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J Biol Chem, Vol. 274, Issue 39, 27567-27572, September 24, 1999
From the The complex eukaryotic initiation factor 3 (eIF3)
was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary
complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1
(TIF32), the p110 subunit of the eIF3 core complex in
yeast. In a screen for Saccharomyces cerevisiae multicopy
suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene
product, named Hcr1p, was able to support, under restrictive
conditions, growth of the rpg1-1
temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An
hcr1 null mutant was viable, but showed slight reduction of
growth when compared with the wild-type strain. Physical interaction
between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation
analysis. The combination of Eukaryotes initiate protein synthesis by dissociation of an 80 S
ribosome into 40 S and 60 S subunits, formation of the
Met-tRNAi·eIF21·GTP
ternary complex and binding of this complex to the 40 S subunit, joining the resulting 43 S preinitiation complex with mRNA, and finally, formation of the 48 S initiation complex at the initiation codon, followed by addition of the 60 S ribosomal subunit. Most of
these processes are aided by eIF3, the most complex of all the
initiation factors described so far (reviewed in Ref. 1). This factor
is believed to prevent premature association of 40 S and 60 S ribosomal
subunits, to stabilize the ternary complex bound to the 40 S ribosomal
subunit, and to promote the binding of the 43 S preinitiation complex
to mRNA (2-7). In the yeast Saccharomyces cerevisiae,
eIF3 is thought to form a core complex composed of five subunits, Rpg1p
(Tif32p), Nip1p, Prt1p, Tif34p, and Tif35p (8-15). All of them have
homologues in the mammalian eIF3 complex. This core complex was found
to be associated with additional proteins, among them Sui1p and Gcd10p
(16, 17). However, the latter was shown to be involved in specific
initiator methionyl-tRNA modification during its maturation, and its
role as an eIF3-associated protein (if any) is unclear (18).
Interestingly, Hinnebusch and co-workers (9, 19, 20) recently reported that yeast eIF3 binds the joining factor eIF5; this suggests a novel
function for eIF3 in start codon recognition, GTP hydrolysis by eIF2,
and 60 S ribosomal subunit joining. A similar model has recently also
been postulated for mammals (21).
Genetic approaches in yeast resulted in cloning of several genes coding
for translation initiation factors or their subunits (eIF4B, eIF2,
eIF2B, and eIF3) (for reviews, see Refs. 1, 22, and 23). For instance,
a multicopy suppressor screen done with a temperature-sensitive allele
of the known eIF3 subunit Tif34p resulted in identification of another
eIF3 subunit, eIF3-p33 (Tif35p) (13, 14). Another example is the
STM1/TIF3 gene, the protein product of which was identified
as a multicopy suppressor of a temperature-sensitive mutation in eIF4A
and shown to represent the yeast homologue of mammalian eIF4B (24). We
recently reported the characterization of the temperature-sensitive
(ts) mutant of the large subunit of yeast eIF3, Rpg1p (Tif32p) (10).
When rpg1-1 ts mutant cells are incubated at a
restrictive temperature (37 °C) or the conditional mutant cells are
depleted of Rpg1p, translation ceases, and cells accumulate in the
G1 phase of the cell cycle and do not respond to
To learn more about the large subunit of yeast eIF3 and its
physiological interactions, a multicopy suppressor study was carried out to identify proteins that can functionally complement the temperature-sensitive phenotype of the rpg1-1 ts
allele. Here, we present biochemical and genetic data demonstrating
that the nonessential S. cerevisiae HCR1 gene
(high copy suppressor of RPG1) codes
for a protein that appears to be the yeast homologue of the p35 subunit
of mammalian eIF3 and that, in high dosage, partially suppresses the
growth defect of the rpg1-1 ts mutant caused by
incubation at the restrictive temperature. Moreover, both Hcr1 and Rpg1
proteins were shown to interact with each other, which in light of the
previously described interaction between their mammalian homologues,
eIF3-p35 and eIF3-p170 (29), suggests a possible involvement of Hcr1p
in translation initiation.
