònoma de Barcelona,
Bellaterra 08193, Spain and the ¶ Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, North Carolina
27710
Received for publication, November 30, 2000, and in revised form, January 31, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vivo 32P-labeled yeast
proteins from wild type and ppz1 ppz2 phosphatase mutants
were resolved by bidimensional electrophoresis. A prominent
phosphoprotein, which in ppz mutants showed a marked shift
to acidic regions, was identified by mixed peptide sequencing as the
translation elongation factor 1B The elongation step of protein synthesis involves the binding of
aminoacyl-tRNA to the ribosomal "A" site, formation of a peptide
bond, and translocation of the newly formed peptidyl-tRNA to the
"P" site. The elongation factor 1 (EF1)1 is responsible for the
GTP-dependent binding of aminoacylated tRNA to the
ribosomal A site in polypeptide chain elongation and participates in
proofreading of the codon-anticodon match (1)
In the budding yeast Saccharomyces cerevisiae, EF1 consist
of different subunits: EF1A (formerly EF1 The function of EF1B The yeast Ppz phosphatases are encoded by genes PPZ1 and
PPZ2 (10, 11) and represent a novel type of Ser/Thr
phosphatases characterized by a catalytic carboxyl-terminal half
related to type 1 phosphatase. These phosphatases are involved in a
variety of cell processes, including maintenance of cell integrity, in connection with the Pkc1/Mpk1 mitogen-activated protein kinase pathway
(11, 12), regulation of salt tolerance (13), and regulation of cell
cycle at the G1/S transition (14). In all cases, the
function of Ppz1 appears to be more important than that of Ppz2.
Recently, we have identified the halotolerant determinant Hal3 as a
negative regulatory subunit of Ppz1 that modulates the diverse
physiological functions of the phosphatase (15).
As an attempt to better understand the physiological role of the
Ppz phosphatases, we have performed a two-dimensional
electrophoretic analysis of proteins from in vivo
32P-labeled wild type and ppz strains, in
search for polypeptides that might display an altered phosphorylation
state in the absence of the phosphatases. This approach has led us to
establish a previously unsuspected link between the Ppz phosphatases
and the translation elongation factor 1B Strains and Growth Conditions--
Escherichia coli
strains NM522 or DH5 were used as a host for DNA cloning. Bacterial
cells were grown at 37 °C in LB medium containing 50 µg/ml
ampicillin, when needed, for plasmid selection. Yeast cells were grown
at 28 °C in YPD medium or, when indicated, in synthetic minimal (SD)
or complete minimal (CM) medium (16). Mutant yeast strains used in this
work derive from JA-100 (MATa PPZ1 PPZ2 ura3-52
leu2-3,112 trp1-1 his4 can-1r). Construction of JA-101
(MATa ppz1::URA3), JA-103
(MATa ppz2::TRP1), and
JA-105 (MATa ppz1::URA3
ppz2::TRP1) has been described previously
(14). Strain EDN75 (MATa
ppz1::Kan) was made by replacing the
entire PPZ1 ORF with the KanMX4 module (17),
using oligonucleotides PPZ1/SFH_5' (5'-CCA CTC TCT GCT TAT CTT TCC TTC
CTT TTC AAA ATG GGC AGC TGA AGC TTC GTA CGC-3') and PPZ1/SFH_3' (5'-CTG
TTG AGA TTC GTT ATC ATT TGT GAT GCT TGT TTC CAT CAT AGG CCA CTA GTG GAT
CTG-3').
Recombinant DNA Techniques and Plasmid
Construction--
E. coli cells were transformed using the
standard calcium chloride method (18). S. cerevisiae cells
were transformed by a modification of the lithium acetate method (19),
which includes treatment with dimethyl sulfoxide. Restriction
digestions, DNA ligations, and other standard recombinant DNA
techniques were performed essentially as described previously (18).
A vector for high copy expression of EF1B
Mutation of the phosphorylatable Ser-86 to Ala was made by sequential
PCR. In a first step, the EF1B
High copy number expression of Ppz1 and Hal3 was accomplished by
transforming cells with plasmid YEp181-PPZ1 (21) or YEp351-HAL3 (15).
