(Received for publication, February 27, 1997)
From the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine of Yeshiva University, Jack and Pearl Resnick Campus, Bronx, New York 10461
Eukaryotic translation initiation factor 5 (eIF5) interacts in vitro with the 40 S initiation complex (40 S·AUG·Met-tRNAf·eIF2·GTP) to mediate the hydrolysis of ribosome-bound GTP. In Saccharomyces cerevisiae, eIF5 is encoded by a single copy essential gene, TIF5, that encodes a protein of 45,346 daltons. To understand the function of eIF5 in vivo, we constructed a conditional mutant yeast strain in which a functional but a rapidly degradable form of eIF5 fusion protein was synthesized from the repressible GAL promoter. Depletion of eIF5 from this mutant yeast strain resulted in inhibition of both cell growth and the rate of in vivo protein synthesis. Analysis of the polysome profiles of eIF5-depleted cells showed greatly diminished polysomes with simultaneous increase in free ribosomes. Furthermore, lysates of cells depleted of eIF5 were dependent on exogenously added yeast eIF5 for efficient translation of mRNAs in vitro. This is the first demonstration that the TIF5 gene encodes a protein involved in initiation of translation in eukaryotic cells. Additionally, we show that rat eIF5 can functionally substitute yeast eIF5 in translation of mRNAs in vitro as well as in complementing in vivo a genetic disruption in the chromosomal copy of TIF5.
Eukaryotic translation initiation factor 5 (eIF5),1 in conjunction with GTP and other initiation factors, plays an essential role in initiation of protein synthesis (for reviews, see Refs. 1-5). In vitro studies using purified initiation factors have shown that the overall initiation reaction proceeds with the initial binding of the initiator Met-tRNAf as the Met-tRNAf·eIF2·GTP ternary complex to a 40 S ribosomal subunit followed by the positioning of the 40 S preinitiation complex (40 S·eIF3·Met-tRNAf·eIF2·GTP) at the initiation AUG codon of the mRNA to form the 40 S initiation complex (40 S·eIF3·mRNA·Met-tRNAf·eIF2·GTP). The initiation factor eIF5 then interacts with the 40 S initiation complex to mediate the hydrolysis of ribosome-bound GTP. Hydrolysis of GTP causes the release of eIF2·GDP (and Pi) from the 40 S ribosomal initiation complex which is essential for the subsequent joining of the 60 S ribosomal subunit to the 40 S complex to form a functional 80 S initiation complex (80 S·mRNA·Met-tRNAf) that is active in peptidyl transfer (6-9).
eIF5 was purified originally from rabbit reticulocyte lysates on the basis of its ability to stimulate in vitro translation of globin mRNA in a partially reconstituted system (10-12). The purified protein was shown to be essential for the joining of 60 S ribosomal subunits to the 40 S initiation complex to form the 80 S initiation complex (7, 13, 14). In these initial studies the apparent molecular weight of the purified protein varied between 125,000 (10) and 150,000-168,000 (11, 12). In our laboratory, however, using an assay that measured directly the formation of an 80 S initiation complex by the joining of 60 S ribosomal subunits to a preformed AUG-dependent 40 S initiation complex, we purified eIF5 both from mammalian cells (15-18) as well as from the yeast Saccharomyces cerevisiae (19). The purified mammalian and yeast proteins were shown to be monomers that migrated on SDS gels with an apparent molecular weight of about 58,000 and 54,000, respectively (15-19). More recently, we have cloned and expressed the mammalian cDNA encoding functional eIF5 of calculated Mr = 48,926 (20-22). The S. cerevisiae gene encoding eIF5, designated TIF5, has also been characterized and shown to be a single copy essential gene that encodes a protein of 45,346 daltons (23). The purified mammalian protein has also been used to characterize in vitro the eIF5-mediated subunit joining reaction using AUG-dependent 40 S initiation complex as a substrate (8, 9, 17, 18, 24). However, the involvement of either the 49-kDa mammalian eIF5 or the 45-kDa yeast protein in translation of natural mRNAs in vivo or in vitro has not yet been defined. This is relevant in view of previous observations (1-5) that proteins, e.g. eIF5A (25), isolated on the basis of their ability to stimulate partial reactions in the initiation pathway, were later shown not to be involved in translation of mRNAs in vivo.
