Oncogene (1998) 16, 2011 ± 2016
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Interaction of Rho1p target Bni1p with F-actin-binding elongation factor
1a: implication in Rho1p-regulated reorganization of the actin cytoskeleton
in Saccharomyces cerevisiae
Masato Umikawa, Kazuma Tanaka, Takashi Kamei, Kazuya Shimizu, Hiroshi Imamura,
Takuya Sasaki and Yoshimi Takai
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Osaka, Japan
The RHO1 gene encodes a homolog of mammalian
RhoA small G protein in the yeast Saccharomyces
cerevisiae. We have shown that Bni1p is one of the
downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction
with pro®lin, an actin monomer-binding protein. A
Bni1p-binding protein was anity puri®ed from the
yeast cytosol fraction and was identi®ed to be Tef1p/
Tef2p, translation elongation factor 1a (EF1a). EF1a is
an essential component of the protein synthetic machinery and also possesses the actin ®lament (F-actin)binding and -bundling activities. EF1a bound to the 186
amino acids region of Bni1p, located between the FH1
domain, the proline-rich pro®lin-binding domain, and the
FH2 domain, of which function is not known. The
binding of Bni1p to EF1a inhibited its F-actin-binding
and -bundling activities. The BNI1 gene deleted in the
EF1a-binding region did not suppress the bni1 bnr1
mutation in which the actin organization was impaired.
These results suggest that the Rho1p ± Bni1p system
regulates reorganization of the actin cytoskeleton
through the interaction with both EF1a and pro®lin.
Keywords: Rho; actin cytoskeleton; EF1a
Introduction
The Rho family (Rho) belongs to the small G protein
superfamily and consists of the Rho, Rac, and Cdc42
subfamilies (Hall, 1994; Takai et al., l995). Evidence is
accumulating that, through reorganization of the actin
cytoskeleton, Rho regulates various cell functions, such
as cell shape change, formation of stress ®bers and
focal adhesions, cell motility, membrane ru‚ing,
cytokinesis, cell aggregation, cell-to-cell adhesion, and
smooth muscle contraction (Hall, 1994; Takai et al.,
1995). Rho has two interconvertible forms: GDPbound inactive and GTP-bound active forms. The
GTP-bound form interacts with its speci®c downstream
target and performs its cell functions. Many potential
targets of Rho have been identi®ed, but it has not yet
been clari®ed how Rho regulates reorganization of the
actin cytoskeleton through these targets (Machesky
and Hall, 1996).
Cells of the yeast Saccharomyces cerevisiae grow by
budding for cell division and the actin cytoskeleton
Correspondence: Y Takai
Received 13 October 1997; accepted 14 November 1997
plays a pivotal role in the budding and cytokinesis
processes (Drubin, 1991). The budding yeast possesses
the Rho gene family members, including RHO1,
RHO2, RHO3, RHO4, and CDC42 (Cid et al., 1995).
RHO1 is a homolog of the mammalian RhoA gene and
the rho1 mutants are de®cient in the budding process
(Yamochi et al., 1994). Immuno¯uorescence microscopic study indicates that Rho1p is localized at the
growth site, including the presumptive budding site, the
bud tip, and the cytokinesis site (Yamochi et al., 1994).