In Vivo 32P-Labeling, Two-dimensional
Electrophoresis, and Phosphopeptide Analysis--
Yeast cells were
32P-labeled as follows. 50-ml cultures were grown in YPD
(or SD medium lacking leucine or uracil, when containing plasmids)
until an OD660 of 0.5-0.8 was reached. Cells were
collected by centrifugation, washed with 10 ml of low phosphate YPD
medium (22), and finally resuspended in the same medium to achieve an
OD660 of 0.2. Growth was resumed until an OD660
0.8 was reached, and 500 µCi of carrier-free
[32P]orthophosphate was added. Incubation was continued
for 60 min. Cells were then centrifuged, and pellets were resuspended
in 10 ml of ice-cold trichloroacetic acid and incubated on ice for 10 min. After centrifugation, the mixture was vigorously resuspended in 1 ml of dimethylketone, centrifuged, and dried at room temperature. Pellets were then resuspended in 360 µl of lysing buffer (0.1 M Tris-HCl, pH 8, 0.3% SDS, 2.5%
To identify proteins of interest, membranes were stained with Amido
Black and subjected to autoradiography. Protein staining and
phosphorylation patterns were compared using Melanie software (Bio-Rad). Relevant fragments were sliced, removed, digested for 90 min
with 200 µl of 500 mg/ml cyanogen bromide, as described (23), and
subjected to amino acid sequencing in a Applied Biosystems Procise 494 apparatus. The mixed sequenced obtained were run against the
FASTF data base to identify the protein (24).
To identify the nature of the phosphorylated residue(s), strain EDN75
(ppz1::KAN) was transformed with
plasmid p426/TEF5, and cells were labeled with 32P as
above, recovered by centrifugation, and disrupted with glass beads in
25 mM Tris-HCl buffer (pH 7.5), containing 1 mM
EDTA, 1 mM dithiothreitol, 100 mM NaCl, 0.5 mM PMSF, 0.5 mM benzamidine, 1 µg/ml
leupeptin, and 1 µg/ml pepstatin. The homogenate was centrifuged at
100,000 × g for 60 min at 4 °C, and the supernatant
was loaded into a 1-ml glutathione-Sepharose column equilibrated with
the above mentioned buffer. The column was washed with the same buffer plus 250 mM NaCl, and the fusion protein was eluted with 10 mM glutathione. The purified GST-EF1B Immunodetection of Ppz1 Bound to Purified GST-EF1B Nonsense Suppression Assays--
Suppression of nonsense codons
was estimated by using the pUKC series plasmids (26). Plasmid pUKC815
derives from YCp50 and expresses the LacZ gene from the
yeast PGK promoter. Plasmids pUKC817, pUKC818, and pUKC819
are identical to pUKC815, but they carry the nonsense codons UAA, UAG,
and UGA at the beginning of the Sensitivity to Drugs and Growth Assays--
For testing the
sensitivity to various drugs on plates, cultures were grown until an
OD660 of 2 and 100 µl of the culture was spread on YPD
plates. 20 µl of a solution of each drug was placed on sterile disks
on top of the plates, and growth resumed at 30 °C for 2-3 days.
Sensitivity in liquid medium was assessed as follows. Exponential
cultures were diluted up to an OD660 of 0.01. Aliquots of
200 µl were supplemented with 100 µl of medium containing the
appropriate amount of the drug. Growth was resumed for 16-18 h, and
sensitivity to drugs was monitored by measuring the OD660
of the culture.
Yeast EF1B
The identity of the mentioned phosphoprotein was established by
recovering the region of the membrane, followed by digestion with
cyanogen bromide and automated Edman sequencing of the resulting peptide mixture. Analysis of yeast protein data banks indicated that
this protein corresponded to the product of the single-copy, essential
TEF5 gene, encoding the eukaryotic elongation factor 1B
To identify the residue(s) phosphorylated in vivo, a
GST-EF1B Affinity-purified GST-EF1B Evidence for Functional Interactions between Ppz Phosphatases
and EF1B
Changes in sensitivity to paromomycin have been related to altered
translational fidelity. Therefore, we sought to investigate whether the
absence of Ppz phosphatase activity might affect this cellular
function, by evaluating the suppressor capacity of the ppz
mutant strains. To this end, we transformed wild type and ppz1 cells with constructs that express the LacZ
gene from the PGK1 promoter, carrying one of the UAA, UAG,
or UGA stop codons at the beginning of the
It has been reported that high levels of Ppz1 are detrimental for cell
growth, because they lead to an expanded G1/S cell cycle
transition. To know if this effect could be somehow related to the
function of EF1B In this report we demonstrate that, in the yeast S. cerevisiae, translation elongation factor 1B Our data indicate that deletion of the ppz genes and
overexpression of Hal3, a negative regulatory subunit of Ppz1 (15), result in increased phosphorylation of the EF1B We considered that if EF1B Changes in sensitivity to paromomycin have been related to altered
translational fidelity (33, 34), a phenotype also produced by changes
in the dosage of EF1A (35). Recent evidence (8) has been presented
pointing out that mutations in the carboxyl-terminal region of EF1B Further evidence for a functional interaction between EF1B (formerly eEF1
). An equivalent
shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1. Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable
residue and showed that its phosphorylation was increased in
ppz cells. Pull-down experiments using a glutathione
S-transferase (GST)-EF1B
fusion version allowed to
identify Ppz1 as an in vivo interacting protein. Cells
lacking Ppz display a higher tolerance to known translation inhibitors,
such as hygromycin and paromomycin, and enhanced readthrough at all
three nonsense codons, suggesting that translational fidelity might be
affected. Overexpression of a GST-EF1B
fusion counteracted the
growth defect associated to high levels of Ppz1 and this effect was
essentially lost when the phosphorylatable Ser-86 is replaced by Ala.