In this paper, we have used the S. cerevisiae system to further characterize the function of eIF5 in protein synthesis in vivo. We have constructed a conditional eIF5 expression system in which functional eIF5 was expressed from a transcription unit consisting of a ubiquitin gene cassette which acts as a protein destabilizing genetic element fused to the NH2 terminus of the open reading frame of the yeast TIF5 gene under the transcriptional control of GAL10 promoter. Similar expression systems were previously used by Park et al. (26) and Kang and Hershey (25) as an effective way to rapidly deplete yeast cells of protein of interest. The effect of depletion of eIF5 on cell growth as well as protein synthesis in vivo and in vitro were then analyzed. The results of these experiments show that eIF5 plays an essential role in translation of mRNAs in vivo and in vitro. Additionally, the rat cDNA encoding functional eIF5 was expressed in yeast cells lacking TIF5. We demonstrate that the rat protein can functionally substitute for the homologous yeast protein in vivo.
Yeast strains were grown at 30 °C in either YPD medium (1% (w/v) yeast extract, 2% (w/v) Bacto-peptone, and 2% (w/v) dextrose) or in YPGal medium where 2% (w/v) galactose (glucose-free) replaced dextrose as the carbon source. Where indicated, haploid yeast cells were also grown in either synthetic complete medium containing galactose as the sole carbon source, designated SGal media (2% galactose, 0.67% Bacto-yeast nitrogen base without amino acids, 0.2% amino acid mixture) or in SD medium where 2% dextrose replaced galactose as the carbon source. For in vivo [35S]methionine incorporation, the 0.2% amino acid mixture in either the SGal or the SD media did not contain methionine. These methionine-lacking media were designated SGal-Met and SD-Met, respectively. Yeast transformations and tetrad analysis were performed as described by Rose et al. (27). Methods for plasmid and genomic DNA preparations, restriction enzyme digestion, DNA ligation, cloning, and bacterial transformation were according to standard protocols (28).
Construction of Plasmids for Conditional Expression of Yeast eIF5 or Mammalian eIF5A 2.8-kb yeast genomic fragment containing the
entire eIF5 gene (TIF5) (23) was separately cloned into the
XhoI/HindIII sites of centromeric plasmids pRS316
(URA3-based) (29) and pRS315 (LEU2-based) (29) to
yield pRS316-TIF5 and pRS315-TIF5, respectively. In these newly
constructed plasmids the expression of yeast eIF5 is under the
transcriptional control of its endogenous promoter present in the
inserted 2.8-kb fragment. For expression of yeast and mammalian eIF5
under the control of galactose-inducible GAL1 promoter, we
first isolated a 0.6-kb BamHI/EcoRI fragment
containing GAL1-GAL10 promoter from plasmid pBM272 (30), and
cloned it at the same sites of pRS315 to generate a new recombinant
vector, pTM100. For cloning of the yeast eIF5 open reading frame into pTM100 vector, two primers were synthesized bearing the
NH2-terminal and COOH-terminal ends of yeast eIF5 coding
sequences (23) that were flanked by BamHI and
EcoRI restriction sites: NH2 terminus, 5-dGCGCGCGGATCCATGTCTATTAATATTTGTAG-3
; COOH terminus,
5
-dATCCGGAATTCCTATTCGTCGTCTTCTTC-3
. These primers were used in high
fidelity-polymerase chain reaction using Pyrococcus DNA
polymerase I (Stratagene) and the polymerase chain reaction product was
digested with BamHI and EcoRI and cloned into the
same sites of pGEM7Zf(+) vector to yield pGEM-TIF5. DNA sequencing of
the eIF5 coding region in the recombinant plasmid indicated error-free
DNA synthesis. The 1.218-kb TIF5 coding region was then
excised from pGEM-TIF5 by digestion with BamHI and
XbaI and cloned into the same sites of pTM100 vector to
generate the new recombinant plasmid pTM100-TIF5. For expression of
mammalian eIF5 under GAL1 promoter, the recombinant rat eIF5
expression plasmid, pETIF5 (20, 22), was digested with
EcoRI, blunt-ended by treatment with klenow DNA polymerase
in the presence of 4 dNTPs, and then cut with XbaI to
generate the 1.34-kb fragment that contained the entire coding sequence
of rat eIF5 cDNA, starting 42 base pairs upstream of the initiating
ATG codon and 8 base pairs after the termination codon. This fragment
was then cloned into the NotI (blunt-ended)/XbaI
sites of pTM100 vector to yield the new recombinant plasmid
pTM100-EIF5. We also constructed a plasmid for conditional expression
of a rapidly-degradable form of yeast eIF5 fusion protein (see Fig. 1).
