From the Department of Biophysics and Biochemistry,
Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan, the ¶ Laboratory of Eukaryotic Gene Regulation, NICHD,
National Institutes of Health, Bethesda, Maryland 20892, and
PRESTO, Japan Science and Technology Corporation, Kawaguchi,
Saitama 332-0012, Japan
Received for publication, November 8, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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The murine int-6 locus, identified as
a frequent integration site of mouse mammary tumor viruses,
encodes the 48-kDa eIF3e subunit of translation initiation factor eIF3.
Previous studies indicated that the catalytically active core of
budding yeast eIF3 consists of five subunits, all conserved in
eukaryotes, but does not contain a protein closely related to
eIF3e/Int-6. Whereas the budding yeast genome does not encode a protein
closely related to murine Int-6, fission yeast does encode an Int-6
ortholog, designated here Int6. We found that fission yeast Int6/eIF3e
is a cytoplasmic protein associated with 40 S ribosomes. FLAG
epitope-tagged Tif35, a putative core eIF3g subunit, copurified with
Int6 and all five orthologs of core eIF3 subunits. An int6
deletion (int6 Mouse mammary tumor virus
(MMTV)1 integrates into the
mouse genome and frequently causes mammary tumors. The sites of MMTV integration were identified to delineate the molecular basis of the
tumorigenesis (see Refs. 1 and 2 for review). Except for
int-6, the int loci encode growth factors or
transmembrane receptors. Thus, altered expression of these Int proteins
leads to mammary tumorigenesis presumably by affecting signal
transduction pathways controlling cell growth.
By contrast, the molecular basis for tumorigenesis caused by
integration into int-6 is unclear. Int-6 encodes a
ubiquitously expressed, 52-kDa protein, which corresponds to the 48-kDa
eIF3e subunit2 of eukaryotic
translation initiation factor 3 (eIF3) (3). int-6 is the
site of MMTV integration in at least three independently isolated mouse
tumors; in all cases, MMTV integration into an intron of the
int-6 gene resulted in expression of truncated
int-6 mRNA species (4). Therefore, MMTV integration into
int-6 may produce a dominant negative allele, because of
expression of a truncated, unregulatable form of the protein.
Alternatively, the truncation may simply disrupt the biological
function of Int-6, and tumorigenesis would result from reduced
int-6 gene dosage.
Although Int-6 is a stable component of mammalian eIF3, it is not known
whether it is required for the activity of eIF3 in translation
initiation (3). Furthermore, two laboratories (5-7) have localized a
significant proportion of Int-6 in the nucleus, particularly in a
nuclear compartment called the promyelocytic leukemia (PML)
nuclear body (8). Nuclear localization of an Int-6 ortholog was also
reported in Arabidopsis thaliana (9). These results raised
the possibility that integration into int-6 affected the
role of Int-6 in the nucleus but not in the cytoplasm as a component of
eIF3. To identify the biological role of Int-6 and decipher the
consequences of its truncation, it was important to establish the role
of Int-6 in translation initiation as a part of eIF3. Thus far, eIF3
has been purified from mammalian cells (10, 11), wheat germ (12, 13),
A. thaliana (14), and budding yeast Saccharomyces
cerevisiae (15-17). Mammalian eIF3 associates with the 40 S
ribosome and stimulates the binding of both mRNA and
Met-tRNAiMet (10, 11). Purified S. cerevisiae eIF3 was also shown to stimulate Met-tRNAiMet binding to 40 S ribosomes in
vitro (16, 17).
The eIF3 purified from budding yeast consists of five proteins, eIF3a
(TIF32), eIF3b (PRT1), eIF3c (NIP1), eIF3g (TIF35), and eIF3i (TIF34),
homologous to mammalian eIF3 subunits and does not contain an ortholog
of human eIF3e/Int-6 (17, 18). Moreover, the complete S. cerevisiae genome sequence does not encode a protein closely
related to Int-6. Thus, an Int-6-related protein does not seem to be
involved in the functions of eIF3 in the yeast Saccharomyces. Besides Int-6, mammalian eIF3 contains three
other noncore subunits, eIF3d (p66), eIF3f (p47), and eIF3h (p40),
which are conserved in plants, Caenorhabditis elegans, and
Drosophila melanogaster but absent in S. cerevisiae (19). Thus, it is possible that the activities of eIF3
from higher eukaryotes are modulated by association of the five core
subunits with Int-6 and the other eIF3 subunits not found in S. cerevisiae. Interestingly, the recent progress in sequencing the
entire genome of fission yeast Schizosaccharomyces pombe has
revealed that it encodes the four orthologs of noncore eIF3 subunits,
in addition to orthologs of the five core subunits (14). Thus, S. pombe eIF3 may resemble mammalian eIF3 in containing several
polypeptides in addition to the five core subunits present in budding yeast.