Therefore, the Ppz phosphatases appear to regulate the phosphorylation
state of EF1B
in yeast, and this may result in modification of the
translational accuracy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) is encoded by two
different genes (TEF1 and TEF2), and it binds
aminoacyl-tRNA in a GTP-dependent manner. The exchange of
GDP for GTP on EF1A is stimulated by a member of the guanine nucleotide
exchange factor family, EF1B
, which is encoded by a single gene
(TEF5). An additional subunit of uncertain function is
encoded by genes TEF3 and TEF4. Although lack of
TEF3 and TEF4 results in no observable defects in
translation (2), lack of TEF5 or simultaneous deletion of
TEF1 and TEF2 is lethal (3, 4). Components of EF1
have been shown to be phosphorylated in vitro by diverse
protein kinases in species different from yeast (5-7).
on EF1A has been shown to be critical for an
efficient and accurate translation. For example, cells with increased
expression of the EF1A subunit can bypass the lethality of cells
lacking EF1B
. However, these cells present a number of defects,
including higher sensitivity to inhibitors of translation elongation
and changes in translational fidelity (4). Alterations in translational
fidelity have been also produced by specific mutations in EF1B
, as
it has been documented by evaluation of sensitivity to drugs such as
paromomycin and analysis of translational fidelity at nonsense codons
(8). The fidelity of translation may be related, at least in part, to
the requirement for nucleotide exchange, as it has been tested by
mutations in the GTP-binding motif of yeast EF1A (9).
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
as a GST fusion protein
was constructed as follows. The entire EF1B
ORF was amplified by PCR
with oligonucleotides EFb5'
(5'-CGGGATCCCCATGGCATCCACCGATTTCTC-3') and EFb3'
(5'-GCGGATCCTTATAATTTTTGCATAGCAG-3'). The
underlined residues denote the engineered
BamHI site used to clone the EF1B
ORF in-frame with the
GST sequence of plasmid p426TEG2, to yield p426/TEF5. Plasmid p426TEG2
is based on the high copy number yeast vector pRS426 (20) and allows
expression of GST fusion proteins under the control of the
TEF1 promoter. For low level expression of EF1B
, the
1.6-kbp GST-EF1B
fusion sequence was excised from p426/TEF5 with
KpnI and SacI and cloned in the centromeric
plasmid pRS416. The promoter region of TEF5 was amplified by
PCR with oligonucleotides Efbprom_5' (5'-CGC GAG CTC AAT
ACC GAC AGC TTT TGA C-3') and Efbprom_3' (5'-CGC GAG CTC
CAT TAT GTG TGT ATA TAT TCG-3'). The artificial SacI site
(underlined) was used to clone the promoter downstream of
the hybrid ORF to yield pRS/TEF5. Plasmid DNA was recovered and
sequenced to identify clones with the correct orientation and lacking
unwanted mutations.
ORF was amplified in two separate
reactions by using the pair of primers EFb5'/EF1bSA3 (5'-CTTCTTCATCGTCGGCACCGAATAAATCG-3'), and EFb3'/EF1bSA5 (5'-CGATTTATTCGGTGCCGACGATGAAGAAG-3'). The residues in boldface denote the modification introduced to change Ser-86
to Ala. In a second step, the 637- and 370-bp fragments produced in
these PCR reactions were mixed, and the entire EF1B
ORF amplified by
using oligonucleotides EFb5' and EFb3'. The amplification fragment was
cloned into the BamHI site of p426TEG2, to yield
p426/TEF5(S86A), and sequenced.
-mercaptoethanol)
supplemented with protease and phosphatase inhibitors (150 µM NaVO4, 1 µM microcystine, 1 mM benzamidine, 1 mM PMSF), and homogenates
were prepared with the aid of glass beads. Samples were boiled for
10 s then cooled on ice for 1 min, and 30 µl of RNase A solution
(200 units/ml de Rnase A in 0.5 M Tris-HCl pH 7, 50 mM MgCl2 buffer) and 6 µl of a 1 unit/µl
solution of DNase (Promega) were added. Samples were incubated for 2 min at 4 °C, supplemented with 750 mg of urea and 200 µl of a
solution containing 4% CHAPS, 4.75 M urea, and 5%
-mercaptoethanol and further incubated for 10 min at room temperature. After centrifugation at 750 × g for 10 min, supernatants were centrifuged at 13,000 rpm in a
microcentrifuge and the second supernatants (50-200 µg of
proteins) were then subjected to two-dimensional electrophoresis and
transferred to membranes essentially as described earlier
(23).