Yeast Strains, Media, and General Techniques--
The
following strains were used in this work: W303
(MATa, ade2-1, trp1-1,
can1-100, leu2-3,112, his3-11,15,
ura3), YLV314U (MATa,
ura3::URA3::rpg1-1,
trp1-1::TRP1::rpg1- Plasmid and Strain Constructions--
To construct strains
YLVH10 and YLVH11, a SpeI-StuI fragment from the
original PAS29 library clone named YEpLVHCR2.18 was ligated into
SpeI-StuI-digested YEplac181 (34), producing
YEpLVHCR1, and this construct was introduced into the W303 and YLV314U
strains, respectively. Similarly, to produce a YLVH12 strain, the
former library fragment was ligated into
SpeI-StuI-digested YEplac112 (34), and a
resulting construct (YEpLVHCR1-1) was transformed into the YLV041 strain.
YCpLVHCR1-cMyc is a CEN4,LEU2 plasmid that contains the
Myc-tagged HCR1 gene under its endogenous promoter. This
plasmid was constructed in two steps. First, an HCR1
fragment generated by polymerase chain reaction using the original
library clone YEpLVHCR2.18 as a template was digested using
SalI-KpnI polymerase chain reaction-incorporated artificial sites (primers HCR1-SalI
(5'-CTGCAGGTCGACTCTAGTGCA-3') and HCR1-KpnI
(5'-CTATAGGTACCTTAGCGGCCGCCCATAAAGTCGTCATCACCAGG-3')) and ligated into SalI-KpnI of YEpLVHCR1,
producing YEpLVHCR1-N, which also contained a NotI
restriction site inserted by polymerase chain reaction immediately in
front of the stop codon of HCR1. The second step of plasmid
construction consisted of an in-frame insertion of a
NotI-digested Myc tag into the NotI site of
YEpLVHCR1-N. The resulting YEpLVHCR1-cMyc was used for transformation
of the haploid yeast strain W303 to generate strain YLVH10-Me
expressing the Myc-tagged Hcr1 protein in high copy number in order to
test the function of the Myc-tagged HCR1 gene. Strain
YLVH10-Mc was produced by introduction of YCpLVHCR1-cMyc, a construct
generated by insertion of the 1.5-kb SalI-KpnI
fragment carrying the Myc-tagged HCR1 gene into
SalI-KpnI-cut YCplac111 (34).
Disruption of the HCR1 Gene--
A gene disruption was carried
out in a diploid S. cerevisiae strain using the one-step
transplacement method described previously (35). For the disruption of
HCR1, the plasmid YEpHCR1-D was constructed as follows. A
1.2-kb NdeI-NheI fragment representing the entire
coding sequence and part of the promoter region of HCR1 was
replaced by a 2.2-kb NdeI-XbaI fragment of pJJ250
(36) containing LEU2 in the original library clone
YEpLVHCR2.18. The SpeI-SpeI fragment of the
former plasmid containing the LEU2 gene was isolated and
transformed into the diploid strain W303 to produce strain
YLVH13d (see Fig. 1A). The resulting strain was
then sporulated, and tetrads were dissected. Integration into the
HCR1 locus and disruption of only one HCR1 copy
were confirmed by Southern analysis.
Western Blot Analysis and Co-immunoprecipitations--
Crude
cell extracts or immunoprecipitates were fractionated by
SDS-polyacrylamide gel electrophoresis (37) using the Mini-Protean system (Bio-Rad) and immunoblotted onto nitrocellulose (38). Immunodetection of proteins was carried out using monoclonal antibody directed against Rpg1p (10) and anti-Myc hybridoma supernatant antibody. As a secondary antibody, anti-mouse IgG antibodies conjugated with alkaline phosphatase or with horseradish peroxidase, respectively, were used. Proteins were visualized either using alkaline phosphatase substrates (nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) in 100 mM Tris-HCl, 100 mM NaCl, and
5 mM MgCl2 (pH 9.5) or as described in the ECL
manual (Amersham Pharmacia Biotech). Preparation of crude cell extracts
as well as immunoprecipitates used was described previously (10).