In this study, we examined the role of the S. pombe protein,
Int6, closely related to murine Int-6. We found that Int6 is predominantly localized in the cytoplasm, unlike mammalian and plant
Int-6. We also provide evidence that Int6 is a stable component of
fission yeast eIF3 and is functionally homologous to human Int-6
protein. Unlike tif35+, which encodes an
essential core eIF3g ortholog, int6+ is
dispensable. The int6 Plasmid and Yeast Strains--
The DNA segment containing the
int6+ open reading frame (ORF) was amplified by
polymerase chain reaction with oligonucleotides 1 and 2 (Table
I) from genomic DNA, digested with
NdeI and XhoI, and subcloned into the
NdeI and SalI sites of pREP41 (20) or pREP41HA N
(21), producing pREP-int6 or pRHA-int6, respectively. The
tif35+ ORF was similarly amplified with
oligonucleotides 3 and 4, digested with NdeI and
BglII, and subcloned into the NdeI and
BamHI sites of pRF41, a derivative of pREP41 containing the
coding sequence for the FLAG epitope, inserting it between the
BamHI and SmaI sites of pREP41. The resultant
pREP41-tif35-FLAG plasmid encodes eIF3g (Tif35) FLAG-tagged at its C
terminus under the control of the nmt1 thiamine-repressible
promoter.
The DNA segment encompassing the human int-6 ORF was
amplified by polymerase chain reaction with oligonucleotide 5 and a T7-promoter primer from
pETp483 (the T7 promoter
sequence lies upstream of the human int-6 ORF in pETp48,
with a BamHI site in between) and digested with
BamHI. The resulting 1.3-kilobase BamHI
fragment was used to replace the S. pombe int-6+
ORF in pRHA-int6 to generate pRHA-hInt-6.
S. pombe strains JW348 (h90 leu1-32 ade6-M216
int6-GFP (+kanMX6)), JW350 (h90 leu1-32 ade6-M216
int6-3HA (+kanMX6)), and JW346 (h90 leu1-32
ade6-M216 int6 Immunoprecipitation--
Immunoprecipitation of HA-Int6 and
FLAG-Tif35 was performed as previously described (24), using anti-HA
mouse monoclonal antibody 16B12 (BabCO) and anti-FLAG mouse monoclonal
antibody M2 (Sigma), respectively.
Purification of the Putative eIF3 Complex Containing
FLAG-Tif35/eIF3g--
Transformants of JW350 (int6-3HA)
carrying pREP-tif35-FLAG, or pREP41 as a control, were grown in 2 liters of EMM-leu-B1 to A600 = ~10 and
harvested by centrifugation. After washing with sterilized water, cells
(~10 g) were suspended in 15 ml of buffer A (25) and homogenized with
a French Press (Spectronic Unicam). After clearing the extract by two
rounds of centrifugation (13,000 × g for 20 min and
23,000 × g for 20 min), all ~15 ml of WCE (~750 mg
of total protein) were centrifuged in quick seal tubes in a Beckman
70.1 Ti rotor at 55,000 rpm for 2 h at 4 °C. The ribosomal pellets were suspended in 5 ml of buffer B (same as buffer A described by (25), except containing 350 mM KCl), followed by
centrifugation in polycarbonate tubes in a Beckman TLA 100.4 rotor at
80,000 rpm for 1 h at 4 °C. The resulting ribosomal salt wash
(RSW) fraction was immediately mixed with 100 µl of M2 anti-FLAG
affinity resin (Sigma) for 2 h at 4 °C. After washing four
times with 1 ml of buffer B, the bound proteins were eluted in 200 µl
of buffer B containing 0.4 mg/ml FLAG peptide (Sigma). The resulting
FL-Tif35 preparation contained ~30 ng/µl of putative eIF3 complex.
Polysome Profile Analysis--
S. pombe cells were
treated for polysome analysis exactly as described for S. cerevisiae (23). For immunoblot analysis of the gradient samples,
the WCEs were layered on 15-40% sucrose gradients, centrifuged at
39,000 rpm for 4.5 h, and separated into twenty 0.6-ml fractions.
A portion of the first fifteen fractions from the top was precipitated
with 10% trichloroacetic acid, followed by SDS-PAGE and immunoblotting.
Mass Spectrometry--
We resolved the FLAG-Tif35 complex by
SDS-PAGE, and each of the five peptides (except p40 and p39 analyzed
together) was subjected to in gel trypsin digestion and
matrix-assisted laser desorption-ionization-time-of-flight mass
spectrometry at Integrative Proteomics, Inc. (Toronto, Canada). The
observed mass spectrum of its tryptic peptides was compared with the
predicted spectra of all the proteins in the data base using the
ProFound program.
The Phenotypes of S. pombe Cells Lacking the int6+
Gene--
Fission yeast S. pombe encodes a hypothetical
protein (GenBankTM accession number CAA22813) homologous to
murine eIF3e/Int6. This protein, designated Int-6 here, is 45%
identical and 57% similar to murine Int6. Because this extent of
similarity is only slightly less than that between murine Int-6 and
related proteins in C. elegans (52% identical) and
D. melanogaster (62% identical), we suspected that Int6
is the S. pombe homolog of mammalian Int-6.
