| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, April
21, 1995; and in revised form, July 18, 1995) From the
Translation initiation factor eIF1A is required in vitro for maximal rates of protein synthesis in mammalian systems. It
functions primarily by dissociating ribosomes and stabilizing 40 S
preinitiation complexes. To better elucidate its precise role in
promoting the translation initiation process, the yeast form of eIF1A
has been identified in Saccharomyces cerevisiae and purified
to homogenity on the basis of its cross-reaction with antibodies
prepared against mammalian eIF1A. The apparent mass of yeast eIF1A (22
kDa) resembles that of the mammalian homolog (20 kDa), and the yeast
factor is active in stimulating methionyl-puromycin synthesis in an
assay composed of mammalian components. The gene encoding yeast eIF1A,
named TIF11, was cloned and shown to be single copy. TIF11 encodes a protein comprising 153 amino acids (17.4 kDa); the
deduced amino acid sequence exhibits 65% identity with the sequence of
human eIF1A. Both human and yeast eIF1A contain clusters of positive
residues at the N terminus and negative residues at the C terminus.
Deletion/disruption of TIF11 demonstrates that eIF1A is
essential for cell growth. Expression of human eIF1A cDNA rescues the
growth defect of TIF11-disrupted cells, indicating that the
structure/function of yeast and mammalian eIF1A is highly conserved.
The initiation phase of protein synthesis in eukaryotic cells is
promoted by a large number of proteins called initiation factors (eIF) ( The
process of translation initiation appears to be very conserved between
eukaryotes as distantly related as mammals and the yeast, Saccharomyces cerevisiae. This conclusion is based on
similarities of mRNA structure and the basic mechanism of protein
synthesis(9) . Particularly striking are the structural
similarities between yeast and mammalian initiation factors, which
share amino acid sequence identities ranging from 26 to 71%. This
strong conservation has made it possible to recognize several yeast
genes as encoding specific initiation factors based on their sequence
homology to mammalian cDNAs: eIF2 The recent cloning of the mammalian eIF1A cDNA has provided
structural information about the factor(21) . The failure to
stimulate translation rates by overexpression of the cDNA in
transiently transfected mammalian cells suggests that eIF1A is not
limiting for protein synthesis(22) . However, because of the
intrinsic complexity of the mammalian system and the limited ability to
manipulate specific gene expression, we decided to study eIF1A function
in yeast. We report here the purification and biochemical
characterization of yeast eIF1A, the cloning of its gene, TIF11 (for translation initiation factor 1A), and in vitro and in vivo characterization of the factor.
A yeast
genomic library in
Figure 1:
Gel electrophoretic and
Western immunoblot analyses. Yeast cell fractions were subjected to 15%
SDS-PAGE and Western immunoblotting as described under ``Materials
and Methods.'' The arrows on the left identify
where purified yeast eIF1A migrates. Molecular mass markers are shown
on the right in kilodaltons. Panel A, protein
fractions derived from wild type yeast strain W303-1A: lane1, total cell lysate (approximately 30 µg); lane2, low salt post-ribosomal supernatant fraction (S100); lane3, high salt post-ribosomal supernatant (HSW); lane4, ribosomes following pelleting from the high
salt buffer; lane5, same as lane1 except treated with preimmune serum. For lanes2-4, the same proportion of the preparation was
added for each of the subcellular fractions. Panel B, yeast
eIF1A was purified as described under ``Materials and
Methods.'' Fractions from the Mono Q column were subjected to 15%
SDS-PAGE and staining with Coomassie Blue. Lane1,
protein loaded onto the Mono Q column; lanes2-5, column fractions
11-14.
Since the
immunoreactive protein distributes in both the S100 and high
salt-washed fractions, the lysate was raised to 500 mM KCl
before initially pelleting the ribosomes. This high salt post-ribosomal
supernatant (in effect, S100 + HSW) supplemented with protease
inhibitors was fractionated by fast protein liquid chromatography Mono
S and Mono Q chromatography as described under ``Materials and
Methods.'' Putative eIF1A elutes from the Mono Q column at about
430 mM KCl as detected by Coomassie Blue staining (Fig. 1B). Approximately 10 µg of yeast eIF1A were
isolated from a 0.8-liter culture of cells (
Figure 2:
Activity of yeast eIF1A. The indicated
amounts of purified human recombinant (
Figure 3:
Sequence of TIF11. The DNA
sequence of the HindIII-EcoRI fragment, which
contains the coding region of TIF11 and its flanking
sequences, was determined as described under ``Materials and
Methods.'' The derived amino acid sequence for eIF1A is aligned
below. Residue numbers for nucleotides and amino acids are shown on the right. The reported sequences as well as another 1.3 kb of DNA
sequence upstream are available from GenBank under accession number
U11585.
