Rpg1p/Tif32p, a Subunit of Translation Initiation Factor 3,
Interacts with Actin-Associated Protein Sla2p
Jan Palecek,*,
† Jiri Hasek,‡,1
and Helmut Ruis*
*Vienna Biocenter, Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Dr. Bohrgasse 9, A-1030
Vienna, Austria; †Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135,
Brno 612 65, Czech Republic; and ‡Institute of Microbiology, Academy of Sciences of the Czech Republic,
Videnska 1083, 142 20 Praha 4, Czech Republic
Received March 12, 2001
The yeast two-hybrid system was used to screen for
proteins that interact in vivo with Saccharomyces cerevisiae
Rpg1p/Tif32p, the large subunit of the translation
initiation factor 3 core complex (eIF3). Eight positive
clones encoding portions of the SLA2/END4/
MOP2 gene were isolated. They overlapped in the
region of amino acids 318–550. Subsequent deletion
analysis of Sla2p showed that amino acids 318–373
were essential for the two-hybrid protein–protein interaction.
The N-terminal part of Rpg1p (aa 1–615) was
essential and sufficient for the Rpg1p–Sla2p interaction.
A coimmunoprecipitation assay provided additional
evidence for the physical interaction of Rpg1p/
Tif32p with Sla2p in vivo. Using immunofluorescence
microscopy, Rpg1p and Sla2p proteins were colocalized
at the patch associated with the tip of emerging
bud. Considering the essential role of Rpg1p as the
large subunit of the eIF3 core complex and the association
of Sla2p with the actin cytoskeleton, a putative
role of the Rpg1p–Sla2p interaction in localized translation
is discussed. © 2001 Academic Press
Key Words: yeast Saccharomyces cerevisiae; twohybrid
system; coimmunoprecipitation; protein–
protein interaction; translation initiation factor 3;
Rpg1p/Tif32p; actin associated cytoskeleton; Sla2p/
End4p/Mop2p; localized translation.
Association of specific mRNP particles and polysomes
with the cytoskeleton has been suggested as the
basis of a mechanism generating an asymmetric protein
distribution by compartmentalization of the protein
synthesis in the cell (1, 2). There is considerable
evidence that at least a subfraction of mRNA, translation
factors, and polysomes is co-localized or associated
with cytoskeletal structures (3). It has been shown
that, for example, the levels of initiation factors eIF-2,
eIF-3, eIF-4A, and eIF-4B are enriched in the cytoskeletal
fraction of HeLa cells compared to the soluble
fraction (4). In addition, using fluorescence microscopy,
eIF-2 and the elongation factor EF-2 were localized
along microfilament bundles of cultured mouse embryo
fibroblasts (5, 6). Another elongation factor (EF-1␣)
colocalized with the actin cytoskeleton in mammalian
(7) and amoebae (8) cells.
Recent data imply that in budding yeast, Saccharomyces
cerevisiae, the actin cytoskeleton plays an active
role in generation of the asymmetric distribution of
specific proteins (9, 10). Yeast may employ similar
mRNA localization mechanisms as higher eukaryotes
(11). With respect to association of polysomes with the
cytoskeleton, the yeast ribosomal protein, Rpp2bp/
Rpl45p, was found in a two-hybrid search for actininteracting
proteins (12). Another two-hybrid search
(13) has revealed complex formation between the cytoskeletal
assembly protein Sla1 and the prion-forming
domain of the release factor Sup35 (eRF3). It was
shown that yeast elongation factor 1 alpha, Tef1p/
Tef2p, binds to Bni1p protein, a downstream target of
Rho1p-Bni1p-mediated pathway functioning in reorganization
of the actin cytoskeleton (14). All these interactions
may be involved in the intracellular localization
of translational complexes in yeast.
Here we characterize a direct physical interaction of
Rpg1p with actin-associated protein Sla2p using the
yeast two-hybrid system. Our immunofluorescence
data suggest that portions of both proteins colocalize at
specific cellular domains, especially at the patch associated
with the tip of emerging bud.