First we examined the effect on cell growth of deleting the
int6+ gene. We transformed a diploid S. pombe strain with an int6 deletion construct containing
a selectable kanamycin resistance gene (kan) in place of the
int6+ ORF and confirmed the deletion of one
allele of chromosomal int6+ by Southern blot
analysis. When the resulting heterozygous diploid strain was allowed to
sporulate, we observed normal formation of tetrads with four viable
ascospores. As expected, two spores in every tetrad were
KanR, indicating the presence of the
int6
We found that the haploid int6
Next, we examined the intracellular localization of Int6. For this
purpose, we constructed a haploid yeast strain (JW346) encoding the
int6 protein fused to GFP, in place of the wild type int6 protein (see "Materials and Methods"). JW346 grew
indistinguishably from its isogenic wild type parent (JY450) both in
rich and minimal media (data not shown), indicating that GFP-Int6 is
functional. As shown in Fig. 2,
fluorescence was observed throughout the cytoplasm and was absent from
the nucleus. Therefore, Int6 is predominantly localized in the
cytoplasm.
Int6 Is Part of a High Molecular Mass Complex That Associates with
40 S Ribosomes--
Because eIF3 associates with 40 S ribosomes, it
was important to examine whether Int6 is associated with 40 S ribosomes
in S. pombe cells. We constructed strain JW350 containing an
HA epitope-tagged int6 allele (int6-3HA) in
place of the chromosomal wild type allele. HA-Int6 is functional,
because JW350 grew indistinguishably from the wild type both in rich
and minimal media (data not shown). As a control, we analyzed the
S. cerevisiae strain KAY50 encoding an HA-tagged form of
eIF3i subunit TIF34 (HA-TIF34), because it was shown that eIF3 is
associated with 40 S subunits in budding yeast (27). Cell extracts
prepared from these strains in the presence of cycloheximide were
resolved by sucrose gradient velocity sedimentation, and the resulting
A254 absorbance profiles indicated that the
ribosomal species in S. pombe cosedimented exactly with the
corresponding ribosomal species in S. cerevisiae (Fig. 3, A and B,
upper panels). Immunoblot analysis of the gradient fractions showed that the majority of HA-Int6 sedimented with the bulk of 40 S
subunits in S. pombe extracts, consistent with its physical association with 43-48 S preinitiation complexes (Fig. 3A,
lanes 7 and 8). As expected, a large fraction
(~50%) of the HA-TIF34 in S. cerevisiae extracts
sedimented at exactly the same location (Fig. 3B,
lanes 7 and 8).
We also found that a subpopulation of both HA-Int6 (Fig. 3A,
lanes 4 and 5) and HA-TIF34 (Fig. 3B,
lanes 4 and 5) sedimented in fractions just
preceding the 40 S ribosome, suggesting that these proteins are present
in high molecular mass complexes. Immunoblotting of the S. cerevisiae fractions with antibodies against the budding yeast
eIF3b (PRT1) supports the notion that the high molecular mass complex
containing HA-TIF34 at fractions 4-5 is the eIF3 complex of ~600 kDa
(17, 18, 28). As expected, the S. cerevisiae eIF2 complex of
124 kDa sedimented in fractions closer to the top of the gradient, with
a peak in fraction 3, whereas the bulk of eIF1 (13 kDa) peaked in
fraction 2 (Fig. 3B, bottom two panels). Thus,
the sedimentation pattern of HA-Int6 strongly suggests that Int6 is
associated with a high molecular mass complex, most likely eIF3, which
interacts tightly with the 40 S ribosome.
HA-Int6/eIF3e Resides in a Multisubunit Complex Containing
FLAG-Tif35/eIF3g and Four Other Proteins Homologous to Core eIF3
Subunits--
Next, we attempted to establish a physical interaction
between Int6 and eIF3 subunits in S. pombe. We focused first
on a predicted S. pombe protein of 31.4 kDa, designated
Tif35/eIF3g (GenBankTM accession number CAA18400), that is
an ortholog of the 32-kDa subunit of S. cerevisiae eIF3
encoded by TIF35 (18, 29, 30) and of the 44-kDa subunit of
mammalian eIF3 (31) (Table II). We found
that deletion of tif35+ in S. pombe
is lethal, indicating that tif35+ is an
essential gene, as expected for a core eIF3 subunit (data not shown;
see "Materials and Methods"). We constructed a plasmid (pREP-tif35-FLAG) encoding C-terminally FLAG-tagged Tif35 under a
thiamine-repressible promoter and showed that it complemented the
lethality associated with tif35
Encouraged by this finding, we attempted to affinity purify the
S. pombe eIF3 complex. We prepared a RSW fraction (that
should be enriched for initiation factors) from the transformant of
JW350 (int6-3HA) carrying pREP-tif35-FLAG after growth in
minimal medium lacking thiamine. The FLAG-Tif35-containing complex was
purified from the RSW with anti-FLAG affinity resin. As shown in Fig.