Figure 4:
Comparison of yeast and human eIF1A
sequences. Human (upper) and yeast (lower) eIF1A
sequences were aligned using the program BESTFIT from the GCG software
package on a VAX computer. Sequence identities are shown by verticallines between the two sequences; similar
residues, marked by twodots, are defined as: VIL,
DE, KR, NQ, FYW, and ST. Every 10th residue has a dotabove it. The sequences exhibit 65% identity and 76%
similarity.
The number of TIF11 genes was
investigated by Southern blot analyses of genomic DNA. The generation
of a single
Figure 5:
Disruption of TIF11. Panel
A, restriction map of the TIF11 region and scheme of the
gene disruption/replacement with HIS3 as described under
``Materials and Methods.'' The panel shows
restriction sites above and below the gene and its
flanking sequences. The TIF11 and HIS3 coding regions
are shown as filled and openrectangles,
respectively. Panel B, spores from eight individual tetrads
from CMD1 were dissected, arranged vertically, on a YPD plate as
indicated by letters on the left, and allowed to
germinate and grow at 30 °C for 48 h. The panel shows a
computer scan of a photograph of the plate.
To
determine the phenotype of a null mutant that lacks eIF1A, independent
isolates of CMD1 were sporulated and tetrads were dissected. A
2 To distinguish
between these two possibilities, the entire TIF11 open reading
frame (but lacking the CIF gene homolog) was placed under
control of the glucose-repressible GAL1 promotor as described
under ``Materials and Methods.'' The resulting plasmid pW-y1A
was transformed into the diploid strain CMD1. Since pW-y1A carries TRP1 as the selectable marker, transformants were selected on
a SD-trp plate and named CMD2. This strain was subsequently sporulated
followed by tetrad dissection on galactose plates. Due to random
plasmid segregation, the ratio of viable to nonviable spores was 2:2,
3:1, or 4:0 (Fig. 6A). Co-segregation of the
His
Figure 6:
Tetrad analysis of CMD2 spores. Panel A, spores from 9 tetrads of CMD2 were analyzed as
described in Fig. 5B except that growth was on YPG
plates. Panel B, following tetrad dissections of CMD2 (panelA), one tetrad giving four viable spore
colonies was selected, and the four colonies were streaked onto YPG and
YPD plates and onto SG and SD plates with the omission of histidine or
tryptophan as indicated in the figure. The plates were incubated at 30
°C for 2-4 days, photographed, and computer
scanned.
If eIF1A is essential for cell viability, the
His
Figure 7:
Tetrad analysis of CMD3 spores. Panel
A, spores from 9 tetrads of CMD3 were analyzed as described in Fig. 5B except that growth was on YPG plates. Panel
B, following tetrad dissections of CMD3 (panelA), one tetrad giving four viable spore colonies was
selected and analyzed as described in Fig. 6B.
To show that strain CM2 actually synthesizes human eIF1A
but not yeast eIF1A, Western blot analysis was carried out with
polyclonal anti-human eIF1A antibody affinity purified with yeast
eIF1A. The results (Fig. 8) clearly show that under galactose
growth conditions, the S30 lysate prepared from strain CM2 (lane2) contains human eIF1A (
Figure 8:
Western blot analysis of human and yeast
eIF1A. Strains W303-1A and CM2 were lysed in 10 mM Tris-HCl, pH 7.5, 2 mM phenylmethanesulfonyl fluoride,
and 10 mM dithiothreitol and centrifuged at 29,000
We report here the purification of a yeast initiation factor
called eIF1A and the cloning of its gene, TIF11. Several lines
of evidence indicate that the yeast protein corresponds to the
mammalian homolog, eIF1A. 1) The apparent mass determined by SDS-PAGE
is similar for the yeast (22 kDa) and mammalian (20 kDa) protein. 2)
The amino acid sequence of yeast eIF1A exhibits 65% identity and 76%
similarity to human eIF1A, and both proteins have basic N termini and
acidic C termini. 3) Highly specific antibodies to mammalian eIF1A
cross-react with the yeast protein. 4) Yeast eIF1A substitutes for
mammalian eIF1A in the in vitro assay for methionyl-puromycin
synthesis. 5) The human eIF1A cDNA confers growth to yeast cells