For this purpose, the coding region of TIF5 was first fused
in-frame to a lacI-flu segment present in the plasmid
pGEM-flu (26) as follows. The vector pGEM-TIF5 was digested with
BamHI and SphI and the 1.235-kb BamHI (blunt-ended)/SphI fragment containing the entire
TIF5 coding region was cloned into the PstI
(blunt-ended)/SphI site of the vector pGEM-flu to yield
pGEM-flu-TIF5. This new vector was then cut with BamHI and
SphI and the resulting 1.34-kb lacI-flu-TIF5 fragment was fused in-frame to the ubiquitin gene, UBI4 (26) under the control of the GAL10 promoter as follows. The
plasmid pUB23 which contains the UBI4 gene was cut with
EcoRI and BamHI and the resulting
EcoRI/BamHI fragment that encodes URA3
upstream activation sequence of the GAL promoter
(UASGal)-ubiquitin (Ub)-X (where
X is the trinucleotide codon for arginine) was ligated with
the BamHI/SphI fragment that encodes the
lacI-flu-TIF5 gene derived from the pGEM-flu vector. The
resulting EcoRI/SphI fragment was then cloned
into the EcoRI/SphI site of the centromeric
HIS3 vector pSE362 (26) to generate pUB-TIF5R
(Fig. 1). Expression of yeast eIF5 from the GAL10 promoter
in this plasmid initially generates a NH2-terminal
ubiquitinylated eIF5 fusion protein, which is rapidly processed in
yeast cells by a deubiquitination enzyme to yield free ubiquitin plus
an eIF5 fusion protein having arginine (R) as the
NH2-terminal amino acid (see Fig. 1).
Construction of Haploid Yeast Strains for Expression of Yeast and Mammalian eIF5 Proteins
The haploid yeast strain TMY101 (see
Table I), containing the chromosomal copy of the TIF5
gene disrupted with TRP1 marker gene and harboring the
plasmid pRS316-TIF5 that expresses yeast eIF5 from its own promoter,
was constructed from the diploid strain W303(a/) following the
one-step gene disruption method of Rothstein (31) as described for a
similar disruption procedure with URA3 gene by Chakravarti
and Maitra (23). The newly generated diploid strain
(tif5::TRP1/TIF5) was then transformed with
plasmid pRS316-TIF5 and Ura+ transformants were selected.
One of the transformants was then sporulated at 30 °C, and the
resulting tetrads were dissected and spores were germinated on YPD
plates followed by selection on SD-Trp-Ura plates. Only spores
containing the tif5::TRP1 gene and harboring the
plasmid, pRS316-TIF5 will germinate to yield Trp+Ura+ colonies. This haploid strain, named
TMY101 (Table I), was the host strain used for all plasmid shuffling
experiments described in this study. The genotype of the
TIF5 loci on the chromosome and on the plasmid of TMY101 was
confirmed by Southern blot analysis (23) using relevant DNA restriction
fragments as probes. For construction of haploid yeast strains for
conditional expression of ubiguitinated eIF5 fusion protein, the strain
TMY101 was transformed with LEU2-plasmid pRS315-TIF5, and
Trp+Ura+Leu+ transformants were
selected on SD plates and replica-plated onto another SD plate which
contained uracil and 5-FOA to promote the loss of the URA3
plasmid, pRS316-TIF5. The yeast strain recovered from the 5-FOA plate,
designated TMY203, has the disrupted chromosomal copy of
TIF5 but now harboring pRS315-TIF5 as the complementing plasmid. This strain was then transformed with pUB-TIF5R that contains
the TIF5 gene fused to the ubiquitin gene cassette (see Fig.
1). The resulting Leu+His+Ura+
transformants were then selected and grown in SGal-His-Ura media to
promote the loss of the LEU2-plasmid, pRS315-TIF5 by
segregation. The newly generated yeast strain, which has the disrupted
chromosomal copy of the TIF5 gene, but harboring only the
plasmid, pUB-TIF5R, was selected on appropriate SGal media. This
strain, designated TMY201R, expresses R-eIF5 fusion protein (arginine
at the NH2 terminus of the mature protein (Fig. 1).
|
The haploid yeast strains W303
or TMY201R were grown in YP medium containing 2% galactose (YPGal) to
early logarithmic phase. The cells were harvested and 3 A600 units of cells were resuspended in 100 ml
of YPD medium supplemented with 0.4 mg/ml adenine sulfate and grown for
about 10 h at 30 °C. Following addition of cycloheximide (50 µg/ml), cultures were rapidly chilled in an ice-water bath and
centrifuged. The cells were washed twice with LHB buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 30 mM MgCl2, and 50 µg/ml cycloheximide). The
washed cells were then suspended in 1.2 ml of the LHB buffer and lysed
by vortexing with an equal volume of glass beads. After adding an
additional 0.5 ml of the same buffer, the cell lysates were clarified
by centrifugation first at 12,000 × g for 10 min, followed by recentrifugation at 17,600 × g for another
10 min. Equivalent amounts of A260 absorbing
material (approximately 10 A260 units in 200 µl) were layered on 11 ml of 7-47% (w/v) sucrose gradients in
either a low salt buffer (10 mM Tris acetate, pH 7.0, 12 mM MgCl2, 50 mM NH4Cl)
or in a high salt buffer (10 mM Tris-HCl, pH 7.5, 15 mM MgCl2, 800 mM KCl (32)) and
centrifuged at 40,000 rpm for 2.5 h at 4 °C in a Beckman SW41
rotor. The gradients were fractionated in an ISCO density gradient
fractionator and the absorbance profile at 254 nm was analyzed in an
ISCO UA-5 absorbance monitor.