Isolation of Multicopy Suppressors of the rpg1-1
Temperature-sensitive Mutant--
To isolate proteins that interact
with the eIF3 complex, we set out to identify multicopy suppressors of
the rpg1-1 ts allele. A yeast genomic library
(kindly provided by Alexander Schleiffer) constructed in the high copy
number vector YEplac181 (34) was introduced into the recipient strain
YLV314U (11), carrying the chromosomally integrated
rpg1-1 ts allele. After overnight incubation of
the plates at a permissive temperature (25 °C), the transformants
were incubated for up to 7 days at a restrictive temperature
(37 °C). A total number of 34 transformants growing at this
temperature were identified, the rescued plasmids of which were able to
suppress the growth at 37 °C of the rpg1-1 ts
mutant after back-transformation into the YLV314U strain. Out of these plasmids, 19 contained the wild-type RPG1 gene, indicating
that our screen was saturated. Restriction analysis of the remaining 15 suppressor plasmids revealed five different clones, all containing a
1.52-kb SpeI fragment. The insert of one of the plasmids
from each clone was sequenced, and the sequence was compared with
sequences in the yeast genome data base. All suppressor clones
contained DNA from chromosome XII corresponding to the hypothetical
open reading frame region YLR192C, which we then named
HCR1 (high copy suppressor of
RPG1) (Fig.
1A). Further subcloning into
the plasmid YEplac181 confirmed the minimal region required for
suppression to be a 1.2-kb SpeI-StuI DNA fragment
exclusively containing the open reading frame of
YLR192C/HCR1 (strain YLVH11) (Figs. 1A and 2). To
show that suppression was due to the presence of multiple copies of the
HCR1 gene, the 1.2-kb SpeI-StuI DNA fragment was inserted into the centromeric plasmid YCplac111 (34). The
presence of this plasmid in the mutant strain YLV314U did not suppress
the mutant phenotype (data not shown). Moreover, expression of the
rpg1-1 ts allele on a multicopy plasmid did not
lead to suppression of growth arrest of the
rpg1-1 ts mutant strain at the restrictive
temperature.2 Thus, it is
highly unlikely that overexpression of Hcr1p suppressed the
rpg1-1 ts mutation by an effect on the expression
of the mutant rpg1-1 ts gene. This was documented
by Northern blot analysis carried out with the YLVH11, YLV314U, and
W303 strains grown overnight at 25 °C and subsequently shifted to
37 °C for 12 h, where no significant alteration in
RPG1 transcript level was observed (data not shown). These
findings strongly suggest that Hcr1p is indeed a specific multicopy
suppressor of the rpg1-1 ts mutant.
Hcr1p May Be a Homologue of the Human p35 Subunit of eIF3--
As
mentioned above, the DNA fragment that suppresses the
rpg1-1 ts mutation is 1239 nucleotides long and
contains the HCR1 open reading frame. The predicted ATG
codon of HCR1 is preceded by an in-frame stop codon at
position Suppression of the rpg1-1 ts Mutation by HCR1--
Overexpression
of Hcr1p leads to suppression of the temperature-sensitive phenotype of
the rpg1-1 ts mutant (Fig.
2). However, the suppression seems not to
be complete since the doubling time of the suppressed
rpg1-1 ts cells was estimated to range from 8 to
10 h, compared with 1.5 h for the wild-type cells (Fig.
3). We have previously reported that the
majority of the rpg1-1 ts mutant cells accumulate
in the G1 phase of the cell cycle as unbudded cell doublets
upon shift to the restrictive temperature, displaying a marked cell
separation defect (11). As judged from morphological and flow
cytometric analyses, the rpg1-1 ts mutant cells
carrying the high copy gene are delayed in G1, having
similar morphological characteristics as the
rpg1-1 ts cells themselves (data not shown). Whereas the rpg1-1 ts mutant cells contain
~15% budded cells (11), we have observed >60% budded cells in the
HCR1-suppressed population. This suggests that
overexpression of Hcr1p somehow helps the mutant cells to overcome the
G1 arrest and to reinitiate budding. No effect was observed
when HCR1 was overexpressed in the wild-type W303 strain
(strain YLVH10). YLVH10 cells proliferated with the same generation
time as W303 cells, and no morphological abnormalities were seen.