was the only
radioactive band when analyzed by SDS-PAGE. The protein (0.5 mg) was
digested with endolysyl peptidase C (20 µg/ml), which cuts
carboxyl-terminal to lysine residues. Digests were acidified with TFA,
and applied to a reverse phase column (Waters Nova-Pak C18, 3.9 × 150 mm) that had been equilibrated in 0.1% TFA (buffer A). The flow
rate was maintained at 1 ml/min. The column was washed for 10 min with buffer A before peptides were eluted with a linear gradient of acetonitrile (0-80% in 80 min) in buffer A. Fractions (1 ml) were collected, and peptides containing 32P were identified by
measuring Cerenkov radiation. Those fractions were pooled and
evaporated to dryness before the peptides were immobilized to Immobilon
membranes (Millipore) following the manufacturer's instructions. The
32P-labeled peptides generated from in vivo
labeled EF1B
were identified with a vapor phase amino acid sequencer
(Applied Biosystems Procise 494). Phosphorylated residues within
phosphopeptides were located by determining the cycles in which
32P was released when samples were subjected to sequential
Edman degradation under conditions that optimize recovery of
32P (25).
Fusion
Protein--
Wild type strain JA-100 and the ppz1 mutant
strain EDN75 were transformed with the empty plasmid p426TEG2 and
plasmid p426/TEF5. Cells were grown in synthetic medium lacking uracil
up to an OD660 of 2, recovered by centrifugation, and
disrupted with the aid of glass beads in 50 mM Tris-HCl
buffer (pH 8.0), containing 1 mM EDTA, 2 mM
dithiothreitol, 150 mM NaCl, 0.5 mM PMSF, 0.5 mM benzamidine, 1 µg/ml leupeptin and 1 µg/ml
pepstatin. A crude extract was prepared by centrifugation at 750 × g, and 1 mg of protein was incubated with 100 µl of
glutathione-Sepharose beads for 90 min at 4 °C. Beads were washed
with the above mentioned buffer, resuspended in SDS-sample buffer, and
boiled, and the supernatant was loaded in a 8% SDS-polyacrylamide gel.
After transfer to Immobilon-P membranes (Millipore), the presence of
Ppz1 was assessed by immunoblotting with anti-Ppz1 antibodies as
described previously (15).
-galactosidase coding sequence.
Yeast strains were transformed with these plasmids and grown up to an
OD660 of 0.5-1.0 in SD medium lacking uracil, and the
-galactosidase activity was determined as described previously
(27).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Is a Phosphoprotein in Vivo Whose Phosphorylation
State Is Affected by Ppz Phosphatases--
To evaluate the influence
of lack of Ppz phosphatases in the cellular phosphorylation pattern,
wild type and ppz1 ppz2 yeast cells were
32P-labeled and total cell extracts prepared and subjected
to bidimensional electrophoresis. Global analysis of the distribution
of phosphoproteins did not show a remarkable overall modification of
the pattern. However, a clear shift to more acidic regions of a
phosphoprotein of about 22.5 kDa and focusing at pI 4.3 was observed in
the double mutant ppz1 ppz2 compared with the wild type
yeast cells (Fig. 1). The acidic shift of
this protein was also observed when the gel was silver-stained,
indicating that it was a relatively abundant component. Because
shifting to more acidic regions is often associated to increased
phosphate content, this result was considered as indicating that this
protein could be a target for the Ppz phosphatases. To further test
this possibility, a similar experiment was carried out using wild type
cells that contained a high copy plasmid carrying the HAL3
gene, which codes for a negative regulator of Ppz1 (15). As shown in
Fig. 1, overexpression of HAL3 resulted in a pattern identical to the one observed in ppz mutants, supporting the
notion that the shift was the result of lack of Ppz activity.
View larger version (81K):
[in a new window]
Fig. 1.
The absence of Ppz phosphatase activity
affects the phosphorylation state of
EF1B . Wild type strain JA-100
(WT), strain JA-105 (ppz1 ppz2), or wild type
cells overexpressing the gene encoding the negative regulatory subunit
HAL3 (HAL3) were 32P-labeled, and
phosphoproteins were analyzed by two-dimensional gel electrophoresis.
IEF indicates isoelectric focusing, and
SDS denotes the second dimension electrophoresis
(SDS-PAGE). Silver staining of the gels is shown in the upper
panel, whereas the lower panel displays the
corresponding autoradiograms.
(3). Therefore, yeast EF1B
is a phosphoprotein in vivo, and its phosphorylation state may be modified by the Ppz phosphatases.
fusion protein was expressed from plasmid p426/TEF5 in
32P-labeled yeast cells. The product was affinity purified
by glutathione-Sepharose chromatography and digested with endolysyl
peptidase C. Phosphopeptide mapping by HPLC analysis indicated that all
in vivo phosphorylation sites are located in a single
endolysyl peptidase C fragment (Fig. 2).