4B, lane 2, Coomassie staining of the purified complex
eluted from the resin revealed seven major polypeptides (p105, p85,
p70, p40, p39, p32, and p20) and several other minor constituents (p64, p60, p46, p43, and p36). None of these proteins were contained in the
fraction prepared from a control untagged tif35+
strain (lane 1). Immunoblotting of the purified complex
revealed that the 60-kDa minor constituent (p60) reacted with
anti-HA antibodies (lanes 3 and 4), whereas p39,
p32, and p20 reacted with anti-FLAG antibodies (lanes 5 and
6). These results suggest that p60 and p39 are Int6-HA and
FL-Tif35, respectively, and that p32 and p20 are degradation products
of FL-Tif35. Identification of p60 as HA-Int6 was supported by
preparing the FL-Tif35 complex from a control strain that carries
pREP-tif35-FLAG but contains untagged int6+ and
finding a 58-kDa protein instead of p60 in SDS-PAGE (data not shown).
Because functional eIF3 complexes purified from S. cerevisiae contained only five core subunits eIF3a, eIF3b, eIF3c,
eIF3g, and eIF3i (16, 17), we suspected that the five major
polypeptides in the FL-Tif35-containing complex, p105, p85, p70, p40,
and p39 would correspond to the predicted S. pombe proteins,
eIF3a, eIF3b, eIF3c, and eIF3i, in addition to
(FL-)Tif35/eIF3g itself (see the Introduction). To test
this idea directly, we processed each of the five polypeptides
for mass spectrometry (see "Materials and Methods"), and
we identified all five S. pombe orthologs of S. cerevisiae core eIF3 subunits and the heat shock
protein Hsp70 (Table II). Copurification of Hsp70 with
FL-Tif35 was unexpected but occurs frequently in affinity
purification of protein complexes from different
organisms.4 These results
suggest strongly that the FLAG-Tif35-containing complex corresponds to
S. pombe eIF3.
The presence of HA-Int6 in a purified complex containing FL-Tif35
indicates that the HA-Int6/FL-Tif35 interaction we observed in cell
extracts (Fig. 4A) was tight enough to withstand the high salt buffer used to strip initiation factors from the ribosomes in
preparing the RSW. The fact that the amount of Int6 was
substoichiometric compared with the amounts of the five core subunits,
including FL-Tif35, may indicate that only a portion of eIF3 contains
Int6. However, it is possible that Int6 is a stoichiometric component of eIF3 but was partially removed from the core complex during the RSW
preparation. Based on the results shown in Figs. 3 and 4 and Table II, we propose that Int6 is
physically associated with at least a portion of the eIF3 complexes in
S. pombe.
Deletion of int6+ Diminishes Translation Initiation in
Minimal Medium--
As shown in Fig. 1, deletion of
int6+ was not lethal and, instead, showed
pleiotropic phenotypes including slow growth in EMM and defects in
mitosis and meiosis. To determine whether the int6 Genetic Evidence That S. pombe Int6 Is Homologous to Mammalian
Int-6--
Finally, we examined whether S. pombe Int6 was
functionally homologous to the mammalian Int-6/eIF3e protein. We
constructed plasmid pRHA-hInt-6 for expression of HA-tagged human Int-6
under the thiamine-repressible promoter and investigated whether this plasmid could complement the slow growth phenotype associated with the
int6
We also examined whether the human Int-6 encoded by pRHA-hInt-6
can complement the nuclear partitioning defect we observed in Fig.
1B. We grew the int6 In this study, we characterized a fission yeast protein Int6
closely related to murine Int-6 protein, encoded by a frequent MMTV
integration site. We showed that Int6 is cytoplasmic (Fig. 2) and
associates in vivo with 40 S ribosomes (Fig. 3). Affinity purification of the putative eIF3 complex of S. pombe
directed against FLAG epitope-tagged Tif35/eIF3g revealed the presence of Int6 and the five proteins homologous to the core subunits of
budding yeast eIF3 (Fig. 4 and Table II). This is the first evidence
that fission yeast eIF3 contains the five core proteins that comprise
budding yeast eIF3 (17, 18) and also occur in mammalian eIF3 (19). In
addition to the five core subunit orthologs, we also identified the
fission yeast ortholog of eIF3f (PIR accession number T40490, 33 kDa)
as the 36-kDa polypeptide in the FLAG-Tif35 complex shown in Fig.