Abbreviations used: ECL, enhanced chemiluminescence; eIF, eukaryotic
translation initiation factor; HRP, horseradish peroxidase;
MCS, multiple cloning sites; ORF, open reading frame; PCR, polymerase
chain reaction; SDS–PAGE, sodium dodecyl sulfate–
polyacrylamide gel electrophoresis; X-Gal, 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside;
WT, wild type.
1
To whom correspondence should be addressed. Fax: ϩϩ4202-
4752501. E-mail: hasek@biomed.cas.cz.
Biochemical and Biophysical Research Communications 282, 1244–1250 (2001)
doi:10.1006/bbrc.2001.4721, available online at http://www.idealibrary.com on
12440006-291X/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
MATERIALS AND METHODS
Cell strains and growth media. The following strains were used
in this work: FY1679 (MATa/MAT␣ ura3/ura3 trp1/ϩ leu2/ϩ his3/
ϩ), L40 (his3 trp1 leu2 ade2 lexA-HIS3:LYS2 lexA-lacZ:URA3 gal4
(15)), RH2887 (MATa lys2 leu2 ura3 his3 trp1 bar1 (16)), RH3395
(MATa lys2 leu2 ura3 his3 bar1 end4⌬::HIS3 end4⌬376-501:TRP1
(16)), WAY205 (MATa lys2 leu2 ura3 his3 trp1 bar1 end4⌬::HIS3 (A.
Wesp)), WAY223 (MATa lys2 leu2 ura3 his3 bar1 end4⌬::HIS3
end4⌬114-367:TRP1 (A. Wesp)).
Yeast was grown in YPD and/or YPUADT rich medium (1% yeast
extract, 2% peptone, 20 mg/L uracil, adenine and tryptophan, 2%
glucose), and/or synthetic complete SCϪ medium (0.67% yeast nitrogen
base without amino acids, 2% glucose); SC-TϪ
,LϪ
, ϩX-Gal was
SCϪ medium without tryptophan, leucine and with 40 mg/L X-Gal
(5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside). Yeast transformations
were performed by the lithium acetate method (17) and/or
by the method described by Chen et al. (18).
Construction of plasmids. The plasmid containing the lexA DNAbinding
domain fused with the entire coding region of Rpg1p was
constructed as follows (constructs are shown diagrammatically in
Fig. 1C). The BamHI–PstI fragment from plasmid pYCpAW20 was
inserted into the multicloning site of pBTM116 (constructed by P.
Bartel and S. Fields) in frame (blunting the overhangs of BamHI
cleavage sites) to generate plasmid pLBP. pLCP plasmid was created
by removing the SmaI–ClaI (in the N-terminal sequence) fragment
of pLBP. pLBN was created by removing the NdeI–PstI fragment of
pLBP. The EcoRI–PstI fragment from plasmid pYCpAW20 was inserted
into the multicloning site of pBTM116 in frame to generate
pLEP. Every new ORF fusion was controlled by DNA sequencing and
the expression of the protein was checked by Western blots (19).
Construction of plasmids containing the Gal4 DNA-activating domain
fused to the entire coding region (pGCE) and/or deletion versions
of the SLA2 gene (pGLE and pGSE) was described elsewhere
(16). Primers introducing BamHI-sites 2 bp upstream of the initiator
methionine and downstream of the stop codon, respectively, were
used to amplify the SLA2 coding region of the sla2⌬318-373 allele
(16). This PCR product was digested with BamHI, gel purified and
ligated into BamHI-digested vector pGAD424 (15) to give pGME
plasmid. Each plasmid complemented the growth defect of sla2⌬
mutant (WAY205) at 35°C (16).
Standard techniques were used for plasmid isolation, plasmid
analysis, and restriction fragment cloning (20). PCR was performed
in 100-␮l reactions using Vent polymerase (New England Biolabs,
Beverly, MA) under conditions recommended by the manufacturer
(21). DNA sequencing was accomplished by means of the dideoxy
nucleotide chain termination method (22) using a T7 Sequencing Kit
(Pharmacia). Autoradiography was performed with FUJI RX X-ray
film (Tokyo, Japan), and the films were developed with an AGFA
automatic film processor.