Phosphoamino acid analysis of both the entire labeled fusion protein or
the relevant chromatographic fractions revealed that all radioactivity
was bound to Ser residue(s) (Fig. 2). Further analysis of the
radioactive endolysyl peptidase C fragment by measuring recovery of
32P during Edman sequencing proved that radioactivity was
associated to a single residue that was determined to be Ser-86.
View larger version (19K):
[in a new window]
Fig. 2.
Phosphoamino acid analysis and phosphopeptide
map of yeast EF1B . A, EF1B
was expressed as a GST fusion protein using plasmid p426/TEF5,
affinity-purified from 32P-labeled ppz mutant
cells and eluted with 10 mM glutathione. The sample was
digested with endolysyl peptidase C that cuts carboxyl-terminal to Lys
residues. Peptides were resolved by C18 reverse-phase HPLC and eluted
with a gradient (dashed line) from 0 to 60%
acetonitrile/0.1% TFA over 80 min. The radioactivity associated to the
eluted peptides was monitored by Cerenkov counting. B,
phosphoamino acid analysis. Lane 1, 32P-labeled
purified GST-EF1B
; lane 2, HPLC-purified phosphopeptide
F36 (fraction 36). Amino acid symbols denote the mobility of
unlabeled phosphoamino acid standards. C, Amino acid
sequence of the region surrounding Ser-86 in S. cerevisiae
EF1B
.
from Yeast Cells Contains Bound
Ppz1--
The possibility that the PPZ phosphatases might interact
in vivo with the elongation factor was approached by using
an affinity system based in the expression of EF1B
in yeast as a GST
fusion protein. This recombinant protein was affinity-purified, and the presence of accompanying proteins was evaluated by SDS-PAGE. When the
presence of Ppz1 was tested by immunoblot in these samples (Fig.
3), it was found that affinity-purified
EF1B
contained bound Ppz1. This finding indicates that Ppz1 and
EF1B
can interact in vivo and leads to the possibility
that Ppz1 could directly dephosphorylate EF1B
. In fact, when the
GST-EF1B
fusion protein was expressed in 32P-labeled
cells from the low copy plasmid pRS/TEF5, an increase in radioactive
phosphate content of about 2-fold was observed in ppz cells
when compared with the wild type strain (data not shown). However, our
attempts to in vitro dephosphorylate the in vivo
32P-labeled translation factor using available
bacterially expressed Ppz1 have been so far unsuccessful.
View larger version (26K):
[in a new window]
Fig. 3.
Immunodetection of Ppz1 bound to yeast
GST-EF1B . The fusion protein GST-EF1B
was expressed in wild type (PPZ1+) or EDN75 cells
(PPZ1
) and affinity-purified from 1 mg of total yeast
extracts through a glutathione-Sepharose matrix. An additional control
in which wild type cells were transformed with the empty p426 plasmid
was also included. After extensive washing the samples were resuspended
in sample buffer, electrophoresed in SDS-polyacrylamide gels, and
transferred to membranes. The presence of interacting Ppz1 was tested
by immunoblot using available anti-Ppz1 antibodies developed against
recombinant GST-Ppz1 protein. Note that these antibodies also recognize
the GST moiety of GST-EF1B
.
--
The observation that the Ppz phosphatases may affect
the in vivo phosphorylation state of EF1B
prompted us to
analyze phenotypes related to changes in the function of this protein.
It has been recently reported that mutations in the conserved carboxyl
terminus of EF1B
alter the sensitivity of yeast cells to translation
elongation inhibitors. We tested the sensitivity of wild type and
phosphatase-deficient strains to paromomycin, hygromycin B, and
cycloheximide by both liquid cultures and the halo assay. As shown in
Fig. 4, deletion of both phosphatase
genes clearly increased the tolerance of yeast cells to paromomycin and
hygromycin, whereas tolerance was not modified in the case of
cycloheximide. A rather strong effect was also observed when only the
PPZ1 gene was absent (Fig. 4), whereas lack of
PPZ2 resulted in minor changes (not shown).
View larger version (31K):
[in a new window]
Fig. 4.
ppz mutants are hypertolerant to inhibitors
of translational fidelity. Upper panel, YPD medium
containing the indicated concentrations of drug was inoculated (initial
OD660 of 0.007) with wild type JA-100 ( ), JA-101
(ppz1,
), or JA-105 (ppz1 ppz2,
) cells.
Cultures were grown for 18 h, and the density of the cultures was
then measured. Relative growth was calculated as the ratio between
growth in the presence or the absence of added drug and expressed as a
percentage. Data are mean ± S.E. from four independent
experiments performed by triplicate. Lower panel, lawns of
JA-100 (WT), JA-101 (ppz1), or JA-105 (ppz1
ppz2) were prepared on plates with sterile filters containing 30 mM hygromycin B (Hygro) or 0.025 mM
cycloheximide (Cyclo). Growth was monitored after 3 days.
-galactosidase coding
region. Therefore, only when these codons are suppressed can the
enzymatic activity be detected. As shown in Fig.