To test the allele specificity of the suppressor effect, we subcloned
the HCR1 gene into the plasmid YEplac192 (34) and transformed the resulting construct into the Rpg1p depletion strain YLV041 containing a methionine-suppressible copy of RPG1
(10), producing strain YLVH12. The cells of this strain were then
tested for growth on medium containing methionine, which shuts off
transcription of the RPG1 gene from the MET3
promoter. Since no suppression was observed (data not shown), we
conclude that high dosage HCR1-driven complementation is
rpg1-1 ts allele-specific. This also demonstrates that Hcr1p is not able to functionally replace Rpg1p. In addition, overexpressed Hcr1p is unable to suppress the ts growth defect of
prt1-1, another translation initiation mutant
tested (data not shown). The rpg1-1 ts allele
specificity observed may indicate that Hcr1p directly interacts
with Rpg1p.
Hcr1p Interacts with Rpg1p in Vitro--
The physical interaction
between Tif34p and its multicopy suppressor eIF3-p33 was suggested to
play an important role in the functional rescue observed in this case
(13). In addition, the recently identified homologue of Hcr1p, the p35
subunit of human eIF3, has been shown to interact with another eIF3
subunit, p170, actually representing the mammalian homologue of yeast
Rpg1p (29). To test whether the rpg1-1 ts
multicopy suppressor Hcr1p could interact with Rpg1p, we constructed a
single copy expression plasmid for the c-Myc-tagged version of Hcr1p
and introduced it into the wild-type W303 strain, generating strain
YLVH10-Mc. The fusion protein exhibiting an apparent molecular mass of
~55 kDa was expressed in the YLVH10-Mc strain (Fig.
4A, lane 1). Three
additional bands migrating with a smaller size likely represent
proteolytic breakdown products. The tagged protein was shown to
complement the ts defect of the rpg1-1 ts mutant,
demonstrating that it is functional (data not shown). Crude extracts
from strains expressing a tagged or an untagged version of Hcr1p were
prepared and used for immunoprecipitation experiments with antibodies
directed against Rpg1p or Myc-tagged Hcr1p. The resulting immune
complexes were separated by SDS-polyacrylamide gel electrophoresis and
probed by immunoblot analysis using either anti-Rpg1p or anti-Myc
antibodies. As demonstrated in Fig. 4 (B, lane
5), immunoprecipitation of Myc-tagged Hcr1p resulted in
coprecipitation of Rpg1p. Conversely, immunoprecipitation of Rpg1p
using anti-Rpg1p monoclonal antibody coprecipitated Hcr1p (Fig.
4A, lane 3). The 50-kDa bands seen in Fig.
4A correspond to the IgG heavy chain. In control
experiments, anti-Myc antibody immunoprecipitated neither HCR1 nor RPG1 gene products of the untagged Hcr1p
strain (Fig. 4, A and B, lanes 6).
Similarly, in an immunoprecipitate obtained with anti-Rpg1p antibody,
untagged Hcr1p was not detectable by anti-Myc antiserum (Fig.
4A, lane 4). Thus, these data indicate that both
proteins interact directly and/or reside on the same multiprotein
complex.
HCR1 Is Not Essential for Cell Proliferation--
To determine
whether the HCR1 gene is essential for cell viability, one
copy of the entire coding sequence in a diploid wild-type strain (W303)
was replaced by the LEU2 gene (Fig. 1A; see
"Experimental Procedures"). Correct integration was verified by
Southern blot analysis (data not shown). After sporulation and tetrad
dissection, all four spores in each tetrad were able to form a colony,
and the LEU2 gene segregated 2+:2
To further investigate the functional relation between both Hcr1 and
Rpg1 proteins, we investigated a possible synthetic interaction of the
hcr1 null mutant with the rpg1-1 ts
mutation. The idea of synthetic lethality is based on the observation
that certain double mutants are inviable under conditions in which the
parental single mutants are viable (41), which suggests a biochemical function in the same pathway. Several different combinations of translation factor mutations were found to be lethal or to show a
synthetic enhancement (42). Cells of YLVH13 (hcr1) and
YLV314U (rpg1-1) strains were crossed, and the
resulting diploid strain, YLVH14d, was sporulated. After
dissection of tetrads and outgrowth of the spores, the resulting
strains were evaluated for amino acid auxotrophy and for temperature
sensitivity. This revealed that the combination of the hcr1
null allele with the rpg1-1 ts mutant allele did
not result in an inviable double mutant (data not shown). However,
comparison of the growth characteristics at the semipermissive temperature (33 °C) of the double mutant YLVH14 and the
rpg1-1 ts mutant itself (strain YLV314U) showed a
significant decrease in the growth rate of the double mutant YLVH14
(Fig. 5). This could be caused by a
synthetic enhancement of the rpg1-1 ts phenotype defective in translation initiation at 33 °C or, alternatively, may
be caused by an additive effect of the hcr1 deletion on
protein synthesis already affected by the rpg1-1
ts mutation. Since a polysome profile of the
rpg1-1 ts mutant grown at 33 °C itself shows a
severe reduction in the polysome/monosome ratio and remarkable accumulation of the 80 S ribosomes, we were not able to detect further
changes when looking at the polysome profile of the double mutant
YLVH14. Nevertheless, the data presented here indicate the existence of
a genetic interaction between these two proteins.