4B (data not shown). Thus, physical interactions between the
core and noncore subunits appear to be conserved between the fission
yeast and mammalian eIF3 complexes. Furthermore, the complementation of
the int6 Despite the evidence for physical interaction between Int6 and the
putative core eIF3 subunits, int6+ is not an
essential gene, and we observed only a moderate reduction in the rate
of protein synthesis initiation in int6 We found that int6 In S. cerevisiae, reduced protein synthesis rates caused by
mutations in eIF3 subunit genes TIF34, PRT1, or
TIF32/RPG1 result in cell cycle arrest primarily in the
G1 phase, although the tif34 mutation
additionally impedes the G2/M transition (29, 35, 36). In
contrast, int6 While this manuscript was under review, two articles were published
reporting the characterization of the Int-6 ortholog in S. pombe (38, 39). Crane et al. (38) proposed that
S. pombe Int-6 is a part of eIF3, based on
coimmunoprecipitation of epitope-tagged eIF3i and eIF3b orthologs with
Int-6-HA. We demonstrated this point by affinity purification (Fig.
4B) and subsequent immunoblot (Fig. 4B) and mass
spectrometry (Table II) analyses of a FLAG-Tif35/eIF3g-containing complex. This purified complex contained seven of the nine eIF3 subunit
orthologs encoded in fission yeast. We also showed that the amount of
polysomes engaged in protein synthesis was reduced significantly in the
int6 Finally, what are the implications of our results for MMTV tumor
biology? Our hypothesis that the deletion of
int6+ has an impact on translation of a specific
subset of mRNAs is consistent with the generation of tumors, which
would contain normal rates of protein synthesis but display protein
expression profiles different from that of normal cell lines. If
unequal nuclear division observed for S. pombe int6) mutant was viable but grew slowly in
minimal medium. This slow growth phenotype was accompanied by a
reduction in the amount of polyribosomes engaged in translation and was
complemented by expression of human Int-6 protein. These findings
support the idea that human and Schizosaccharomyces pombe
Int-6 homologs are involved in translation. Interestingly, haploid
int6
cells showed unequal nuclear partitioning, possibly
because of a defect in tubulin function, and diploid
int6
cells formed abnormal spores. We propose that Int6
is not an essential subunit of eIF3 but might be involved in regulating
the activity of eIF3 for translation of specific mRNAs in S. pombe.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mutant grew slowly in minimal
medium, however, and this slow growth phenotype was accompanied by
reduction in the amount of polyribosomes engaged in translation. Our
results implicate Int-6 homologs in translation initiation and lay the groundwork for investigating the cytoplasmic function of Int-6 using
fission yeast as a model organism.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
List of oligonucleotides used in this study
(+kanMX6)) were derived from JY450 (h90 leu1-32 ade6-M216). The int6+
disruptant (int6
) was constructed using
oligonucleotides 6 and 7, based on the polymerase chain
reaction-based gene targeting method for S. pombe (22). The
int6 alleles tagged with 3-HA or GFP coding sequences,
int6-3HA or int6-GFP, respectively, were constructed similarly with oligonucleotides 7 and 8. For the
disruption of tif35+, a DNA fragment containing
most of the tif35+ ORF was replaced with a
ura4+ cassette in a plasmid carrying the
2.3-kilobase HindIII-BglII tif35+ fragment. Diploid JZ489 was transformed
with this construct, and disruption of a single allele of
tif35+ was confirmed by Southern blotting.
Subsequent tetrad analysis indicated that tif35
is
inviable. The heterozygous diploid was transformed with pREP-tif35-FLAG
and sporulated for the isolation of a tif35
haploid
strain carrying pREP41-tif35-FLAG. This strain was able to grow only on
the thiamine-depleted medium in which the tif35-FLAG allele
would be expressed. S. cerevisiae strain KAY50
(TIF34-HA) was described previously (23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
::kan allele in 50% of the ascospores. These results indicate that int6+is
not essential (data not shown).
strain designated JW346
grew normally in rich medium (YES) with a doubling time of ~3-4 h at
30 °C in liquid medium but grew slowly in minimal medium (EMM) at
30 °C with a doubling time of ~7 h compared to ~3 h for the isogenic wild type strain JY450. This reduction in the growth rate was
exacerbated by prolonged incubation in EMM for >24 h. When haploid
int6
cells were shifted to minimal medium, we observed that a large fraction (>70%) of these cells became smaller and a
significant fraction (~5-10%) of them exhibited displacement of
cell wall material (arrowhead in Fig.
1A) after only 6 h of incubation. In a substantial number of cells, the nucleus was displaced
from the medial region, and during mitosis, divided nuclei did not
separate well (asterisk in Fig. 1B). As a
consequence, after 24 h in EMM, ~16% of septated
int6
cells had undergone unequal nuclear partitioning,
such that one daughter received two nuclei and the other received none
(I in Fig. 1B) or that one of the daughter nuclei
was lodged in the middle of the new septum (II in Fig.