5, ppz1 mutants displayed, in
all cases, a higher
-galactosidase activity than wild type cells (3- to 4-fold of increase), indicating a higher suppressor capacity. Experiments performed in parallel with ppz1 ppz2 mutants
produced similar results (data not shown).
View larger version (25K):
[in a new window]
Fig. 5.
Nonsense suppressor capacity of
Ppz1-deficient yeast cells. Wild type JA-100 (filled
bars) and Ppz1-deficient EDN75 (dashed bars) strains
were transformed with plasmids pUKC817 (p17), pUKC818
(p18), and pUKC819 (p19) that carry the nonsense
codons UAA, UAG, and UGA, respectively, at the beginning of the
-galactosidase coding sequence. Cells were grown up to an
OD660 of 0.5-1, and
-galactosidase activity was
determined using the chromogenic substrate
o-nitrophenil-
-D-galactose. Data are
mean ± S.E. from four independent clones determined by
triplicate.
, we transformed wild type cells with a high copy
plasmid containing the PPZ1 gene, and then we introduced either an empty p426TEG2 vector, the p426/TEF5 plasmid, or the same
construct carrying a version of EF1B
in which the phosphorylatable Ser-86 had been changed to Ala. Although high levels of EF1B
did not
modify the growth rate of a strain with normal levels of Ppz1 (data not
shown), the presence of an excess of the factor results in a clear
improvement in cell growth of cells overexpressing Ppz1 (Fig.
6). However, this effect was completely
lost when the S86A version of EF1B
was expressed. Immunoblot
experiments determined that the amount of wild type and mutated
versions of EF1B
were virtually identical, exhausting the
possibility of an artifact due to difference in expression
levels. A further test was performed by transforming wild type cells
with the mentioned constructs and determining paromomycin tolerance.
Overexpression of the wild type version of EF1B
increased tolerance
to the drug, and this effect was fully abolished when Ser-86 was
replaced by Ala. These results provide evidence of functional changes
as a result of the absence of the phosphorylation site in EF1B
.
View larger version (16K):
[in a new window]
Fig. 6.
Overexpression of EF1B
attenuates the cell growth defect due to high levels of
Ppz1. Wild type JA-100 cells carrying a high copy number plasmid
with the PPZ1 gene were transformed with an empty p426
plasmid (
), plasmid p426/TEF5 (
), or p426/TEF5(S86A) (
).
Positive clones were exponentially grown in SD medium lacking leucine
and uracil and then diluted in the same medium up to an
OD660 of 0.02. Growth was resumed and monitored at
different times. Data are mean ± S.E. from at least eight
independent clones.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a
phosphoprotein. Phosphorylation site mapping and sequence analysis
indicates that the Ser-86 is the only phosphorylatable residue in this
protein, at least under standard growth conditions. It is remarkable
that data base search reveals that the equivalent Ser residue (as well
as its acidic environment) is also found in a large variety of
organisms, including Drosophila melanogaster,
Caenorhabditis elegans, mouse, and human. Phosphorylation of
EF1B
has been reported in Artemia salina (5), wheat (6),
and reticulocyte (7). In the former case, phosphorylation was ascribed
to Ser-89, which is equivalent to Ser-86 in yeast EF1B
.
Interestingly, phosphorylation has been correlated to changes in its
catalytic nucleotide exchange activity, although reports are somewhat
contradictory (5, 7).
protein,
specifically at Ser-86. These results would be compatible with a role
of Ppz1 in regulating the phosphorylation state of the translation
factor and, possibly, its function. We also show here evidence that
affinity-purified yeast EF1B
contains significant amounts of bound
Ppz1, by using an approach that was pivotal in the past to identify the
Hal3 protein as a subunit of Ppz1 (15). This could be taken as an indication that Ppz1 could be able to directly dephosphorylate EF1B
.
However, we have been unable to detect direct dephosphorylation of
either in vivo labeled or CK-2 in vitro
phosphorylated EF1B
in the presence of bacterially expressed Ppz1.
Although at this point we cannot provide direct evidence for the
translation factor being a substrate for the phosphatase, this
possibility formally remains. For instance, the phosphatase might
require accessory proteins (absent in our in vitro assay) to
effectively use EF1B
as substrate. In this regard, there is a large
body of evidence for the requirement of specific regulatory subunits
(targeting subunits) for Ser/Thr phosphatases to localize at specific
subcellular sites or to use a given phosphoprotein as an effective
substrate (28, 29). It must be noted that dephosphorylation events have been previously related to the control of the accuracy of protein synthesis, as it is the case of the Ppq1/Sal6 Ser/Thr protein phosphatase (30, 31), the closest structural homologue of the Ppz
phosphatases. However, the possible role of this phosphatase has not
been worked out.