The strain TMY201R was grown in YPGal
supplemented with adenine sulfate (0.4 mg/ml) overnight at 30 °C
(A600 1.0). Cells were harvested and
suspended in YPD medium containing adenine sulfate (0.4 mg/ml) such
that the initial absorbance at 600 nm was about 0.03 and grown at
30 °C for about 18 h (about 2 generations in YPD medium) when
growth was nearly completely arrested. Cells were then harvested and
cell-free translation extracts were prepared and
mRNA-dependent cell-free translation was performed
using [35S]methionine as the labeled amino acid as
described by Hussain and Leibowitz (33).
Total RNA of S. cerevisiae was isolated as described by Rose et al.
(27) while poly(A)+ RNA was isolated from total yeast RNA
by chromatography on oligo(dT)-cellulose columns (Pharmacia Biotech
Inc.) using the protocol supplied by the manufacturer. Luciferase
mRNA was purchased from Promega and capped at the 5 end with
vaccinia guanylylmethyltransferase as described (34). The vaccinia
enzyme was a kind gift of Dr. Stewart Shuman of Sloan-Kettering Cancer
Institute, New York. Purified eIF5 from rabbit reticulocyte lysates as
well as recombinant rat eIF5 were isolated as described (18, 22). Yeast
eIF5 was purified from S. cerevisiae strain BJ926, as
described by Chakravarti et al. (19). The purified yeast
eIF5 preparation exhibited two polypeptide bands of 54 and 56 kDa upon
SDS-polyacrylamide gel electrophoresis. Each of the two polypeptides
was found to contain eIF5 activity and they were immunologically
related to each other (19). The preparation of specific rabbit antisera
against purified rabbit and yeast eIF5 and their affinity purification
were as described (17, 19). The procedure used for preparation of yeast
cell lysates for immunoblot analysis was adapted from that described by
Sachs and Davis (35).
To understand the function of eIF5 in the yeast S. cerevisiae, we depleted yeast cells of endogenous eIF5 and measured the effects of such depletion on cell growth as well as protein synthesis in vivo and in vitro. We initially constructed a haploid yeast strain in which the chromosomal copy of the TIF5 gene was inactivated by disruption with TRP1 marker gene and the essential eIF5 function was provided by maintenance of a centromeric plasmid that contained the open reading frame of the TIF5 gene under the transcriptional control of the GAL1 promoter. This yeast strain, designated TMY201 (Table I), as expected, grew on YPGal plates but did not form detectable colonies on YPD plates (data not shown). To deplete yeast cells of eIF5, TMY201 cells were grown in galactose-containing medium to early exponential growth phase and then transferred to glucose medium to turn off transcription of the TIF5 gene from the GAL1 promoter and thus, in essence turning off synthesis of new eIF5 protein in cells. Surprisingly, we observed that under these conditions, cell growth continued normally for five to six generations before declining. Immunoblot analysis of cell lysates showed that while the level of eIF5 progressively decreased with time following transfer from galactose to glucose medium, a significant level of eIF5 was still detectable in cell lysates even after six generations of growth (data not shown). Presumably, the rate of degradation of pre-existing eIF5 protein was not rapid enough to deplete yeast cells of detectable levels of eIF5 within one or two generations. In view of the observation (9, 17) that eIF5 acts catalytically in initiation of protein synthesis in vitro, the presence of very low levels of the protein in yeast cells may be sufficient to sustain slow cell growth for many generations even in the absence of new eIF5 synthesis. Furthermore, because depletion of eIF5 in TMY201 cells required several generations of growth under repressing conditions, we were concerned of the possibility that many secondary effects might be induced under these conditions that could complicate interpretation of results obtained with such eIF5-depleted cells.