Two-hybrid analysis. Yeast strain L40 with two reporter genes
(lexA-lacZ and lexA-HIS3) was transformed with pLBP and consequently
with a yeast genomic library (23). The transformants were
streaked on SC-TϪ
,LϪ
,HϪ
plates. After incubation at 30°C for 1 week
colonies were restreaked on selective SC-TϪ
,LϪ
,HϪ
and SC-
TϪ
,LϪ
,ϩX-Gal plates. Plasmids were recovered from colonies that
both grew on plates without histidine and turned the chromogenic
substrate X-Gal blue after incubation at 30°C for 3 days. The recovered
plasmids were reintroduced into the L40 strain harboring
pLBP. LacZ expression was quantitatively examined by measuring
the activity of ␤-galactosidase by the method described by Miller
(24).
Protein extracts, Western blotting and in vivo binding assay.
Yeast protein extracts were prepared from exponentially growing
cells as described elsewhere (16). Precleared extracts used for coprecipitation
experiments were diluted with lysis buffer (0.1 M NaCl, 20
mM Mes pH 6.5, 5 mM MgCl2, 1% Nonidet NP40) to give a final
protein concentration of 1–2 mg per 0.5 ml. 5 ␮l of rabbit antiserum
directed against the extreme C-terminal peptide of the Sla2p protein
((16); kindly provided by Andreas Wesp, Basel, Switzerland) was
added and extracts were shaken for 1 h at 4°C. Immune complexes
were collected after adding 50 ␮l of 50% protein G-Sepharose (Pharmacia
LKB Biotechnology, Inc., Piscataway, NJ) per sample and
incubating for 1 h at 4°C. Protein G-Sepharose beads were washed
three times with lysis buffer and proteins were released into 50 ␮l
SDS-sample buffer by heating the beads at 95°C for 5 min. 10 ␮l
supernatant was separated by SDS–PAGE (25) and blotted to nitrocellulose
membrane (Schleicher and Schuell) using standard procedures
(19). The Rpg1p protein was detected using rabbit anti-Rpg1p
antiserum (1:30,000 dilution). The level of Sla2p protein was checked
with the Sla2p specific antibody. HRP-coupled donkey anti-rabbit Ig
(Amersham Corp.) at 1:10,000 dilution was used as secondary antibody.
Blots were developed using the ECL system (Pierce, Rockford,
IL).
Immunofluorescence microscopy. Yeast cells (FY1679 strain)
were fixed with 3.7% (w/v) formaldehyde for 45 min and processed for
immunofluorescence microscopy as described elsewhere (26). Monoclonal
Rpg1p-specific antibody PK1/1 (27) was applied as an ascitic
fluid finally diluted 50-fold in 1% (w/v) BSA/PEMI at 25°C for 60 min.
Cy3-labeled goat anti-mouse Ig antibodies (GAM-Cy3; Sigma) diluted
1:200 were used as secondary antibodies. Antibody against the
C-terminal domain of Sla2p ((28); kindly provided by David Drubin,
UC Berkeley, CA) and the secondary FITC-labeled goat anti-rabbit
IgG antibody (Sigma, diluted 1:200) were used to visualize Sla2p. To
diminish background fluorescence in yeast cells due to unbound
antibodies to a minimum, all incubation and washing steps were
done in suspension (26).
Samples were investigated with the Olympus BX-60 fluorescence
microscope using standard filter sets. Images were recorded with
1280 ϫ 1024 pixel resolution using Fluoview cooled CCD camera
with the use of analySIS imaging software.
RESULTS
Rpg1p/Tif32p interacts with Sla2p in the two-hybrid
assay. A screen using the two-hybrid system was undertaken
for genes encoding Rpg1p/Tif32p-binding proteins.