was a target (either direct or indirect)
for Ppz1, it could be possible to establish some sort of functional
connection between both proteins. Deletion of TEF5 is lethal
for the cell, and high copy expression of TEF2 suppresses the lethal phenotype of tef5 mutants (4). However, these
cells are markedly sensitive to translational inhibitors, such as
paromomycin and hygromycin B. It is remarkable that lack of Ppz
phosphatases also results in a change in sensitivity to these
compounds, although in this case yielding more tolerant cells. Because
these drugs are aminoglycosides known to enter the yeast cell driven by
the membrane potential, which is mostly maintained by the function of
the membrane H+-ATPase (32), we considered the possibility
that the increased tolerance could be an indirect effect due to altered
proton efflux. However, this was ruled out by determining this
parameter in wild type and ppz mutants and finding
essentially identical values (data not shown).
results in increased sensitivity to translation inhibitors and that
this effect was accompanied by enhanced translational fidelity
(i.e. reduced readthrough at nonsense codons). These observations are in keeping with our finding that cells lacking Ppz
phosphatases, which are more tolerant to certain translation inhibitors, show an increased readthrough at nonsense codons, most
likely due to a decrease in translational fidelity.
and Ppz1
comes from the observation that overexpression of the translation
factor strongly attenuates the growth defect, due to a delayed
G1/S transition, of cells containing an excess of Ppz1
activity. Although we showed in the past that this defect correlates
with a delay in G1/S cyclin mRNA expression (14), immunoblot analysis of the protein level of different cyclins reveals
that, at least in the case of Clb5, further post-transcriptional alterations (i.e. at the translation level) could
exist.2 Remarkably, a
non-phosphorylatable version of EF1B
was unable to counteract the
effect of an excess of Ppz1, suggesting that in vivo
modulation of the phosphorylation state of the factor is somehow
involved in the regulation of its function. It has been reported that,
when expressed from the powerful GAL promoter, a
carboxyl-terminal fragment of EF1B
, lacking Ser-86, was sufficient for normal growth and did not display dramatically altered drug or
temperature sensitivity (8). Furthermore, a strain containing a S86A
version of EF1B
as the only source for the factor is
viable.3 Therefore, it must
be concluded that regulation of EF1B
by phospho-dephosphorylation at
Ser-86 (which, at least in part, would involve Ppz1) must affect the
function of the translation factor in a subtle way. From our data, it
can be hypothesized that changes in the phosphorylation state of
EF1B
would result in altered nucleotide exchange on EF1A. However,
alternative mechanisms cannot be excluded, because it has been
postulated that EF1B
may have additional regulatory effects on EF1A
(9). In any case, our data provides further support to the notion that
phospho-dephosphorylation mechanisms are relevant for a proper
regulation of protein synthesis.
![]() |
ACKNOWLEDGEMENTS |
---|
The skillful technical help of Anna Vilalta and Yolanda Prado are acknowledged. We thank T. G. Kinzy, M. Remacha, and J. P. García-Ballesta for fruitful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant PB98-0565-C4-02 (to J. A.), by Grants HL19242 and DK52378A (to T. H.), and by a Joint Research Project sponsored by the Commission for Cultural, Educational, and Scientific Exchange between the United States and Spain (to J. A and T. H.). The generous support of Applied Biosystems to the Haystead laboratory is acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A recipient of a predoctoral fellowship from the Ministerio de Educación y Cultura, Spain. Present address: Cell Signaling Unit, Dept. de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra (UPF), E-08003 Barcelona, Spain.
To whom correspondence should be addressed: Tel.:
34-93-5812182; Fax: 34-93-5812006; E-mail: Joaquin.Arino@uab.es.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010824200
2 E. Nadal, R. P. Fadden, A. Ruiz, T. Haystead, and J. Ariño, unpublished results.
3 L. Valente and T. G. Kinzy, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EF1, elongation factor 1; Ppz, protein phosphatase z; SD medium, synthetic minimal medium; CM medium, complete minimal medium; CHAPS, 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate; GST, glutathione S-transferase; ORF, open reading frame; PMSF, phenylmethylsulfonyl fluoride; TFA, trifluoroacetic acid; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); PCR, polymerase chain reaction; kbp, kilobase pair(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hinnenbusch, A., and Liebmann, S. W. (1991) in The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol. I (Broach, J. R. , Jones, E. W. , and Pringle, J. R., eds) , pp. 627-735, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
2. | Kinzy, T. G., Ripmaster, T. L., and Woolford, J. L., Jr. (1994) Nucleic Acids Res. 22, 2703-2707[Abstract] |
3. | Hiraga, K., Suzuki, K., Tsuchiya, E., and Miyakawa, T. (1993) FEBS Lett. 316, 165-169[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Kinzy, T. G.,
and Woolford, J. L., Jr.
(1995)
Genetics
141,
481-489 |
5. |
Janssen, G. M.,
Maessen, G. D.,
Amons, R.,
and Möller, W.