To further lower the cellular level of eIF5 and to ensure more rapid
depletion of eIF5 from yeast cells following transfer to repressing
conditions, we constructed a haploid yeast strain TMY201R as described
under "Experimental Procedures" in which the chromosomal copy of
the TIF5 gene was inactivated with TRP1 marker gene and the
strain was kept viable by maintenance of the URA3-centromeric plasmid that harbors a conditional eIF5
expression system in which eIF5 was expressed from a genetic cassette
designed to destabilize the eIF5 protein. In such a system, constructed by utilizing a strategy first used by Park et al. (26) and
later by Kang and Hershey (25), eIF5 was expressed from a transcription unit consisting of a ubiquitin gene cassette
(Ub-X-lacI-TIF5) which acts as a protein destabilizing
element fused to the NH2 terminus of the open reading frame
of the TIF5 gene under the transcriptional control of
GAL10 promoter (Fig. 1). The eIF5 fusion protein synthesized from this construct contains ubiquitin at the
NH2 terminus followed by a 31-amino acid segment of the
lacI repressor which acts as a recognition element for
ubiquitin-dependent protein degradation by
NH2-terminal recognition pathway (36, 37). Deubiquitination
of this eIF5 fusion protein exposes arginine (R) as the NH2
terminus amino acid (see Fig. 1) and should lead to rapid degradation
of the protein by the N-end rule (36, 37). As expected the strain
TMY201R grew well on plates containing galactose as the carbon source
while it did not form detectable colonies on plates containing glucose
as the sole source of carbon (data not shown). When growth of the
strain TMY201R was monitored in liquid cultures containing galactose as
the carbon source, it grew with doubling time of 3.4 h comparable
with 3.2 h for the wild-type W303 strain (Fig.
2, panel A). However, when the exponentially
growing cultures of these strains were shifted from galactose to
glucose-containing medium to repress transcription of the
TIF5 gene from the GAL10 promoter, the strain
W303
, in which eIF5 expression occurred from its own promoter, as
expected, grew well with a doubling time of about 2 h (Fig. 2,
panel B). In contrast, the growth rate of TMY201R, in which
expression of eIF5 is under the control of GAL10 promoter,
began to decrease after about one generation and the growth rate was
drastically reduced in about two generations (17 h in glucose medium)
(Fig. 2, panel B).
The level of eIF5 protein in cell lysates following the shift from
galactose to glucose medium was also analyzed using affinity-purified anti-yeast eIF5 antibodies as probes. Fig. 2 (lower panel)
shows that the level of eIF5 in cell lysates prepared from
non-repressed W303 cells remained constant during the growth period.
In contrast, there was a rapid disappearance of R-eIF5 fusion protein
in strain TMY201R. After about 17 h there was no detectable level
of R-eIF5 fusion protein (Fig. 2, lower panel). These
results demonstrate that in strain TMY201R, which synthesizes eIF5
fusion protein having Arg as the NH2 terminus, a more rapid
depletion of eIF5 can be achieved following repression of transcription
of the TIF5 gene cassette from the GAL10
promoter, and this depletion of eIF5 level in cells correlated well
with the decrease in the rate of cell growth.
We used strain TMY201R to investigate the effect of
depletion of eIF5 on protein synthesis in yeast cells. For this
purpose, an exponentially growing culture of TMY201R cells was
transferred from galactose to glucose-containing medium and the rate of
protein synthesis was then monitored by measuring the incorporation of [35S]methionine into protein with 5-min pulses of the
labeled amino acid into 1 A600 unit of cells
over the growth period. Fig. 3 shows that there was a
clear correlation between the rate of protein synthesis in
vivo with the growth rate (shown in Fig. 2) of TMY201R cells.
During the first 7.5 h of growth following the shift from galactose to glucose medium, as TMY201R cells continued to grow exponentially due to the presence of pre-existing eIF5 in cells, the
rate of protein synthesis remained fairly constant. However, between
7.5 and 10 h, there was a marked decrease in the rate of protein
synthesis. After about 17-18 h (~2 generations in glucose medium),
the protein synthesis rate was only about 20% of the initial rate.
This is also the time when yeast cells were nearly completely depleted
of eIF5 as judged by immunoblot analysis (data not shown here, see Fig.
2). In contrast, when TMY201R cells were allowed to grow in galactose
medium, or wild-type W303 cells were grown in glucose (SD) medium,
the rate of protein synthesis remained fairly constant during the
entire exponential growth period of about 18 h (Fig. 3). These
results show that eIF5 is required for translation in yeast cells.
For a more detailed analysis of protein synthesis inhibition in
eIF5-depleted cells, the polyribosome content of exponentially growing
cultures of yeast cells containing eIF5 (wild-type W303 cells or
TMY201R cells growing in galactose medium) and growth-arrested TMY201R
cells in YPD medium that are depleted of eIF5 was analyzed by sucrose
gradient centrifugation (Fig. 4). The gradient profile from eIF5-depleted TMY201R cells growing in glucose (YPD) medium showed
a marked relative reduction in the amount of large polyribosomes with a
proportionate increase in the amount of monoribosomes (Fig. 4,
panel E) when compared with wild-type W303
cells growing
in YPD medium (Fig. 4, panel B) or TMY201R cells growing in
galactose medium (Fig. 4, panel D). When equivalent amounts
of cell extracts were also analyzed in sucrose gradients containing 0.8 M KCl, the majority of the accumulated monoribosomes in
eIF5-depleted TMY201R cells were found to be dissociated into subunits
(Fig. 4, panel F), indicating that they are neither bound to
mRNA nor arise due to nonspecific RNase activity in extracts. The
observed decrease in polyribosome content with an increase in the
amount of free 80 S ribosomes and free 60 S and 40 S ribosomal subunits when eIF5 was depleted, indicates a block in translation initiation, and not in the elongation or termination of protein synthesis.