These results suggest that Rho1p regulates the
processes of bud formation through reorganization of
the actin cytoskeleton. Concerning the downstream
targets of Rho1p, at least three molecules have thus far
been identi®ed: Pkc1p (Protein kinase C 1), which
regulates the MAP kinase cascade (Nonaka et al.,
1995; Kamada et al., 1996); 1,3-b-glucan synthase,
which is involved in cell wall synthesis (Drgonova et
al., 1996; Qadota et al., 1996); and Bni1p (Bud neck
involved 1), which is involved in reorganization of the
actin cytoskeleton (Kohno et al., 1996). Bni1p has also
been shown to be a potential target of Cdc42p, Rho3p,
and Rho4p (Evangelista et al., 1997; Imamura et al.,
unpublished results). BNI1 and its related genes in
other organisms, including diaphanous (Castrillon and
Wasserman, 1994) and cappuccino (Emmons et al.,
1995) in Drosophila, FigA of Aspergillus (Marhoul and
Adams, 1995), and fus1 of S. pombe (Petersen et al.,
1995), have been shown to be involved in cytokinesis,
the establishment of cell polarity, or normal cell
morphology. These proteins share conserved domains
named FH1 and FH2 (Formin Homology) (Castrillon
and Wasserman, 1994). Very recently, the Rho-Bni1p
system has been shown to be conserved in mammalian
cells and regulate reorganization of the actin cytoskeleton (Watanabe et al., 1997). It has moreover been
shown that Bni1p and Bnr1p, the product of its related
gene, BNR1 (BNI1 Related), regulate reorganization of
the actin cytoskeleton by interacting with pro®lin, an
actin monomer-binding protein, at their proline-rich
FH1 domains (Evangelista et al., 1997; Imamura et al.,
1997). Since pro®lin is implicated in polymerization of
actin (Sohn and Goldschmidt-Clermont, 1994), the
Rho1p ± Bni1p system is likely to be involved in the
regulation of polymerization of actin. However, it
remains to be clari®ed how the Rho1p ± Bni1p system
regulates reorganization of ®lamentous actin (F-actin),
another important process for reorganization of the
actin cytoskeleton.
In this study, we attempted to isolate a Bni1pbinding protein and identi®ed it to be EF1a, known to
be a component of eukaryotic protein synthetic
Bni1p-EF1a interactions
M Umikawa et al
2012
machinery. EF1a has been shown to possess various
biochemical or biological activities, including the Factin-binding and -bundling activities (Demma et al.,
1990; Yang et al., 1990), the microtuble-severing
activity (Shiina et al., 1994), and the activity making
®broblasts highly susceptible to transformation
(Tatsuka et al., 1992). EF1a has furthermore been
shown to be colocalized with the actin cytoskeleton in
mammalian (Edmonds et al., 1996) and amoebae
(Dharmawardhane et al., 1991) cells. However, the
physiological signi®cance of the interaction of EF1a
with F-actin remained obscure. Our results suggest that
EF1a is involved in the Rho1p ± Bnilp-mediated
reorganization of the actin cytoskeleton.
Yang et al., 1990), we further characterized these
Bni1p-Tef1p/Tef2p interactions. For this purpose, we
puri®ed the His10-tagged human EF1a (His10-EF1a)
(Shiina et al., 1994), whose amino acid sequence is
81% identical and 97% homologous to that of yeast
Tef1p/Tef2p. His10-EF1a bound to MBP±Bni1p(1229 ±
1650) as yeast Tef1p/Tef2p did (Figure 2A). The
concentration of His10-EF1a, which gave a halfmaximal binding to MBP-Bni1p(1229 ± 1650), was
about 0.3 mM. The region of Bni1p which bound to
His10-EF1a was delimited and it was found that the 186
amino acids region between the FH1 and FH2 domains
bound to His10-EF1a (Figure 2B and C).
Inhibition by Bni1p of the F-actin-binding and -bundling
activities of EF1a
Results
Isolation of EF1a as a Bni1p-binding protein
A Bni1p-binding protein was anity puri®ed from the
yeast cytosol fraction using maltose-binding protein
(MBP)-Bni1p(1229 ± 1650), which contained the FH1
and FH2 domains, as a ligand. A protein with a Mr of
about 47 kDa was detected as a major band (Figure 1).
This protein was isolated in a large amount, peptides
were generated by proteolytic digestion and amino acid
sequences of two of these peptides were determined.