In a high dosage suppressor screen of the
rpg1-1 ts mutation, a novel nonessential S. cerevisiae gene named HCR1 was isolated. The screen
appeared to be saturated and highly specific as illustrated by the fact
that, besides the RPG1 gene (19 clones), 15 additional clones were isolated, all of which contained the HCR1 gene.
In high dosage, the Hcr1 protein suppresses the
rpg1-1 ts mutant, but does not rescue the
rpg1 null mutant (a Rpg1p-depleted strain); hence, it does
not bypass the requirement of Rpg1p for growth. The deletion of the
HCR1 gene resulted in a slight reduction of the growth rate
and a significant reduction of the mating ability of the corresponding
strain. In an immunoprecipitation experiment, Hcr1p was shown to
associate with Rpg1p. Furthermore, we demonstrate that the deletion of
HCR1 in combination with the rpg1-1 ts
mutation leads to a synthetic enhancement of the slow growth phenotype of the rpg1-1 ts mutant at the semipermissive
temperature. These data indicate that the two proteins interact both
physically and genetically. Taken together with the facts that Hcr1p
shares ~27% identity and 42% similarity with the p35 subunit of
human eIF3 and that the latter protein was shown to associate with
eIF3-p170 (29), i.e. the mammalian homologue of yeast Rpg1p
(5), we suggest that Hcr1p represents the yeast homologue of
eIF3-p35.
At present, we can only speculate about the mechanism by which Hcr1p
suppresses the rpg1-1 ts mutation. Enhanced
levels of the Hcr1 protein may stabilize just Rpg1p or the entire
multisubunit eIF3 complex at the restrictive temperature by specific
protein-protein interaction or may stimulate one or several of the eIF3
activities in the cell, i.e. binding of the ternary complex
Met-tRNAi·eIF2·GTP or mRNA to the 40 S ribosome.
This would be somewhat reminiscent of Tif35p, another subunit of yeast
eIF3. This protein was identified as a multicopy suppressor of a
tif34 ts mutation and shown to directly interact with Tif34p
(13). Tif34p plays a central role in the assembly of the subunits of
eIF3 into the multiprotein complex (28). To address, at least partly,
the question of whether Hcr1p works as a factor involved in assembly
and/or maintenance of eIF3, we also tested whether it acts as a
multicopy suppressor of the prt1-1 ts mutation.
This is not the case. This might mean that the suppressor effect is
Rpg1p-specific, i.e. suppression occurs at the level of the
translation initiation block, if applicable, caused by the
rpg1-1 ts mutation.
An alternative possibility is that a high dosage of Hcr1p stabilizes
RPG1 mRNA. In this case, the elevated level of the
rpg1-1 ts protein would result in sufficient Rpg1p activity to allow slow growth of cells. However, Northern blot analysis and the fact that
overexpression of the rpg1-1 ts protein in the
rpg1-1 ts mutant does not rescue growth clearly
contradict this possibility.
When we analyzed the suppression of the rpg1-1 ts
mutation by overexpression of Hcr1p by measuring the growth rates of
the corresponding strains, we noticed that it is rather weak (Fig. 3).
We do not know yet the precise function of Rpg1p and hence the
biochemical defect conferred by the rpg1-1 ts
mutation. However, since Rpg1p is involved in translation initiation,
we assume that Hcr1p overexpression partially corrects the translation
initiation defect. Rpg1p, the largest subunit of the eIF3 core complex,
may have several functions, and the rpg1-1 ts
mutation may impair more than one of them. The weak suppression of
rpg1-1 by HCR1 might then be explained
by suppression of only some of the affected functions of the rpg1-1
protein by Hcr1p. This would be sufficient to overcome the
G1 arrest and to reinitiate budding (likely by enhancing
the rate of the otherwise severely reduced protein synthesis).