Originally, the entire coding region of Rpg1p was
fused to the lexA DNA-binding domain (plasmid
pLBP). Conditional mutant strain YLV041, expressing
RPG1 gene under the control of the MET3 promoter
(29), was transformed with control (pBTM116) and/or
pLBP plasmid, respectively. Cells carrying lexA-Rpg1p
hybrid protein were viable while the control cells exhibited
a lethal phenotype under restrictive conditions
(data not shown). These results and Western blot analysis
of the protein extracts from transformants (data
not shown) confirmed that the lexA-Rpg1p fusion protein
is functional.
Plasmid pLBP and the yeast S. cerevisiae genomic
library, expressed as fusion proteins with the Gal4
transcription-activating domain (23), were introduced
into L40 cells that carried lexA-lacZ and lexA-HIS3
reporters. The plasmids were then recovered from the
colonies that grew on SC-TϪ
,LϪ
,HϪ
selective plates.
After reintroduction of the plasmids, only 8 clones specifically
increased the level of expression of both HIS3
and lacZ reporter gene in L40 cells in a pLBPdependent
manner. Sequence analysis revealed that
each of these eight clones was derived from the SLA2/
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1245
END4 gene originally identified during a screen for
mutants that are defective in the internalization step
of endocytosis (30) and during an independent screen
for synthetic lethals with abp1 null mutant (31).
Among these clones (four of them—pHB21, pHB22,
pHB29 and pHB201—were identical) longer fragments
containing a second coiled-coil domain (aa 575–767)
exhibited higher affinity to Rpg1p than shorter fragments
(Fig. 1A). All SLA2 clones overlapped in the
region of amino acid residues 318–550 (Fig. 1A).
Amino acids 318–373 of Sla2p are essential for the
Sla2p-Rpg1p interaction. The binding affinity of the
full-length Sla2p hybrid protein (Fig. 1B, pGCE) was
similar to the level observed for the shorter fragments
(Fig. 1A; pHB21, pHB2 and pHB26). To identify residues
essential for the two-hybrid interaction between
Rpg1p and Sla2p, we constructed several truncated
mutants (Fig. 1B). The results from the screen suggested
amino acids 318–550 of Sla2p to be sufficient for
the interaction with full-length Rpg1p protein (Fig.
1A). Deletion of amino acids 318–373 abolished the
enhanced expression of the reporter genes (Fig. 1B,
pGME). In contrast, deletion of any part of the central
coiled-coil domain (aa 376–573) enhanced the interaction
(Fig. 1B, pGLE and pGSE). These results suggested
that the domain formed by amino acids 318–373
is essential for the Sla2p–Rpg1p interaction.
N-terminal part of Rpg1p mediates the interaction
with Sla2p. To identify the Rpg1p domain that participates
in the interaction with Sla2p, truncated versions
of Rpg1p were introduced into L40 cells harboring
pGCE plasmid encoding full-length Sla2p.
Deletions in the evolutionary conserved N-terminal
half (29) abolished expression of the reporter genes
(Fig. 1C, pLCP and pLEP), while deletion of the
C-terminal part (aa 615–968) did not disrupt the interaction
(Fig. 1C, pLBN). These results suggested that
the Rpg1p–Sla2p interaction is mediated by the
N-terminal half of Rpg1p/Tif32p.
Rpg1p interacts with Sla2p in vivo. Rpg1p and
Sla2p were also tested for interaction in cell extracts.
Wild-type, sla2⌬376-501, and sla2⌬114-367 strains
were used for immunoprecipitation of Sla2p by the
antiserum directed against the extreme Sla2p
C-terminal peptide. Rpg1p was followed in these immune
complexes by Western blotting analysis using
anti-Rpg1p antiserum. Rpg1p was coimmunoprecipitated
with Sla2p (Fig. 2, lane 1) but not with the
sla2⌬114-367 (Fig. 2, lane 3). A sixfold higher Rpg1p
level was observed in an immune complex obtained
from the sla2⌬376-501 mutant (Fig. 2, lane 2). The
Sla2p level observed in the sla2⌬376-501 mutant was
similar to the wild-type level (data not shown). These
results from in vivo binding experiments are consistent
with the two-hybrid data.