The sequences perfectly matched with those of Tef1p/
Tef2p, which is EF1a (Schirmaier and Philippsen,
1984). Tef2p is a homolog of Tef1p with 100% amino
acid sequence identity. The predicted Mr of Tef1p/
Tef2p (49.9 kDa) was consistent with the Mr of the
Bni1p-binding protein. We concluded that the Bni1pbinding protein with a Mr of about 47 kDa was Tef1p/
Tef2p.
Since EF1a had been shown to possess F-actinbinding and -bundling activities (Demma et al., 1990;
We next examined the e€ect of Bni1p(1328 ± 1513) on
the F-actin-binding and -bundling activities of EF1a.
The F-actin-binding activity was assayed by cosedimentation with F-actin by ultracentrifugation. The Factin-bundling activity was assayed by two methods:
A
a
b
1 2 3 4 5
1 2 3 4 5
MBP-Bni1p
(1229-1650)
MBP
B
His10
-EF1α
His10
-EF1α
1
2
3
4
5
6
7
C
Mr (kDa)
205
1
2
3
4
116
96
46
MBP-Bni1p
(1229-1650)
*
MBP
29
Figure 1 Isolation of a Bni1p-binding protein from yeast. The
yeast cytosol fraction was applied onto an amylose resin column
which was prebound to MBP or MBP-Bni1p(1229 ± 1650). The
bound proteins were eluted and subjected to SDS ± PAGE (5 ±
18% polyacrylamide gradient gel), followed by protein staining
with Coomassie brilliant blue. Lanes 1 and 2, control bu€er; lanes
3 and 4, the cytosol fraction; lanes 1 and 3, MBP; lanes 2 and 4,
MBP-Bni1p(1229 ± 1650). An asterisk indicates a Bni1p-binding
protein. The other bands observed below MBP-Bni1p(1229 ± 1650)
were degradation products of MBP-Bni1p(1229 ± 1650). The
protein markers used were myosin (Mr=205 000), b-galactosidase
(Mr=116 000), phosphorylase B (Mr=96 000), ovalbumin
(Mr=46 000), and carbonic anhydrase (Mr=29 000)
Figure 2 Binding of Bni1p to recombinant EF1a and mapping of
the EF1a-binding region of Bni1p. (A) binding of Bni1p to
recombinant EF1a. Puri®ed His10-EF1a was applied onto an
amylose resin column which was prebound to MBP or MBPBni1p(1229 ± 1650). The bound proteins were eluted and subjected
to SDS ± PAGE (10% polyacrylamide gel), followed by protein
staining with Coomassie brilliant blue or Western blotting with
the anti-poly-L-histidine antibody. (a) staining with Coomassie
brilliant blue. (b) Western blotting with the anti-poly-L-histidine
antibody. Lanes 1 and 2, control bu€er; lanes 3 and 4, His10EF1a; lanes 1 and 3, MBP; lanes 2 and 4, MBP-Bni1p(1229 ±
1650); lane 5, His10-EF1a as a control (5 pmol). (B) mapping of
the EF1a-binding region of Bni1p. Puri®ed His10-EF1a was
applied onto a glutathione Sepharose 4B column which was
prebound to GST fused with various fragments of truncated
Bni1ps. The bound proteins were eluted and subjected to SDS ±
PAGE (10% polyacrylamide gel), followed by Western blotting
with the anti-His6 antibody. Lane 1, GST-Bni1p(1229 ± 1650); lane
2, GST-Bni1p(1229 ± 1513); lane 3, GST-Bni1p(1328 ± 1513); lane
4, GST-Bni1p(1229 ± 1328); lane 5, GST-Bni1p(1426 ± 1953); lane
6, GST; lane 7, His10-EF1a as a control (5 pmol). (C) schematic
representation of the results in (B)
Bni1p-EF1a interactions
M Umikawa et al
(1) viscometry of F-actin solution; and (2) sedimentation of F-actin by ultracentrifugation. His10-EF1a
increased the viscosity of F-actin solution, but this