lacking the TIF11 gene. The strong conservation of primary
structure between the yeast and mammalian homologs suggests that eIF1A
plays an important role in these cells. A particularly striking
structural feature is the highly charged termini of the factor, where
the N terminus is positively charged and the C terminus is negatively
charged. The functions of these domains are not known, but one may
speculate that the N terminus is responsible for the RNA binding
property of eIF1A(27) . The yeast eIF1A sequence exhibits no
significant homology to other proteins (other than human eIF1A) in the
data bases. eIF1A is one of the most conserved proteins among the
initiation factors. Sequence identities between the yeast and mammalian
initiation factors range from 26% for eIF4B (15, 16) and 33% for eIF4 The similar primary structures of yeast,
mammalian, and plant eIF1A are manifested in their biochemical
activities. Either yeast or wheat germ eIF1A (6) functions in
place of mammalian eIF1A in an in vitro assay for initiation
based on mammalian components. Furthermore, the human cDNA encoding
eIF1A complements a yeast strain lacking a functional TIF11 gene, indicating that the human protein functions in vivo with yeast components of the translational machinery. Many other
mammalian cDNAs can relieve a growth defect of cells where the
corresponding yeast gene has been disrupted. For example, the effects
of disruption of SUI2, SUI3, TIF51A/B, and CDC33 are reversed by expressing the cDNAs for eIF2 Most initiation factor proteins
in yeast are essential for cell growth and viability. The only
exceptions known to date are the
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U11585[GenBank].
Volume 270,
Number 39,
Issue of September 29, pp. 22788-22794, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)(for reviews, see (1) and (2) ). One of
these, eIF1A (formerly eIF-4C), has been purified from both mammalian (3, 4, 5) and plant cells (6) and is
essential for maximal in vitro protein synthesis. eIF1A is a
small protein (17-22 kDa) that appears to undergo no
post-translational modification reactions(7) . The initiation
factor is implicated in 80 S ribosome dissociation, stabilizes
initiator Met-tRNA
binding to 40 S ribosomal subunits, and
facilitates mRNA binding to the 40 S preinitiation
complex(2, 8) . Thus, eIF1A has pleiotropic effects at
different steps of the initiation pathway. Purified eIF1A from wheat
germ and rabbit reticulocytes functions interchangeably in
vitro(6) , suggesting that the functional domains are
highly conserved. Nevertheless, a clear understanding of how eIF1A
promotes the initiation phase of protein synthesis is lacking.
(10) ,
eIF2
(11) , eIF2(12) , eIF2B
(13) , eIF4A(14) , eIF4B(15, 16) ,
eIF4
(17) , eIF4(18) , eIF5 (19) , and
eIF5A(20) . In addition, there are numerous examples of yeast
initiation factors functioning in mammalian assays and mammalian
initiation factor cDNAs replacing the corresponding yeast genes.
Therefore, studies on yeast initiation are expected to yield results
that likely are applicable to the initiation process in all eukaryotic
cells.
Strains and Genetic Manipulations
The
genotypes and sources of S. cerevisiae strains used or
constructed in this work are described in Table 1. The diploid
strain W303D was made by mating W303-1A and
W303-1B(23) . Construction of the strains carrying a
disrupted eIF1A gene is described below. Yeast cells were grown in YP
or synthetic minimal medium (S) supplemented with the relevant amino
acids and 2% glucose (D) or 2% galactose (G) as described(24) .
Cultures were grown at 30 °C and were monitored by measuring
optical density at 600 nm in a Beckman spectrophotometer. For
sporulation(24) , cells were grown on YPD plates for 24 h
(containing 6% glucose) and then were sporulated at room temperature on
Spo plates (0.3% potassium acetate, 0.02% raffinose, 10 µg/ml of
each amino acid) specialized for strain W303D. Tetrad dissections and
DNA transformations were carried out by standard
procedures(25) .