Rpg1p partially colocalizes with Sla2p. We have
shown recently that Rpg1p is a microtubuleinteracting
protein (26). To compare Rpg1p localization
with Sla2p distribution by immunofluorescence microscopy
the double-labeling procedure was applied using
the antibody against the C-terminal domain of
Sla2p (28). The individual images of the double-labeled
cells (FY1679 strain) are shown in Fig. 3. In the most of
unbudded cells both proteins displayed a punctuate
distribution. They colocalized at the nucleusassociated
patch that probably corresponds to spindle
pole body (Fig. 3A). In some unbudded cells Rpg1p
co-localized with Sla2p in the cortical domain, probably
at the presumptive bud site (Fig. 3B). In budded cells,
Rpg1p is accumulated in a patch at the very end of
cytoplasmic microtubules reaching the bud tip (26). In
these cells, we found that a portion of Sla2p obviously
colocalized with the patch of Rpg1p near the bud tip
(Fig. 3C). In cells of the other cell cycle stages Sla2p
accumulates in the neck region where the actin filaments
are nucleated before cytokinesis (28) whereas
Rpg1p is usually spread through the cytoplasm displaying
colocalization with anaphase spindle microtubules
(26). In these budded cells no obvious colocalization
of both proteins was observed (data not shown).
More information could be obtained by the confocal
laser scan microscopy of double labeled cells.
DISCUSSION
Based on purification and in vivo binding assays it
has been proposed that five yeast proteins (Rpg1p/
Tif32p, Prt1p, Tif34p, Tif35p and Nip1p), homologous
to human eIF3 subunits, are components of the conserved
core of yeast eIF3 (32–34). In this respect, the
Sla2p has not been classified as a stable component of
the eIF3 complex. In agreement with this, Sla2p was
observed to colocalize only partially with Rpg1p at
specific cellular domains. Furthermore, the immunoprecipitated
Sla2p complex contained only a small portion
of the total cellular Rpg1p (J. Palecek, unpublished
data) suggesting that the Rpg1p–Sla2p
interaction is transient. It has been shown that the
coiled-coil domains of Sla2p influence its distribution
between the cytoplasm and the plasma membrane (28)
due to modulation of Sla2p interactions with different
protein complexes (16, 28). Interestingly, genetic manipulation
of the Sla2p coiled-coil domains significantly
increased the affinity of the protein to Rpg1p
(Figs. 1A, 1B, and 2) suggesting that Rpg1p may specifically
associate with some of these complexes to mediate
its SLA2-related function.
Na et al. (35) have studied a specific function of the
SLA2/END4/MOP2 gene product with respect to expression
of Pma1p (the plasma membrane protontranslocating
ATPase). mop2 mutations isolated in a
search for modifier of pma1 (mop) mutants, conferred
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1246
FIG. 1. Domains required for the Rpg1p-Sla2p interaction. The SLA2 sequences (A, genomic library clones; B, the entire coding region and/or
deletion mutated versions) expressed as fusion proteins with Gal4 transcription activation domain are indicated. Both central (aa 376–573) and
second (aa 575–767) coiled-coil domains (hatched), and talin-like domain (black) is indicated in the SLA2 coding region. Plasmids indicated on the
left were introduced into L40 cells harboring either pLBP (wt RPG1) or pBTM116 (control). (C) The RPG1 sequences expressed as lexA fusion
proteins are represented by lines below the map of RPG1. The evolutionarily conserved domain (crosshatched) is indicated. Plasmids indicated on
the left were introduced into L40 cells harboring either pGCE (wt SLA2) or pGAD424 (control). The activities of ␤-galactosidase (units/mg protein)
and the His3 phenotypes were assayed. ϩ, growth. Ϫ, no growth. B, BamHI. C, ClaI. E, EcoRI. N, NdeI. P, PstI.