activity was inhibited by glutathione S-transferase
(GST)-Bni1p(1328 ± 1513) in a dose-dependent manner
(Figure 3A). The F-actin-bundling activity of a-actinin
was not inhibited by GST-Bni1p(1328 ± 1513) (data not
shown). GST-Bni1p(1328 ± 1513) by itself showed a
negligible e€ect on the viscosity. In the presence of
His10-EF1a, F-actin sedimented with His10-EF1a by
ultracentrifugation at 20 000 g, due to its F-actin
bundling activity (Figure 3B, b). GST-Bni1p(1328 ±
1513), but not GST, inhibited this His10-EF1a-induced
sedimentation of F-actin at 20 000 g (Figure 3B, c and
d). These results indicate that GST-Bni1p(1328 ± 1513)
inhibited the F-actin-bundling activity of EF1a. A
fraction of His10-EF1a was recovered with GSTBni1p(1328 ± 1513) in the 100 000 g supernatant
(Figure 3B, c). Moreover, neither His10-EF1a nor
GST-Bni1p(1328 ± 1513) was detected in the 100 000 g
precipitate, which contained most of the F-actin. These
A
results indicate that GST-Bni1p(1328 ± 1513) inhibited
the F-actin-binding activity of EF1a. We concluded
that the binding of Bni1p to EF1a inhibited both the
F-actin-binding and -bundling activities of EF1a.
Formation of the Bni1p-EF1a-pro®lin ternary complex
We have previously shown that pro®lin binds to the
FH1 domain of Bni1p (Imamura et al., 1997). We
examined whether EF1a, pro®lin, and Bni1p form a
ternary complex. His10-EF1a bound to MBPBni1p(1229 ± 1650) complexed with GST-pro®lin, but
not to GST-pro®lin (Figure 4). This result indicates
that EF1a, pro®lin, and Bni1p form a ternary complex.
Requirement of the EF1a-binding region of Bni1p for the
functions of Bni1p
It should be examined genetically whether the
interaction of Bni1p with Tef1p/Tef2p is of physiological signi®cance. TEF1/TEF2 have not yet been
demonstrated genetically to be involved in the control
of the actin cytoskeleton. However, this would be
dicult to be demonstrated, because a major function
of Tef1p/Tef2p is elongation of a peptide chain in
translation (Schirmaier and Philippsen, 1984). Therefore, we examined the e€ect of deletion of the EF1abinding region of BNI1 on the functions of BNI1. Cells
of the bni1 bnr1 double mutant show temperaturesensitive growth, abnormal morphology and abnormal
distribution of cortical actin patches (Imamura et al.,
1997). pRS316-GAL1-BNI1D(1328 ± 1513), lacking the
EF1a-binding region of BNI1, did not suppress the
temperature-sensitive phenotype (Figure 5). This result
suggests that the interaction of Bni1p with Tef1p/Tef2p
is important for the functions of Bni1p.
A
B
1
2
1
2
B
a
1
2
3
b
1
2
3
His10-EF1α
Actin
MBP-Bni1p
(1229-1650)
c
Actin
1
2
3
d
His10-EF1α
GST-Bni1p
(1328-1513)
1
2
3
His10-EF1α
His10-EF1α
GST-Profilin
GST
Figure 3 Inhibition by Bni1p(1328 ± 1513) of the F-actin-binding
and -bundling activities of EF1a. The F-actin-binding and bundling activities of EF1a were assayed by the two methods in
the presence or absence of EF1a and/or GST-Bni1p(1328 ± 1513).
(A) low shear viscometry. (*), EF1a and GST-Bni1p(1328 ±
1513); (*), EF1a and GST; (~), GST-Bni1p(1328 ± 1513); (~),
GST. (B) sedimentation by ultracentrifugation. (a) control. (b)
EF1a (c) EF1a and GST-Bni1p(1328 ± 1513). (d) EF1a and GST.