Fractionation of Yeast Cell Lysates
Cells
from strain W303-1A were grown in YPD medium to A = 0.5 - 1 and lysed in 20 mM HEPES-KOH, 100 mM potassium acetate, 2 mM
magnesium acetate, 2 mM dithiothreitol, and 0.5 mM phenylmethanesulfonyl fluoride, pH 7.4, by vortexing with glass
beads. The S30 lysate was centrifuged at 100,000
g for
22 min to generate an S100 supernatant and ribosomal pellet. The pellet
fraction was suspended in 20 mM HEPES-KOH, 500 mM KCl, 6 mM magnesium acetate, 2 mM
dithiothreitol, and 0.5 mM phenylmethanesulfonyl fluoride, pH
7.4, followed by centrifugation through a cushion of the same buffer
containing 20% glycerol. The supernatant (called HSW) and washed
ribosome pellet (called Rb) were collected separately and stored frozen
at -70 °C.Purification of Yeast eIF-1A
Strain
W303-1A was grown in 0.8 liters of YPD medium to an A of 1.5. The cells were harvested (15.2 g, wet
weight) and lysed as described above except that additional protease
inhibitors were added to the lysis buffer: aprotinin (2 µg/ml),
leupeptin (0.5 µg/ml), and pepstatin (0.7 µg/ml). The crude S30
extract was adjusted to 500 mM KCl and centrifuged for 2 h at
100,000
g (4 °C). The supernatant was dialyzed at
4 °C against 5 liters of buffer H (20 mM HEPES-KOH, 5%
glycerol, 0.2 mM EDTA, and 7 mM -mercaptoethanol, pH 7.4) containing 100 mM KCl. The
dialysate (106 mg of protein) was applied to a fast protein liquid
chromatography Mono S 10/10 column and eluted with a 176-ml linear
gradient of 150-450 mM KCl in buffer H. The column
fractions containing eIF1A were identified by Western blot analysis.
Peak fractions were dialyzed against 2 liters of buffer H containing
100 mM KCl and applied to a Mono Q 5/5 column. Proteins were
eluted with a 20-ml linear gradient of 100-450 mM KCl in
buffer H. Fractions containing eIF1A were concentrated and adjusted to
100 mM KCl by centrifugation in a Centricon 10 (Amicon)
filtration apparatus. The column fractions were analyzed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie
Blue staining.Western Blot Analysis
Yeast protein
samples were fractionated by 15% SDS-PAGE (26) and subjected to
Western immunoblot analysis as described(22) . Coomassie blue
staining sometimes was used to confirm that the same quantity of
protein was transferred in each lane.Methionyl-Puromycin Synthesis Assay
Each
30-µl reaction mixture contained 20 mM Tris-HCl (pH 7.5),
70 mM KCl, 2 mM magnesium acetate, 30 mM potassium acetate, 10 mM -mercaptoethanol, 16.1 pmol
of [
H]Met-tRNA (specific activity, 70.5 Ci/mmol),
0.8 mM GTP, 1 mM puromycin, 33 µM ApUpG,
0.06 and 0.15 A units, respectively, of 40 S and
60 S rat liver ribosomal subunits, 1.08 µg of HeLa eIF2, 1.44
µg of HeLa eIF3, 0.68 µg of HeLa eIF5, 0.47 µg of HeLa
eIF5A, and either yeast or HeLa recombinant eIF1A as indicated. The
reaction mixtures were incubated for 20 min, and the
methionyl-puromycin formed was analyzed as previously
described(28) .
Cloning the cDNA and Gene Encoding
eIF-1A
Escherichia coli strain Y1090 was used to
grow recombinant phages from a gt11 cDNA library of S.
cerevisiae (Clontech). The eIF1A cDNA was isolated by
immunoscreening the expression library with an affinity-purified
anti-human eIF1A antibody(22) . Membranes were treated with a
1:1,000 dilution of the anti-human eIF1A antibody followed by alkaline
phosphatase-conjugated goat anti-rabbit IgG antibody. Immunoreactive
plaques were identified with the chromogenic substrates nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate, and putative
positive phages were plaque purified. The 600-bp insert from a single
positive phage was excised, cloned into phages M13 mp18 and mp19, grown
in E. coli JM101, and sequenced as described below.
gt11 (Clontech) was screened with the
P-labeled 600-bp insert as previously
described(21) . A 2.4-kb EcoRI DNA fragment was
excised from one of the
gt11 phages and subcloned into the EcoRI site of pSP72 (Promega) to generate pSP72-TIF11.
Restriction enzymes HindIII, BglII, AccI,
and EcoRV were used to digest the 2.4-kb fragment to obtain a
restriction map. The smaller fragments generated by different
combinations of restriction enzymes were then subcloned into M13 mp18
and mp19 vectors and transformed into E. coli strain JM109.
Both DNA strands were sequenced with a Sequenase II kit (U. S.
Biochemical Corp.) according to the manufacturer's instructions.
DNA sequence comparisons with sequences in the GENEMBL data bank were
carried out with the FASTA program.
Construction of Plasmids
Plasmid pW-y1A
was constructed by ligating a blunt-ended SpeI-EcoRV
genomic fragment from pSP72-TIF11, which carries the entire TIF11 open reading frame but not the adjacent CIFI gene (see
``Results''), and pHSX3 (23) blunt ended at the BamHI site just downstream from the GAL1 promoter.