If Hcr1p is involved in translation initiation as suggested by the
suppression of the ts mutation in the RPG1
(TIF32) gene and from its homology to the human eIF3-p35
subunit, one might have expected that the deletion of the
HCR1 gene would be lethal. However, this is not the case.
Other nonessential genes encoding translation initiation factors are
STM1, encoding the yeast homologue of mammalian eIF4B, which
was identified as a multicopy suppressor of the eIF4A ts mutation (24);
and GCN3, encoding a subunit of the nucleotide exchange
factor eIF2B (43). Gcn3p is a regulatory subunit of eIF2B and plays an
important role in up-regulation of translation of GCN4
mRNA under conditions of amino acid starvation (44). In analogy to
Gcn3p, it is tempting to speculate that Hcr1p might be a regulator of
eIF3 activity, acting by binding to Rpg1p under special physiological
conditions, but being dispensable for eIF3 activities under normal
growth conditions. In accordance with this possibility, Hcr1p was found
not to belong to the firmly associated eIF3 complex referred to as the
eIF3 core complex (8, 9). Together with other proteins, among them
Sui1p and maybe Gcd10p, Hcr1p may belong to a group of eIF3-associated
proteins whose functions await elucidation. Moreover, when Hershey and co-workers (29) analyzed human eIF3-p35, they found it to be a very
"sticky" protein, interacting with nearly all subunits of human
eIF3, especially with p66 and the p170 homologue of yeast Rpg1p.
Further experiments are necessary to find the function for Hcr1p in the
translation initiation machinery and to explain the functional
connection with Rpg1p (Tif32p). This may lead to a better understanding
of the function of the p35 subunit of the human eIF3 complex.
We thank Alexander Schleiffer for the
YEplac181-constructed PAS29 yeast genomic library. We also thank
Kate *
This work was supported by a grant from the
Bundesministerium für Wissenschaft und Verkehr, Vienna, Austria
(to H. R.) and by Grant 204/99/1531 from the Grant Agency of the Czech
Republic, Prague, Czech Republic (to J. H.).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.
¶
To whom correspondence should be addressed: Lab. of Eukaryotic
Gene Regulation, NICHD, National Institutes of Health, Bethesda, MD
20892. Tel.: 301-594-7236; Fax: 301-496-8576; E-mail:
lvalasek@aghmac1.nichd.nih.gov.
2
L.Valá The abbreviations used are:
eIF, eukaryotic
initiation factor;
ts, temperature-sensitive;
kb, kilobase(s).
The Saccharomyces cerevisiae HCR1 Gene Encoding a
Homologue of the p35 Subunit of Human Translation Initiation Factor 3 (eIF3) Is a High Copy Suppressor of a Temperature-sensitive Mutation in
the Rpg1p Subunit of Yeast eIF3*
Valáek
§¶,
í
Haek
,§, and§
Vienna Biocenter, Institute of Biochemistry
and Molecular Cell Biology, University of Vienna, A-1030 Vienna,
Austria, the § Ludwig Boltzmann-Forschungsstelle für
Biochemie, A-1030 Vienna, Austria, the Institute of
Microbiology, Academy of Sciences of the Czech Republic, 14220 Prague,
Czech Republic, and the ** Institute of Biochemistry and Molecular
Biology, University of Berne, 3012 Berne, Switzerland
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
hcr1 and rpg1-1 mutations resulted in a synthetic
enhancement of the slow growth phenotype at a semipermissive
temperature. In a computer search, a significant homology to the human
p35 subunit of the eIF3 complex was found. We assume that the yeast
Hcr1 protein participates in translation initiation likely as a protein
associated with the eIF3 complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-pheromone (10, 11). We have shown that the essential S. cerevisiae Rpg1 protein is the homologue of the mammalian
eIF3-p170 protein (5) required for translation initiation in
vivo and in vitro, interacts with Prt1p, and represents
the largest component of the eIF3 core complex. Involvement of Rpg1p in
the process of translation initiation was clearly demonstrated in a
cell-free system dependent on exogenous eIF3 (25): we showed that the
sucrose gradient fraction containing Rpg1p possesses the biochemical
activity ascribed to eIF3, e.g. the restoration of
translation in an extract in which an endogenous eIF3 subunit had been
inactivated. The mammalian homologue of Rpg1p, p170, was proved to
interact directly with the human homologue of Prt1p (26), which is in a
good agreement with our result. In addition, it has been also proposed
to bind eIF4B (7) and to be necessary for binding and stabilization of
the ternary complex (27). We have demonstrated that this protein is not
able to replace yeast Rpg1p in the functional rescue experiment. Up to now, none of the mammalian eIF3 subunits has worked in a similar type
of experiment (26, 28), even though the yeast eIF3 factor functions in
a mammalian methionyl-puromycin assay system (15). In addition to
Prt1p, yeast Rpg1p has recently been shown to interact with Nip1p (9).