1B). These events were not observed with wild type cells
(<0.1% of septated cells) and were observed less frequently (~4%
of septated cells) with int6
cells grown in YES medium. Thus, int6
appears to impair steps in mitosis, especially
the positioning of daughter nuclei. It is noteworthy that mutants deficient in tubulin function exhibit unequal nuclear positioning and
are hypersensitive to microtubule-depolymerizing drugs such as
thiabendazole (26). We found that int6
cells also are
hypersensitive to thiabendazole (data not shown), suggesting that their
nuclear positioning defect might result from defects in tubulin
function. When the diploid int6
cells were subjected to
meiosis and sporulation, >70% of the zygotes formed spores with two
nuclei (asterisk in Fig. 1C) and/or abnormally
shaped spores (arrowhead in Fig. 1C). Thus, the
int6 deletion has pleiotropic phenotypes.
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Fig. 1.
Phenotypes of int6
cells. A, JY450
(int6+) or JW346 (int6
) cells,
cultured in YES, were shifted to EMM and incubated for 6 h,
followed by staining with calcofluor to visualize the septa. The
arrowhead indicates the displaced cell wall material.
b, cells cultured in EMM for 24 h were stained with
DAPI to visualize the nuclei and with a small amount of
calcofluor to visualize the septa. The asterisk indicates an
example of displaced nuclei, and the arrowheads indicate
examples of unequal nuclear partitioning in septated cells.
C, the int6
cells were subjected to
sporulation on synthetic sporulation agar and stained with DAPI.
The asterisk indicates a binucleate fused spore, and the
arrowhead indicates an abnormally shaped spore.
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Fig. 2.
Int6 localizes in the cytoplasm.
Cellular localization of Int6 was examined using the
int6::GFP strain JW348. Cells proliferating in YES
medium were fixed by cold methanol and stained with DAPI.
Fluorescence of GFP (B) represents Int6 localization,
whereas DAPI staining (A) indicates the position of nuclei.
C is the superimposition of the image of A in
red with the image of B in
green.
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Fig. 3.
Int6 is associated with 40 S ribosomes and a
high molecular-mass complex that cosediments with S. cerevisiae eIF3. Twenty A260
units of WCEs prepared from S. pombe strain JW350
(int6-3HA) (A) and S. cerevisiae
strain KAY50 (TIF34-HA) (B) were resolved by
velocity sedimentation on 15-40% sucrose gradients and divided into
twenty 0.6-ml fractions with an ISCO fraction collector while scanning
continuously the absorbance at 254 nm. The top panels depict
the A254 profiles of the gradient fractions, and
the panels beneath show the immunoblot analyses of the corresponding
fractions with mouse monoclonal anti-HA antibodies (BabCO) to detect
S. pombe HA-Int6/eIF3e (A) and S. cerevisiae HA-TIF34/eIF3i (B) and with antibodies
against S. cerevisiae proteins PRT1 (eIF3b) (41), GCD11
(eIF2 ) (42), and SUI1 (eIF1) (43) for B. Different
ribosomal species are labeled. The arrows indicate the
presumed positions of nonribosomal eIF1, eIF2 and eIF3 in S. cerevisiae (B). The asterisk indicates the
presumed position of nonribosomal eIF3 in S. pombe
(A).
in the absence of
thiamine but not in its presence (data not shown). A transformant of
int6-3HA strain JW350 carrying pREP-tif35-FLAG grew as
rapidly as did the parental strain JW350 carrying an empty vector in
minimal medium lacking thiamine, and we confirmed that FLAG-Tif35 was
expressed in WCE prepared from this transformant grown under these
conditions (Fig. 4A,
lanes 2 and 3). As shown in Fig. 4A,
FLAG-Tif35 in the WCE was immunoprecipitated with anti-FLAG antibodies
(panel I, lanes 5 and 6), and HA-Int6
was coimmunoprecipitated only when FLAG-Tif35 was being expressed in
this strain (lanes 4 and 5). Likewise, HA-Int6
and FLAG-Tif35 were coimmunoprecipitated specifically with anti-HA
antibodies (panel II, lane 5). These results
indicate a physical interaction between HA-Int6 and FLAG-Tif35,
presumably in the context of eIF3.
Identification of the five major polypeptides associated with
FLAG-Tif35/eIF3g by MS
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Fig. 4.
Int6 resides in a multisubunit complex with
the S. pombe ortholog of S. cerevisiae
eIF3g subunit Tif35. A, Int6 forms a complex
in vivo with the S. pombe ortholog of S. cerevisiae TIF35. The transformants of int6-3HA strain
JW350 carrying the empty vector pREP41 (lanes 1 and
4) or pREP41-tif35-FLAG (lanes 2 and
5) and the transformant of JY450
(int6+) carrying pREP41-tif35-FLAG (lanes
3 and 6) were grown in thiamine-depleted EMM for
16 h to induce FLAG-Tif35 expression from pREP41-tif35-FLAG. Cell
extracts were prepared and subjected to immunoprecipitation
(CoIP) with an anti-FLAG antibody (panel I) or
anti-HA antibody (panel II). Immnoprecipitates were
separated by SDS-PAGE and examined for the presence of the indicated
proteins by immunoblotting using anti-FLAG or anti-HA antibodies
(lanes 4-6). One-fourteenth of the total crude extracts
were separated and analyzed similarly (lanes 1-3).