For plasmid pW-h1A, two oligonucleotide primers were synthesized to
amplify the coding region of the human eIF1A cDNA in pBluescript-1A
(clone I) (22) (GenBank accession no. L18960(21) ) by
PCR. Primer 1 (P1) is 5`-CCCCTGCAGCCGCCATGGCTCCCAAGAATAAAGG-3`. The
underlined regions are PstI and NcoI sites; P1
corresponds to the region surrounding the initiation codon (-5 to
+20, where +1 is the A of the initiator codon AUG). Primer 2
(P2) is 5`-CCCAAGCTT GAATTCAGAAAAGAT GG-3`. The underlined regions are HindIII and EcoRI sites, respectively; P2 corresponds
to the region 3` of the termination codon (+458 to +471). The
PCR was carried out under standard conditions according to the
manufacturer's protocol (Perkin-Elmer Corp.). The PCR product was
cleaved with NcoI and EcoRI and blunt-ended with
Klenow DNA polymerase; the resulting 470-bp fragment was subcloned into
the blunt-ended BamHI site of pHSX3 to yield pW-h1A. Inserts
with the correct orientation were identified by restriction enzyme
cleavage patterns (results not shown). E. coli strain HB101
was used to propagate the plasmids.Disruption of the Chromosomal TIF11
Gene
To remove the HpaI restriction site in
pSP72-TIF11, the plasmid was altered by digestion with BglII
and BspMI, which flank the HpaI site, and the ends
were filled in with Klenow DNA polymerase followed by blunt-ended
ligation. The resulting recombinant plasmid pSP72a-TIF11 was digested
with SpeI and HpaI to remove a 414-bp fragment
carrying 84% of the TIF11 coding region plus 27 nucleotides
upstream of the AUG. The remaining 4.5-kb fragment was gel purified,
the ends were filled in with Klenow DNA polymerase, and a blunt-ended
DNA containing the HIS3 gene was inserted by ligation to
generate pSP72a-tif11::HIS3. To prepare the HIS3 insert, a
1.75-kb BamHI fragment carrying the entire yeast HIS3 gene was isolated from plasmid pHYH(23) , and the ends
were filled in with Klenow DNA polymerase. pSP72a-tif11::HIS3 was
digested with EcoRI to generate a 3.8-kb fragment carrying tif11::HIS3. The DNA fragment contains 1.4 and 0.6 kb of
flanking DNA 5` and 3` to the TIF11 gene, respectively. The
fragment was transformed into the diploid yeast strain W303D to create
a one-step gene deletion/disruption(29) . Stable His tranformants were selected, and the disruption of one of the TIF11 genes was confirmed by Southern blot analyses (results
not shown).
Detection and Purification of the Yeast Homologue
of eIF1A
Given the fact that translational factors are very
conserved from yeast to humans, it is reasonable to employ an
immunoblot analysis using anti-human eIF1A antibodies to detect the
corresponding yeast eIF1A protein. As shown in Fig. 1A,
a single polypeptide migrating at approximately 22 kDa was detected by
SDS-PAGE in a wild type W303-1A yeast cell lysate. After
fractionating the yeast lysate into a low salt post-ribosomal
supernatant (S100), a high salt-washed ribosomal pellet (Rb), and a
high salt-washed supernatant, followed by Western blot analysis, the
immunoreactive protein was found enriched in the high salt-washed
fraction, although about 20% was present in the S100 fraction (Fig. 1A). This result implies that the immunoreactive
protein weakly associates with ribosomes, as does mammalian eIF1A.
Together, the results suggest that yeast may contain a homolog of
mammalian eIF1A, which is similar in size, shares some of the same
epitopes, and localizes to the same subcellular fractions.
15 g, wet weight). The
protein has an apparent mass of 22 kDa as determined by SDS-PAGE and
represents the major protein in the preparation.
Yeast eIF1A Stimulates Methionyl-Puromycin Synthesis
in Vitro
To obtain further evidence that the 22-kDa protein
is indeed yeast eIF1A, the purified protein was tested in the mammalian
methionyl-puromycin synthesis assay for eIF1A activity. A 3-fold
stimulation of methionyl-puromycin synthesis was obtained, which
compares favorably with stimulations seen with the purified recombinant
human eIF1A (Fig. 2). Both proteins stimulate the assay to
approximately the same extent and require the same amount of protein
for maximal effect. The moles of eIF1A required to saturate the assay
approximate the moles of ribosomes present, suggesting that eIF1A
functions bound to ribosomes. The moles of methionyl-puromycin formed
are quite low compared to the moles of initiation factors added; such
results are routinely obtained in this assay system both for eIF1A and
for other initiation factors (5, 28) and may reflect
more the activity of the ribosomes than that of the initiation factor
being assayed. These results clearly demonstrate that the purified
yeast protein possesses eIF1A activity when tested in vitro.