Interestingly, Nip1p also interacts with Sui1p and eIF5 (19), both of
which are involved in the start codon recognition process previously
not considered to be influenced by eIF3. It is clear that further
experiments are needed to define the complex spectrum of eIF3
functions. Thus, biochemical strategies and genetic studies carried out
with the eIF3 core complex subunits can help to reveal important
interactions between either eIF3 subunits themselves and/or with other
components of the translational apparatus and subsequently to better
understand the function of eIF3 in translation initiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2, ade2-1, can1-100, leu2-3,112,
his3-11,15) (11), YLV041 (MATa, ura3::URA3::MET3-RPG1, ura3,
rpg1-1::LEU2, ade2-1, trp1-1,
can1-100, leu2-3,112, his3-11,15)
(10), JJ-1A (MATa, arg1, thr1), JJ-1C
(MAT, arg1, thr1), YLVH10
(MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15,
ura3, YEpLVHCR1), YLVH11 (MATa, ura3::URA3::rpg1-1,
trp1-1::TRP1::rpg1-2,
ade2-1, can1-100, leu2-3,112, his3-11,15, YEpLVHCR1), YLVH12
(MATa,
ura3::URA3::MET3-RPG1, ura3,
rpg1-1::LEU2, ade2-1, trp1-1,
can1-100, leu2-3,112, his3-11,15, YEpLVHCR1-1), YLVH10-Me (MATa,
ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3,
YEpLVHCR1-cMyc), YLVH10-Mc (MATa, ade2-1, trp1-1, can1-100,
leu2-3,112, his3-11,15, ura3,
YCpLVHCR1-cMyc), YLVH13 (MATa,
hcr1::LEU2, ade2-1, trp1-1,
can1-100, leu2-3,112, his3-11,15,
ura3), and YLVH14 (MATa,
ura3::URA3::rpg1-1, rpg1-2::TRP1, hcr1::LEU2, ade2-1,
can1-100, leu2-3,112, his3-11,15, trp1-1). Strains were grown and subjected to
genetic manipulations as described (30). Flow cytometric DNA analysis
(fluorescence-activated cell sorting) was done as described previously
(31). The DNA sequence of the HCR1 gene was determined by
the dideoxy chain termination method (32) using a T7 sequencing kit
(Amersham Pharmacia Biotech). The quantitative mating efficiency assay
and polysome profile analyses were performed essentially as described previously (10). Northern blot analysis was carried out as described (33).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence characteristics of
HCR1, the multicopy suppressor of the
rpg1-1 ts mutant. A,
schematic drawing of a DNA fragment from the original PAS29 library
clone YEpLVHCR2.18, which suppresses the rpg1-1
ts mutation. The arrows indicate the minimal region required
for suppression containing the HCR1 open reading frame. For
gene disruption, a 1.2-kb NdeI-NheI fragment of
HCR1 was replaced by a 2.2-kb
NdeI-XbaI fragment containing the LEU2
marker. B, sequence comparisons. Shown are similarities of
the deduced amino acid sequences of Hcr1p and the p35 subunit of human
eIF3 viewed using the Clustal alignment method (Lasergene).