(1988)
J. Biol. Chem.
263,
11063-11066 |
6. | Matsumoto, S., Mizoguchi, T., Oizumi, N., Tsuruga, M., Shinozaki, K., Taira, H., and Ejiri, S. (1993) Biosci. Biotechnol. Biochem. 57, 1740-1742[Medline] [Order article via Infotrieve] |
7. | Peters, H. I., Chang, Y. W., and Traugh, J. A. (1995) Eur. J. Biochem. 234, 550-556[Abstract] |
8. |
Carr-Schmid, A.,
Valente, L.,
Loik, V. I.,
Williams, T.,
Starita, L. M.,
and Kinzy, T. G.
(1999)
Mol. Cell. Biol.
19,
5257-5266 |
9. |
Carr-Schmid, A.,
Durko, N.,
Cavallius, J.,
Merrick, W. C.,
and Kinzy, T. G.
(1999)
J. Biol. Chem.
274,
30297-30302 |
10. |
Posas, F.,
Casamayor, A.,
Morral, N.,
and Ariño, J.
(1992)
J. Biol. Chem.
267,
11734-11740 |
11. | Lee, K. S., Hines, L. K., and Levin, D. E. (1993) Mol. Cell. Biol. 13, 5843-5853[Abstract] |
12. | Posas, F., Casamayor, A., and Ariño, J. (1993) FEBS Lett. 318, 282-286[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Posas, F.,
Camps, M.,
and Ariño, J.
(1995)
J. Biol. Chem.
270,
13036-13041 |
14. |
Clotet, J.,
Garí, E.,
Aldea, M.,
and Ariño, J.
(1999)
Mol. Cell. Biol.
19,
2408-2415 |
15. |
De Nadal, E.,
Clotet, J.,
Posas, F.,
Serrano, R.,
Gómez, N.,
and Ariño, J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7357-7362 |
16. | Adams, A., Gottschlings, D. E., Kaiser, C. A., and Stearns, T. (1998) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
17. | Wach, A., Brachat, A., Pöhlmann, R., and Philippsen, P. (1994) Yeast 10, 1793-1808[Medline] [Order article via Infotrieve] |
18. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
19. | Schiestl, R. H., and Gietz, R. D. (1989) Curr. Genet. 16, 339-346[Medline] [Order article via Infotrieve] |
20. | Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H., and Hieter, P. (1992) Gene 110, 119-122[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Clotet, J.,
Posas, F.,
de Nadal, E.,
and Ariño, J.
(1996)
J. Biol. Chem.
271,
26349-26355 |
22. | Warner, J. R. (1991) Methods Enzymol. 194, 423-428[Medline] [Order article via Infotrieve] |
23. |
Alms, G. R.,
Sanz, P.,
Carlson, M.,
and Haystead, T. A.
(1999)
EMBO J.
18,
4157-4168 |
24. |
Damer, C. K.,
Partridge, J.,
Pearson, W. R.,
and Haystead, T. A.
(1998)
J. Biol. Chem.
273,
24396-24405 |
25. |
Russo, G. L.,
Vandenberg, M. T., Yu, I. J.,
Bae, Y. S.,
Franza, B. R., Jr.,
and Marshak, D. R.
(1992)
J. Biol. Chem.
267,
20317-20325 |
26. | Tuite, M. F., Stansfield, I., Eurwilaichitr, L., and Akhmaloka. (1993) Biochem. Soc. Trans. 21, 857-862[Medline] [Order article via Infotrieve] |
27. | Reynolds, A., Lundblad, V., Dorris, D., and Keaveney, M. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , pp. 13.6.1-13.6.6, John Wiley & Sons, New York |
28. | Cohen, P. (1989) Annu. Rev. Biochem. 58, 453-508[CrossRef][Medline] [Order article via Infotrieve] |
29. | Stark, M. J. (1996) Yeast 12, 1647-1675[CrossRef][Medline] [Order article via Infotrieve] |
30. | Chen, M. X., Chen, Y. H., and Cohen, P. T. (1993) Eur. J. Biochem. 218, 689-699[Abstract] |
31. |
Vincent, A.,
Newnam, G.,
and Liebman, S. W.
(1994)
Genetics
138,
597-608 |
32. | Vallejo, C. G., and Serrano, R. (1989) Yeast 5, 307-319[Medline] [Order article via Infotrieve] |
33. | Singh, A., Ursic, D., and Davies, J. (1979) Nature 277, 146-148[Medline] [Order article via Infotrieve] |
34. | Palmer, E., Wilhelm, J. M., and Sherman, F. (1979) Nature 277, 148-150[Medline] [Order article via Infotrieve] |
35. | Song, J. M., Picologlou, S., Grant, C. M., Firoozan, M., Tuite, M. F., and Liebman, S. (1989) Mol. Cell. Biol. 9, 4571-4575[Medline] [Order article via Infotrieve] |