-) and
yeast (o-o) eIF1A were assayed for stimulation of
methionyl-puromycin formation in a mammalian system as described under
``Materials and Methods.''
The Cloning of a cDNA and Genomic DNAs Encoding Yeast
eIF1A
Having demonstrated an antibody-cross-reacting yeast
protein with eIF1A activity, we set about to clone its gene.
Immunoscreening of a gt11 yeast cDNA expression library with an
affinity-purified rabbit anti-human eIF1A polyclonal antibody yielded
one positive clone from approximately 6.5
10
recombinant phage plaques as described under ``Materials and
Methods.'' The phage was plaque-purified, and PCR analysis
revealed the presence of a 600-bp insert, which is sufficient in length
to encode eIF1A. The insert was subcloned into M13 mp18 and mp19 for
sequencing of both strands (see ``Materials and Methods''),
and the region encoding a 153-amino acid protein was identified (the
cDNA sequence is not shown, but see Fig. 3). The 0.6-kb cDNA
insert was P labeled and used as a probe to screen a yeast
genomic library as described under ``Materials and Methods.''
Four positive clones from 6
10 plaques were plaque
purified for further study. Characterization of the four clones by
restriction enzyme mapping and partial sequencing (data not shown)
indicated that they carry portions of the same DNA. A 2.4-kb EcoRI DNA insert was then excised from one of the recombinant
phages, and DNA encoding eIF1A was localized on a 1.1-kb HindIII-EcoRI fragment by Southern blot analysis.
Sequence analysis (Fig. 3) confirmed that the genomic 1.1-kb
clone contains an open reading frame encoding the same 153-residue
protein as found in the cDNA clone. The first in-frame AUG codon is
located at residues 92-94 and possesses the sequence
5`-AUCAUGG-3`, which is compatible with the yeast concensus context,
A(A/U)AAUGU(30) . We have named the gene TIF11 (translation initiation factor 1A) based on the similarity of its encoded protein to human
eIF1A (see below).
Structural Features of eIF1A
Yeast eIF1A
comprises 153 amino acids with a calculated mass of 17.4 kDa. The yeast
and human proteins are very similar throughout the entire structure (Fig. 4). They share 65% sequence identity and 76% similarity,
which reinforces the view that TIF11 encodes yeast eIF1A. The
strong conservation of structure also is seen in the hydrophobicity
profiles (results not shown). Both proteins contain basic N-terminal
domains (29 mol % Lys + Arg in the first 42 amino acids for human
eIF-1A, 30 mol % Lys + Arg in the first 40 amino acids for yeast
eIF1A) and acidic C-terminal domains (54 mol % Asp + Glu in the
C-terminal 28 amino acids for human eIF1A, 54 mol % Asp + Glu in
the C-terminal 37 amino acids for yeast eIF1A). The highly charged
terminal domains may be responsible for the apparent slow mobility of
yeast eIF1A upon SDS-PAGE (apparent mass of 22 kDa versus a
calculated mass of 17.4 kDa). The major difference between the yeast
and mammalian proteins is the ``insertion'' of 8 amino acids
in the yeast protein between residues 131 and 132 in human eIF1A.
Comparison of the predicted amino acid sequence of yeast eIF1A with
protein sequences in the GenBank and EMBL data banks indicate no
significant amino acid sequence homology to other known proteins except
human eIF1A. However, another gene is located upstream from TIF11 in the original 2.4-kb cloned genomic fragment. By searching the
data bases, we determined that this gene encodes a homolog of the CIF1 gene (GenBank accession no. M88172). CIF1 encodes trehalose-6-phosphate synthetase in yeast and is required
for cells to grow on glucose.