eIF-5-dependent Cell-free Yeast Translation System
We used eIF5-depleted TMY201R cells to prepare a yeast
cell-free translation system to demonstrate the requirement of eIF5 for
translation of mRNAs in vitro (Fig. 5).
Cell-free translation extracts prepared (33) from exponentially growing
cultures of wild-type W303 cells in glucose medium was highly active
in translation of total yeast poly(A)+ RNA without any
exogenously added protein factors (Fig. 5, panel A). In
contrast, when similar cell-free extracts were prepared from TMY201R
cells that were initially grown in galactose-containing medium and then
shifted to glucose medium and grown until the growth of cells virtually
stopped (about 18-20 h) due to depletion of eIF5, they were rather
inactive in translation of both total yeast poly(A)+ RNA
(Fig. 5, panel B) as well as luciferase mRNA (Fig.
6). Translation could be restored in these lysates by
the addition of purified yeast eIF5 (Figs. 5B and 6). In the
absence of mRNA, addition of eIF5 had virtually no effect (Fig.
5B and Fig. 6). Recombinant rat eIF5 was also able to
substitute for yeast eIF5 in restoring translation in such
eIF5-depleted cell-free extracts (Fig. 5B). However, the rat
protein was about 50% as effective as an equimolar amount of yeast
eIF5 in restoring translation in eIF5-depleted translation extracts
(Fig. 5, panel B, compare stimulation of translation by 10 ng of each protein factor). In contrast to strong dependence of eIF5
for translation of mRNAs in these cell extracts, extracts prepared
from wild-type yeast cells which were highly active in translation of
poly(A)+ RNA, was not stimulated by exogenously added eIF5
(Fig. 5A), indicating that wild-type yeast extracts
contained saturating levels of eIF5.
Mammalian eIF5 Can Functionally Substitute for the Homologous Yeast Protein in the Yeast S. cerevisiae
Results presented above showed
that recombinant rat eIF5 can substitute for yeast eIF5 in supporting
protein synthesis in in vitro yeast translation system. We
employed the plasmid shuffling technique to determine if the wild-type
mammalian (rat) eIF5 protein can functionally substitute for the
corresponding yeast protein in vivo. For this purpose, we
constructed the haploid S. cerevisiae strain TMY101 as
described under "Experimental Procedures" and Table I. The strain
TMY101 carries inactive TIF5 disrupted with the
TRP1 marker gene and is kept viable by maintenance of a
centromeric URA3 plasmid, pRS316-TIF5 that contains the
yeast wild-type TIF5 gene under the transcriptional control
of its own promoter. For expression of rat eIF5 in TMY101, we cloned
the open reading frame of rat eIF5 cDNA under the control of yeast
galactose-inducible GAL1 promoter in the vector, pTM100 that
contains LEU2 as a selectable marker to yield the plasmid,
pTM100-EIF5 (see "Experimental Procedures"). As controls, we
constructed two additional plasmids, pTM100-TIF5 and pRS315-TIF5, in
which the expression of yeast eIF5 is under control of the
GAL1 promoter and endogenous yeast eIF5 promoter, respectively ("Experimental Procedures"). The host strain TMY101 was transformed individually with each of the three recombinant eIF5
expression plasmids and the parental vector, pRS315.
Trp+Ura+Leu+ transformants were
selected on SGal plates (Fig. 7A, left panel) and replica-plated onto another similar plate which also contained uracil and 5-fluoroorotic acid (5-FOA) to promote the loss of the
original URA3 plasmid, pRS316-TIF5.
Fig. 7 (panel A) shows that cells transformed with control plasmids, pRS315-TIF5 and pTM100-TIF5 (that express yeast eIF5 from endogenous eIF5 promoter and GAL1 promoter, respectively), grew on 5-FOA plates, as expected. Cells transformed with pTM100-EIF5 that express rat eIF5 from GAL1 promoter were also able to lose the URA3 plasmid, pRS316-TIF5, and could thus grow on 5-FOA plates. In contrast, cells transformed with the vector pRS315 failed to grow, as expected. These results show that mammalian eIF5 when expressed from the GAL1 promoter in an extrachromosomally replicating plasmid can substitute for yeast eIF5 function in yeast cells that do not express yeast eIF5.