B, FL-Tif35 complex purified from the RSW contains HA-Int6.
The FLAG-Tif35 complex (+, lanes 2, 4,
and 6) and the control eluate prepared in parallel from a
strain carrying the vector pREP41 ( , lanes 1,
3, and 5) were prepared as described under
"Experimental Procedures." A portion of these samples was subjected
to SDS-PAGE, followed by Coomassie staining (lanes 1 and
2) or immunoblotting with anti-HA or anti-FLAG antibodies
(lanes 3-6). Proteins specifically associated with FL-Tif35
in addition to HA-Int6 were labeled by their sizes in kDa.
View larger version (26K):
[in a new window]
Fig. 5.
The int6
mutation decreases polysome content in
vivo. Yeast strains JW346 (int6
) and the isogenic
wild type strain JY450 were grown in EMM at 30 °C for 12 h to
A600 = ~1.0, and cycloheximide was added to
the cultures for 5 min prior to harvesting the cells. WCEs were
prepared and resolved by velocity sedimentation on 5-45% sucrose
gradients at 39,000 rpm for 2.5 h. The A254
profiles of the gradient samples are shown with the positions of
different ribosomal species indicated. The x axis as drawn
indicates the A254 value of 0. P/M, the ratio of A254 in
the combined polysome fractions to the A254 of
the 80S peak. d. t., doubling time in EMM at 30 °C.
slow
growth phenotype is associated with reduced rate of translation initiation, we analyzed the polysome profiles in cell extracts prepared
from strains JW346 (int6
) and JY450
(int6+) grown in minimal medium. As shown in
Fig. 5, wild type cells had a sizable polysome content, characterized
by a polysome to monosome ratio (P/M) of 4.7 (or
67% polysomes compared with the total amount of ribosomes), whereas
the int-6
mutant contained a reduced amount of
polyribosomes, with a P/M ratio of 1.5 (or 47%
polysomes compared with the total amount of ribosomes). The reduced
P/M ratio in the int6
mutant is
indicative of a moderate reduction in the rate of translation
initiation. In principle, the diminished rate of initiation could be a
secondary consequence of reduced growth rate. However, we showed
previously that a slow growth rate in budding yeast caused by
respiratory deficiency did not alter the P/M
ratio (32).
mutation. As controls, we used the empty vector pREP41HA N, pREP-int6 encoding untagged S. pombe Int6, and
pRHA-int6 encoding HA-tagged S. pombe Int6 (see "Materials
and Methods"). As shown in Fig. 6, the
int6
cells carrying the vector alone grew less rapidly on
EMM than the isogenic wild type cells carrying the same plasmid
(rows 1 and 2). This slow growth phenotype was complemented fully by untagged S. pombe int6 (row
3) or partially by HA-tagged int6 (row 4).
Interestingly, the HA-tagged human int-6 and HA-tagged
S. pombe int6 complemented the slow growth phenotype of
int6
to similar degrees (row 4 and
5). Thus, human Int-6 can substitute at least partially for
the function of S. pombe Int6. Because the slow growth
phenotype of int6
cells is associated with a reduced rate
of translation initiation (Fig. 5), these results suggest that human
Int-6 protein can function in place of S. pombe Int6 in
translation initiation.
View larger version (39K):
[in a new window]
Fig. 6.
Human Int-6/eIF3e can complement the slow
growth phenotype of int6 cells.
Transformants of JY450 (int6+) or JW346
(int6
) carrying the empty vector pREP41HA N (rows
1 and 2) or those of JW346 carrying pREP-int6
(row 3), pRHA-int6 (row 4), or pRHA-hInt-6
(row 5) were grown in SC medium lacking leucine at 30 °C
for 1.5 days. Equal A600 units and 1 × 10
1 or 1 × 10
2 dilutions of these
amounts, were spotted on EMM lacking thiamine and leucine and incubated
at 30 °C for 3 days. W. T., wild type.
cells carrying the vector
alone, pRHA-int6 or pRHA-hInt-6, employed in Fig. 6, in EMM for 24 h and counted the number of septated cells that underwent unequal nuclear partitioning. The frequency of abnormal nuclear partitioning with both pRHA-int6 and pRHA-hInt-6 was reduced to <25% of the level
of the vector control in each of three independent experiments. Thus,
HA-tagged forms of fission yeast and human Int-6 can partially complement the nuclear partitioning defect associated with
int6
. Based on these results, we conclude that human
Int-6 protein can function in place of fission yeast Int6 in mitosis as
well as in translation initiation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
slow growth phenotype by human int-6 suggests that S. pombe Int6 is functionally homologous to
mammalian Int-6 (Fig. 6).
cells (Fig. 5).
Thus, Int6 is dispensable for the essential activities of eIF3.
moe1+, encoding the S. pombe homolog
of human eIF3d subunit p66, is also nonessential (33), although its
association with S. pombe eIF3 remains to be determined.