P-labeled band in every one of the nine
different restriction digestions (results not shown) is consistent with
a single copy gene. However, the analyses do not rule out the
possibility of other genes encoding eIF1A that have diverged
sufficiently to evade detection under the conditions used here.
eIF1A Is Essential for Cell
Viability
Although eIF1A stimulates assays for initiation in vitro, it was not known if it is essential for protein
synthesis and/or cell viability in intact eukaryotic cells. We
therefore generated a null mutant strain in which TIF11 is
substantially deleted and is disrupted by the HIS3 gene. The
plasmid pSP72a-tif11::HIS3 was constructed and was used to disrupt TIF11 in the diploid strain W303D as described under
``Materials and Methods.'' About 84% of the coding region of TIF11 is deleted, and the 3.8-kb tif11::HIS3
EcoRI-linearized fragment contains 1.4 and 0.6 kb of TIF11 flanking sequences separated by the HIS3 gene as shown in Fig. 5A. 10 His transformants were
isolated, and their genomic DNAs were isolated. Southern blot analysis
confirmed the integration of the fragment into a TIF11 gene on
one of the chromosomes (results not shown). Transformants that carry a
disrupted TIF11 gene were named CMD1. A number of independent
isolates of CMD1 exhibit no growth defect at 30 °C when measured
either on plates or in liquid culture (results not shown).
:2
viable to nonviable segregation
pattern was seen after 2 days (Fig. 5B) and all of the
viable spores showed a His
phenotype (results not
shown). This suggests that the phenotype of the tif11::HIS3 allele is lethal. The nonviable spores were further examined after
incubation for a week at 23 °C to confirm that they failed to
germinate and grow. The fact that the tetrad spores generated a
2
:2
segregation pattern and no
His
segregants strongly suggests that eIF1A is
required for cell viability and/or germination.
and Trp
phenotypes further
indicates that TIF11 expressed under galactose induction is
sufficient to support the growth of the tif11::HIS3 haploid
strain (Fig. 6B, upperrow). This
fact rules out the possibility that the
2
:2
segregation pattern seen when
CMD1 was sporulated (Fig. 5B) could be due to
down-regulation of the adjacent CIF homolog gene, since pW-y1A does not
carry the CIF homolog gene. A haploid cell colony containing the tif11::HIS3 allele and plasmid pW-y1A was selected and named
CM1.
Trp
spores will germinate and grow
on plates with galactose medium, which induces eIF1A expression, but
should not grow when transferred to plates with glucose medium. On the
other hand, if eIF1A is required only for germination, the germinated
spore colonies will continue to grow on glucose medium. When the spore
colonies were streaked on glucose-containing SD-his or SD-trp plates,
growth ceased (Fig. 6B, bottomrow).
The results show that TIF11 is required for cell growth and
viability.
Human eIF1A cDNA Complements Yeast
tif11::HIS3
The amino acid sequences of human and S.
cerevisiae eIF-1A share 65% identity and 76% similarity. The fact
that yeast eIF1A functions as well as human eIF1A in the in vitro methionyl-puromycin synthesis assay reconstituted with mammalian
components strongly suggests that the two proteins are functionally
equivalent. Since TIF11 is essential for yeast cell growth, we
are able to use the no-growth phenotype to test if the human cDNA can
replace the yeast gene in vivo. cDNA encoding the entire human
eIF1A open reading frame was placed under control of the yeast GAL1 promotor in plasmid pHSX3 as described under ``Materials and
Methods.'' The resulting plasmid pW-h1A was introduced into the
diploid strain CMD1 to yield CMD3. The diploid strain was sporulated,
and dissected spores were germinated on YPG plates, which allow the
expression of the human form of eIF1A. The appearance of viable to
nonviable spores in the ratio of 2:2, 3:1, and 4:0 (Fig. 7A) resembles that obtained when yeast TIF11 is expressed (Fig. 6A) and therefore suggests that
human eIF1A can function in yeast. To confirm that the human cDNA
functionally replaces TIF11, spore colonies were streaked on
various tester plates. All viable His cells
(indicating disruption of TIF11 on the chromosome) also have a
Trp
phenotype (indicating the presence of the human
cDNA on the plasmid). The His
Trp
cells grow only on galactose but not on glucose-containing plates (Fig. 7B). The growth rate of cells expressing human
eIF1A appears to be somewhat slower compared to cells expressing TIF11 since their colony size is smaller. A
His
Trp
haploid spore colony obtained
from CMD3 was named CM2. The results demonstrate that human eIF1A cDNA
expressed in yeast is able to suppress the lethal phenotype of the tif11::HIS3 allele and support cell growth as the sole source
of eIF1A.