48. The open reading frame encodes a protein containing 265 amino acids with a molecular mass of 29.56 kDa and a pI of 5.03. This
ATG codon is preceded by four adenines and thus should represent an
efficient translation start site (39). The predicted stop codon is
followed by additional in-frame stop codons at positions 847 and 913. The accession number for the DNA sequence in the NCBI Database is
U14913. When the amino acid sequence deduced for Hcr1p was run against
the PROSITE data base (40), sites for protein phosphorylation by
protein kinase C and casein kinase II as well as
N-glycosylation and N-myristoylation modification
sites were identified. When the amino acid sequence was compared with
sequences in the protein data bases, two interesting matches showed up.
The first is the p35 subunit of human translation initiation factor 3 recently characterized by Block et al. (29), displaying
~26% identity and 42% similarity. These authors already noted this
amino acid sequence homology. The other is a protein of unknown
function from Arabidopsis thaliana. Based on this finding, we postulate that Hcr1p represents the S. cerevisiae
homologue of the p35 subunit of human eIF3 (Fig.
1B).
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Fig. 2.
Suppression of the
rpg1-1 ts phenotype by overexpression
of Hcr1p. The YLV314U strain carrying the
rpg1-1 ts mutation was transformed with
HCR1 on a multicopy plasmid (strain YLVH11) and tested for
growth suppression at 37 °C. As controls, the original
temperature-sensitive YLV314U and wild-type W303 strains are shown.
Plates were incubated for 3 days.
View larger version (23K):
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Fig. 3.
Suppression of the
rpg1-1 ts mutant by high copy
expression of HCR1 in liquid medium. Cells were
grown in liquid synthetic complete medium minus Leucin medium at
37 °C (open symbols) or at 25 °C (closed
symbols). W303 (squares) corresponds to a wild-type
(wt) strain hosting an empty YEplac181 vector; YLVH11
(diamonds) to the rpg1-1 ts mutant
strain transformed with HCR1 on a multicopy vector; and
YLV314U (triangles) to the rpg1-1 ts
mutant strain.
View larger version (47K):
[in a new window]
Fig. 4.
Co-immunoprecipitation of Hcr1p with the
Rpg1p subunit of eIF3. Approximately 50 mg of total protein from
crude extracts (extr.) containing either Myc-tagged or
untagged Hcr1p were immunoprecipitated (IP) with either
anti-Rpg1p monoclonal antibody (A and B,
lanes 3 and 4) or anti-Myc antibody (A
and B, lanes 5 and 6). Immune
complexes were then separated by 12.5% SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and probed with
monoclonal antibody against the Myc tag (A) or against Rpg1p
(B). The crude extracts containing tagged or untagged Hcr1p
are shown in A and B (lanes 1 and
2, respectively). Note that Hcr1p is predicted to be a
protein with a molecular mass of ~30 kDa, but that the fusion with
the Myc tag and perhaps some post-translational modifications resulted
in a larger size.
,
indicating that the HCR1 gene is not essential for cell
proliferation (data not shown). The hcr1::LEU2
null mutant strain YLVH13 showed neither heat nor cold sensitivity when
compared with an isogenic wild-type strain. The generation time of the
null mutant grown in YP and 2% glucose calculated from absorbance
measurements of exponentially growing cultures was found to be somewhat
longer (~2.0 h) at both 30 and 37 °C compared with the wild-type
strain generation time of ~1.5 h (data not shown). The mating
efficiency was estimated to be ~10-fold lower than that of the
wild-type strain (data not shown), indicating that mating ability is
reduced in the mutant strain. These results suggest a defect, perhaps on the translational level, in the hcr1-deleted cells;
however, we were not able to prove this by analyzing polysome profiles of the hcr1 null mutant strain (data not shown).
View larger version (13K):
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Fig. 5.
Growth characteristics of the double
mutant hcr1,rpg1-1 strain. Shown is a comparison of the growth characteristics
of the double mutant hcr1,rpg1-1
strain (YLVH14; 
) with the rpg1-1 ts strain
(YLV314U; 
) and the wild-type (wt) strain (W303; 
).
The cells were grown overnight at 25 °C, and then one-half of the
volume of each culture was shifted to 33 °C (B), whereas
the second half remained at 25 °C (A).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
ina Malínská and Dominik
Kadle
ík for great help during the course of this work
and Jan Paleek for critical reading of the manuscript. The
technical assistance of Harald Nierlich is gratefully acknowledged.
FOOTNOTES
ek, unpublished observation.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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