Presumably, the essential functions of S. pombe eIF3 depend
only on the five core subunit orthologs, of which (at least) eIF3i (34)
and Tif35/eIF3g are essential.
cells showed several phenotypes,
including frequent unequal nuclear partitioning during mitosis in
haploids, abnormal spore formation in diploids (Fig. 1), and
hypersensitivity to the microtubule-depolymerizing drug, thiabendazole.
If Int6 is involved in general translation initiation, how can deletion of int6+ lead to such specific phenotypes? One
possible explanation is that a moderate reduction in eIF3 function
caused by int6
severely impaired translation of a subset
of mRNAs with relatively poor initiation regions that compete
poorly for ribosomes and other factors with mRNAs containing
optimal initiation sites. Those mRNAs whose translation was
strongly reduced in int6
cells would include mRNAs
encoding proteins critical for nuclear partitioning or sporulation. An
alternative explanation for the pleiotropic int6
phenotype is that Int6 is involved in gene-specific translational control, e.g. by facilitating recruitment of a specific
subset of mRNAs to the eIF3 bound to 40 S ribosomes.
has more profound effects on the
completion of mitosis (Fig. 1B) than on the start of the
cell cycle, in favor of the model that Int6 is involved specifically in
translational control in G2. In keeping with this idea, deletion of
moe1+ encoding another noncore eIF3 subunit
homolog impairs microtubule function and confers resistance to
microtubule-destabilizing reagents (33), suggesting a link between Moe1
(eIF3d) and Int6 (eIF3e) in mitosis. Recently, one of the
mis (minichromosome instability) mutations in fission yeast
was shown to impair 18 S ribosomal RNA biogenesis, suggesting that a
decrease in general protein synthesis rates would impair chromosome
segregation (37). Understanding the molecular basis for mitotic defects
caused by disruption of the translation machinery will require further examination.
mutant (Fig. 5). These results are consistent with
the polysome analysis of Bandyopadhyay et al. (39), who also
observed a moderately reduced rate of total protein synthesis in the
int6
mutant, as measured by amino acid incorporation. Together, these data provide convincing biochemical evidence that Int6
enhances the rate of translation initiation in vivo. These articles also reported interesting phenotypes associated with overexpression and deletion of int6+.
Overexpression of int6+ conferred multi-drug
resistance, dependent on the transcription activator Pap1 (38), whereas
int6
cells were hypersensitive to caffeine (39). However,
human Int-6 protein did not function in place of fission yeast Int6 to
confer drug resistance when overexpressed, nor did it complement the
caffeine sensitivity of int6
cells. In contrast, the
nuclear partitioning defect we observed in int6
cells
(Fig. 1B) was complemented by expression of human Int-6
protein. Thus, the role of Int-6 in mitosis may be conserved between
fission yeast and mammals.
haploids also happens frequently for mice carrying the MMTV genome
inserted at one allele of int-6, this might increase the
degree of aneuploidy and, hence, the frequency of tumorigenesis (40).
However, it remains possible that disruption of the nuclear function of
Int-6 is responsible for tumorigenesis by altering the transcription
rates of certain genes. PML, protein the major constituent of the PML
nuclear bodies where Int-6 resides, has been implicated as a tumor
suppressor and transcriptional regulator (8). Although fission yeast
Int6 was not localized in the nucleus (Fig. 2), the role of Int6 in activating multidrug resistance appears to be independent of the role
of Int6 in translation (38). Understanding the role of Int-6 by using
S. pombe as a model organism will contribute greatly to
solving this complicated problem.
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ACKNOWLEDGEMENTS |
---|
We are greatly indebted to Henry Levin for critical reading of the manuscript and for sharing materials used for genetics with S. pombe. We thank Yoshiya Kubo for disruption of tif35+, Tomohiro Matsumoto for sharing results prior to publication, Reed Wickner, Tom Dever, and Leos Valasek for comments on the manuscript, and members of the Yamamoto, Hinnebusch, and Levin laboratories for helpful discussion and technical advice.
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FOOTNOTES |
---|
* 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.
§ These authors contributed equally to this work.
** To whom correspondence may be addressed. E-mail: ywatanab@ ims.u-tokyo.ac.jp.
To whom correspondence may be addressed. Present address: Div.
of Biology, Kansas State University, 232 Ackert Hall, Manhattan, KS
66506. Tel.: 785-532-0116; Fax: 775-532-6653. E-mail:
kasano@ksu.edu.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M010188200
2 Nomenclature for eIF3 subunits is based on Ref. 14.
3 J. Clayton and K. Asano, unpublished material.
4 B. Cox, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MMTV, mouse mammary tumor virus; eIF, eukaryotic translation initiation factor; PML, promyelocytic leukemia; ORF, open reading frame; GFP, green fluorescence protein; RSW, ribosomal salt wash; WCE, whole cell extracts; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; EMM, Edinburgh minimal medium; DAPI, 4',6-diamidino-2-phenylindole.
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