20 kDa-protein band) but no
yeast eIF1A, which migrates somewhat more slowly at 22 kDa. In
comparison, a lysate prepared from a strain carrying the wild type TIF11 gene and no human eIF1A cDNA contains only yeast eIF1A (lane3,
22-kDa protein band) as expected. When
the two lysates are combined and analyzed, the two eIF1A bands are
readily resolved (lane1).
g for 10 min; lysate protein from each strain was fractionated
by 15% SDS-PAGE and analyzed by immunoblotting with anti-human eIF1A
antibodies affinity purified with yeast eIF1A as described under
``Materials and Methods.'' The figure shows a computer scan
of the blot. Lane1, wild type strain W303-1A
and strain CM2 expressing human eIF1A (10 µg each); lane2, strainW303-1A (10 µg); lane3, strain CM2 (10 µg).
(17) ,
representing the least conserved proteins, to 65% for eIF4A (31) and 71% for eIF2(32) , the latter being the
most conserved initiation factor known at this time. Thus, initiation
factors are nearly as conserved as ribosomal proteins where the
sequence identity between all cognate yeast and mammalian ribosomal
proteins is 60%, with individual proteins falling in the range from 40
to 88%(33) .
,
eIF2, eIF5A, and eIF4,
respectively(16, 23) . (
)Only in the case
of eIF4A does the mammalian cDNA fail to relieve disruption of TIF1 and TIF2(31) .-subunit of eIF2B (GCN3) (34) and eIF4B (TIF3)(15, 16) , where
cells grow in the absence of the protein, albeit more slowly. The
cloning of TIF11 and the demonstration that it is essential
for cell growth will allow us to address the function of this protein
by genetic and biochemical studies. Both approaches likely will be
required to explain the pleiotropic effects of this small yet essential
initiation factor.
))
We thank Susan MacMillan for performing the
methionyl-puromycin synthesis assays, Elizabeth Shuster for advice on
sporulation procedures, and Charles Moehle for helpful comments on the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. L. Tai, I. Snoek, M. A. H. Luttik, M. J. H. Almering, M. C. Walsh, J. T. Pronk, and J.-M. Daran Correlation between transcript profiles and fitness of deletion mutants in anaerobic chemostat cultures of Saccharomyces cerevisiae Microbiology, March 1, 2007; 153(3): 877 - 886. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Majumdar, A. Bandyopadhyay, and U. Maitra Mammalian Translation Initiation Factor eIF1 Functions with eIF1A and eIF3 in the Formation of a Stable 40 S Preinitiation Complex J. Biol. Chem., February 14, 2003; 278(8): 6580 - 6587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Li and D. W. Hoffman Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy Protein Sci., December 1, 2001; 10(12): 2426 - 2438. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. E. Bean, W. H. Dvorachek , Jr., E. L. Braun, A. Errett, G. S. Saenz, M. D. Giles, M. Werner-Washburne, M. A. Nelson, and D. O. Natvig Analysis of the pdx-1 (snz-1/sno-1) Region of the Neurospora crassa Genome: Correlation of Pyridoxine-Requiring Phenotypes With Mutations in Two Structural Genes Genetics, March 1, 2001; 157(3): 1067 - 1075. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. K. Choi, D. S. Olsen, A. Roll-Mecak, A. Martung, K. L. Remo, S. K. Burley, A. G. Hinnebusch, and T. E. Dever Physical and Functional Interaction between the Eukaryotic Orthologs of Prokaryotic Translation Initiation Factors IF1 and IF2 Mol. Cell. Biol., October 1, 2000; 20(19): 7183 - 7191. [Abstract] [Full Text]
|
|
|
|
|
|
A. Garay-Arroyo, J. M. Colmenero-Flores, A. Garciarrubio, and A. A. Covarrubias Highly Hydrophilic Proteins in Prokaryotes and Eukaryotes Are Common during Conditions of Water Deficit J. Biol. Chem., February 25, 2000; 275(8): 5668 - 5674. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. C. Kyrpides and C. R. Woese Universally conserved translation initiation factors PNAS, January 6, 1998; 95(1): 224 - 228. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Asano, H.-P. Vornlocher, N. J. Richter-Cook, W. C. Merrick, A. G. Hinnebusch, and J. W. B. Hershey Structure of cDNAs Encoding Human Eukaryotic Initiation Factor 3 Subunits. POSSIBLE ROLES IN RNA BINDING AND MACROMOLECULAR ASSEMBLY J. Biol. Chem., October 24, 1997; 272(43): 27042 - 27052. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Chaudhuri, K. Si, and U. Maitra Function of Eukaryotic Translation Initiation Factor 1A (eIF1A) (Formerly Called eIF-4C) in Initiation of Protein Synthesis J. Biol. Chem., March 21, 1997; 272(12): 7883 - 7891. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