Further confirmation of the above conclusion came from the following
observations. First, all three yeast strains recovered individually
from 5-FOA plates and harboring plasmids pTM100-TIF5 (strain TMY201),
pTM100-EIF5 (strain TMY202), and pRS315-TIF5 (strain TMY203) required
uracil for growth, thereby confirming that these strains have lost the
original URA3 plasmid, pRS316-TIF5 (data not shown). Second,
all strains except the one (TMY203) expressing yeast eIF5 from its own
promoter in the plasmid pRS315-TIF5 failed to grow on plates containing
glucose as the sole carbon source (Fig. 7, panel B). Final
confirmation that the growth of the yeast strain harboring the plasmid
pTM100-EIF5 (strain TMY202) is due solely to expression of rat eIF5
that came from immunoblot analysis using monospecific anti-rabbit and
anti-yeast eIF5 antibodies as probes (Fig. 7, panel C).
These antibodies are highly specific for their own antigen and do not
cross-react either with heterologous eIF5 or any other yeast protein
(Fig. 7C, lanes a and b, upper and
lower panels). When lysates of strain TMY202 which express rat eIF5 from the GAL1 promoter in plasmid pTM100-EIF5 were
subjected to immunoblot analysis, a strong immunoreactive polypeptide
band (apparent Mr = 58,000) was observed only
with anti-rabbit eIF5 antibodies (Fig. 7C, upper panel, lane
f). Anti-yeast eIF5-antibodies did not recognize any polypeptide
in this cell lysate (Fig. 7C, lower panel, lane f). As
expected, lysates of strain TMY201 harboring plasmid pTM100-TIF5 and
wild-type strain W303 reacted strongly only with anti-yeast eIF5
antibodies yielding an immunoreactive polypeptide band corresponding to
the molecular mass of yeast eIF5 (56 and 54 kDa doublets) (lanes
e and c of Fig. 7C, lower panel).
Anti-mammalian eIF5 antibodies did not recognize any polypeptide in
these cell lysates (lanes e and c of Fig.
7C, upper panel). However, a yeast strain carrying null
mutations in the chromosomal copy of TIF5 and harboring both
plasmids, pRS316-TIF5 and pTM100-EIF5, expressed both mammalian and
yeast eIF5 (lane d of Fig. 7C, upper and
lower panels). These results firmly establish that yeast
cells without intact endogenous eIF5 loci but expressing mammalian eIF5 can maintain cell growth and viability. Thus, mammalian eIF5 expressed from an extrachromosomally replicating plasmid can functionally substitute for the homologous yeast protein in vivo although
such cells did not grow as rapidly as either the wild-type W303
cells or the strain TMY201 overexpressing yeast eIF5 from the
GAL1 promoter (Fig. 7, panel D), i.e.
doubling time of 4.3 h versus 2.2 and 2.7 h,
respectively. It should be noted that comparison of the steady state
level of eIF5 by immunoblotting of cell lysates showed that while the
molar concentration of eIF5 in strain TMY201 in which yeast eIF5 was
expressed from the GAL1 promoter was nearly 4-fold higher
relative to wild-type W303
cells, the concentration of rat eIF5
expressed from the GAL1 promoter in strain TMY202 was
similar to that of the W303
strain (data not shown).
eIF5 was isolated in our laboratory both from mammalian cell extracts (15-18) as well as from the yeast S. cerevisiae (19) based on an in vitro assay that measured the ability of the factor to mediate the joining of 60 S ribosomal subunits to a preformed AUG-dependent 40 S initiation complex (40 S·AUG·Met-tRNAf·eIF2·GTP) to from the functional 80 S initiation complex. Studies with the purified factor as well as subsequent cloning and characterization of the mammalian cDNA and yeast gene encoding eIF5 (20-23) have led to the demonstration that both mammalian and yeast proteins are monomers of calculated Mr 48,926 and 45,346, respectively. For reasons summarized below, it is, however, important to show that the 45-49-kDa eIF5, purified on the basis of in vitro partial initiation reaction assay, is indeed involved in the translation of natural mRNAs in vivo and in vitro. First, prior to our work, eIF5 was isolated from mammalian cells in several laboratories (11-14) based on its ability to stimulate globin mRNA-directed protein synthesis in a partially reconstituted translation system. The purified factor was reported to be a monomer of about 150,000-160,000 kDa (10-14) and shown to be specifically required for the joining of the 60 S ribosomal subunits to the 40 S initiation complex formed with natural mRNA to form the 80 S initiation complex. Second, there are reports in literature of several protein factors that were isolated from rabbit reticulocyte lysates based on the in vitro activity of each protein in stimulating a partial reaction in the initiation pathway (for review, see Refs. 1-5). Although these protein factors were called translation initiation factors, the role of many of these proteins, e.g. eIF5A (formerly called eIF4D), eIF6, CoeIF2A, CoeIF2B, in initiation of protein synthesis in vivo is far from clear. For example, initiation factor eIF5A, a 16-18-kDa highly conserved protein in eukaryotes (38, 40, 41), was isolated from ribosomal salt-wash proteins of rabbit reticulocytes based on its activity to stimulate the formation of methionyl puromycin by interaction of Met-tRNAf bound to the 80 S initiation complex with puromycin (12, 41, 42). Based on the activity of this protein in the above in vitro assay, eIF5A was thought to be a translation initiation factor required for the synthesis of the first peptide bond following 80 S initiation complex formation. However, subsequent cloning of the S. cerevisiae genes encoding eIF5A (43, 44) and studies on protein synthesis in vivo in eIF5A-depleted yeast cells (25) showed that while eIF5A is an important protein required for yeast cell growth and viability, it may not be a translation factor required for global protein synthesis in yeast cells. These observations lend strong support to the view that the requirement of a protein isolated on the basis of an in vitro partial reaction assay must be demonstrated for protein synthesis in vivo before it can be regarded as a translation factor.