Vol. 282, No. 5, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
hypersensitivity to the translational inhibitor cycloheximide.
A phenotype similar to that observed for
mop2 mutants was followed with sla2⌬ deletion mutants
defective in actin-related function(s) but not with
a sla2⌬318-373 strain (J. Palecek, unpublished results).
These data suggest that the Rpg1p-Sla2p complex
is not essential for the SLA2-related function in
Pma1p specific processing. Recently, a sla2 mutant
was identified during a screen for mutants exhibiting
defects in the decay of several mRNAs. A role of the
actin cytoskeleton in the decay of specific mRNAs was
proposed in this context (36).
Disruption of the Rpg1p interaction domain of Sla2p
(sla2⌬318-373) is not lethal for cells. In addition, the
sla2⌬318-373 mutant cells exhibit a normal polarization
of the actin cytoskeleton (16) and deletion of even
longer Sla2p fragments did not result in a defective
cellular localization of the protein (28). One can assume
that the interaction between Sla2p and Rpg1p is
neither required for the essential role of Rpg1p in the
initiation of translation nor for actin-nucleation activity
of Sla2p (nor for its localization). Our immunofluorescence
data indicate that the transient contact of
Rpg1p with Sla2p may be involved in the intracellular
localization of the eIF3 complex.
Recent data suggest that Saccharomyces cerevisiae
may employ an analogous mRNA localization mechanisms
as higher eukaryotes (11). The actin cytoskeleton
was proposed to play an active role in generation of
the asymmetric distribution of specific proteins (9, 10)
(e.g., Ash1 protein localizes preferentially to the presumptive
daughter nucleus, where it inhibits matingtype
switching (37)). An interaction of a yeast translation
factor with the actin cytoskeleton (Tef1p–Bni1p)
was hypothesized to assist in the localized translation
of specific ASH1 mRNA (14, 38). Interestingly, at the
time of the revision of the manuscript Lin et al. (39) has
demonstrated a direct physical interaction between a
human Rpg1p homologue and the intermediate filaFIG.
2. Rpg1p/Tif32p can coprecipitate with the Sla2p immune
complex. The Sla2p and associated proteins were precipitated by
antiserum directed against the extreme C-terminal peptide of the
Sla2p protein, from extracts prepared from wild-type (lane 1),
end4⌬376-501 (lane 2), and end4⌬114-367 (lane 3) strains, respectively.
Rpg1 protein was detected by specific antibody on Western
blots and quantified by Image QuaNT software.
FIG. 3. Rpg1p/Tif32p partially colocalizes with Sla2p. The exponentially growing cells (strain FY1679) were fixed and double-stained
with the monoclonal anti-Rpg1p antibody PK1/1 and the polyclonal antibody against C-terminal domain of Sla2p. DAPI was used to visualize
DNA. Arrows point to sites on individual images where both proteins seem to colocalize. Bar, 5 ␮m.
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1248
ment protein cytokeratin 7. Their data support our
idea that the largest subunit of the eIF3 could serve as
a bridging molecule between eIF3 and cytoskeleton. To
test a role of Sla2p–Rpg1p interaction in such processes
(i.e., in a localized translation of ASH1 and/or of
mitochondria-specific mRNAs) additional studies are
required.
ACKNOWLEDGMENTS
This work was supported by Grant S5808 from the Fonds fur
Fo¨rderung der wissenschaftlichen Forschung, Vienna, Austria. J.H.
was funded by Grant 204/99/1531 from GACR, Prague, Czech Republic.
The competent technical assistance by Mirka Matz and
Harald Nierlich is gratefully acknowledged. We also thank Pavel
Kovarik, Leosˇ Vala´sˇek, and Vladimı´r Reiser for helpful advice
throughout this project. We thank P. James and R. Sternglanz for
the two-hybrid vectors and L40 strain, to Andreas Wesp for strains
and plasmids carrying SLA2/END4 alleles and for antiserum
against Sla2/End4 protein.
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