(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) ()(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.
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(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.
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 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.
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 (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.
Figure 2:
Activity of yeast eIF1A. The indicated
amounts of purified human recombinant (-
) and
yeast (o-o) eIF1A were assayed for stimulation of
methionyl-puromycin formation in a mammalian system as described under
``Materials and Methods.''
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 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.
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
2:2
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.
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 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.
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
HisTrp
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.
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 (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).
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 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).
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(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) .
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,
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) .
Most initiation factor proteins
in yeast are essential for cell growth and viability. The only
exceptions known to date are the -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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U11585[GenBank].