In this paper, we have used the S. cerevisiae system to
investigate the function of the 45-kDa yeast eIF5 in protein synthesis in vivo and in vitro. We have used a conditional
eIF5 expression system to rapidly deplete eIF5 from yeast cells. Data
presented in this paper clearly show that as yeast cells were depleted
of eIF5, the rate of protein synthesis in these cells was inhibited in
parallel. Analysis of the polysome profiles of eIF5-depleted cells
showed greatly diminished polysomes with the simultaneous increase in
80 S ribosomes (Fig. 4), which suggests that eIF5 plays an essential
role in the initiation phase of protein synthesis. Furthermore, lysates
of cells depleted of eIF5 were inactive in translation of mRNAs
in vitro. Addition of 45-kDa purified yeast eIF5 was able to
restore translation in these cell lysates. These results show that the
TIF5 gene product, a protein of 45,346 daltons, is a
translation factor involved in initiation of protein synthesis. Furthermore, in agreement with the 39% identity and 60% homology in
amino acid sequence between mammalian and yeast eIF5 (21), we observed
that rat eIF5 can functionally substitute for yeast eIF5 in restoring
in vitro translation of mRNAs in eIF5-depleted yeast
cell-free translation extracts (Fig. 5). In accordance with these
results, we also observed that in TIF5 yeast cells in
which no functional eIF5 was synthesized due to disruption of the
essential chromosomal copy of the TIF5 gene, expression of
either rat or S. cerevisiae eIF5 from centromeric plasmids
under the transcriptional control of the GAL1 promoter can
support growth and viability of yeast cells. It is to be noted,
however, that the growth rate of
TIF5 yeast cells
expressing mammalian eIF5 from the GAL1 promoter is about
40-50% of that exhibited by either the wild-type W303
strain or by
a yeast strain expressing yeast eIF5 from the GAL1 promoter.
This difference in growth rate between the two eIF5 expression systems
presumably reflects subtle evolutionary differences between the
components of the translation machinery of the unicellular yeast and
the multicellular mammalian systems. We should also point out that
among many translation initiation factors that have been tested for the
ability of their cDNAs to functionally substitute for the
corresponding yeast gene in yeast cells, only eIF4A has not been found
to complement (for review, see Ref. 5). These observations lend strong
support to the view that the basic mechanism of initiation of
translation is highly conserved from the unicellular yeast to mammals.
Thus, the information gathered from molecular genetic studies of
translation in the yeast S. cerevisiae will complement
in vitro biochemical studies in the mammalian system leading
to a better understanding of the process of translation and its
regulation in eukaryotic cells.
Finally, availability of both a cell-free yeast translation system
dependent on exogenous eIF5 for efficient in vitro
translation of mRNAs and a TIF5 yeast strain in which
functional eIF5 is produced in vivo from the TIF5
gene under the control of GAL promoter should be powerful
tools to analyze structure-function studies of eIF5 in vivo
and in vitro.
We are grateful to Dr. Eun-Chung Park, Massachusetts General Hospital, for the kind gift of plasmids pGEM-flu, pUB23, and pSE362 used in this study. We are also indebted to Dr. Philip Hieter, Johns Hopkins University School of Medicine, Baltimore, MD, for providing us with the yeast plasmids, pRS315 and pRS316, and Dr. Michael J. Leibowitz, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, for help in preparation of yeast cell-free translation extracts. We thank Dr. Stewart Shuman, Sloan-Kettering Cancer Institute, NY, and Dr. Jayanta Chaudhuri of this institution for critically reading the manuscript.