Lane 1, 20 000 g precipitate; lane 2, 100 000 g precipitate; lane 3,
100 000 g supernatant
Figure 4 Formation of the Bni1p-EF1a-pro®lin ternary complex.
Puri®ed His10-EF1a was applied onto a glutathione Sepharose 4B
column which was prebound to GST-pro®lin or GST-pro®lin
complexed with MBP-Bni1p(1229 ± 1650). The bound proteins
were eluted and subjected to SDS ± PAGE (10% polyacrylamide
gel), followed by protein staining with Coomassie brilliant blue.
(A) GST-pro®lin. (B) GST-pro®lin complexed with MBPBni1p(1229 ± 1650). Lane 1, control bu€er; lane 2, His10-EF1a
2013
Bni1p-EF1a interactions
M Umikawa et al
2014
Figure 5 Requirement of the EF1a-binding region of Bni1p for
the functions of Bni1p. Cells of HIY11 (bni1 bnr1) were
transformed with pRS316-GAL1 [Vector], pRS316-GAL1-BNI1
[BNI1], or pRS316-GAL1-BNI1D(1328 ± 1513) [BNI1D(1328 ±
1513)]. A transformant obtained from each transformation was
streaked onto a YPGalAU plate medium and incubated for 4
days at 338C
Discussion
In the present and previous (Imamura et al., 1997)
works, we have shown that Bni1p directly interacts with
both pro®lin and EF1a. Bni1p interacts at its FH1
domain with pro®lin (Imamura et al., 1997), which is
implicated in polymerization of actin (Sohn and
Goldschmidt-Clermont, 1994). Pro®lin has been shown
to stimulate the ADP/ATP exchange reaction of actin
(Sohn and Goldschmidt-Clermont, 1994). However, the
interaction of pro®lin with MBP-Bni1p(1229 ± 1650) did
not show any e€ect on this activity (Umikawa et al.,
unpublished results). EF1a bound to the region just
downstream of the FH1 domain of Bni1p, suggesting
that EF1a is required for reorganization of the actin
cytoskeleton regulated by the FH1 domain-pro®lin
action. The formation of the ternary complex composed of Bni1p, pro®lin, and EF1a seems to be
consistent with this possibility. Recently, a novel
protein, Bud6p/Aip3p, has been shown to interact with
the C-terminal region of Bni1p by the two-hybrid
method (Evangelista et al., 1997). The bud6/aip3
mutant shows the phenotypes similar to those of the
bni1 mutant (Zahner et al., 1996). Moreover, Bud6p/
Aip3p has been independently isolated by the twohybrid screening method as a protein which binds to
actin (Amberg et al., 1997). Although biochemical
characterization of Bud6p/Aip3p has not yet been
reported, it is possible that Bud6p/Aip3p is an F-actinbinding protein. EF1a may also be involved in the
bundling of the Bud6p/Aip3p-bound F-actin.
It is currently not known how the Bni1p-EF1a
interactions are regulated. It has been shown that
overexpression of Bni1p lacking the N-terminal,
Rho1p-binding region induces abnormal organization
of the actin cytoskeleton (Evangelista et al., 1997). This
result suggests that Rho1p is involved in the regulation
of the Bni1p-mediated reorganization of the actin
cytoskeleton. It is interesting to examine whether
Rho1p regulates the interaction of Bni1p with pro®lin
and/or EF1a. For this purpose, we have attempted to
express and purify full-length Bni1p in yeast, insect,
and mammalian cell systems. However, we have not
yet succeeded in purifying it due to its insolubility and
proteolytic degradation.
Our genetic result suggests that the interaction of
Bni1p with EF1a is important for the functions of
Bni1p. EF1a has recently been isolated as a gene whose
overexpression causes aberrant cell morphology in the
®ssion yeast Schizosaccharomyces pombe (D Hirata,
personal communication). This result genetically
indicates that EF1a is involved in the cytoskeletal
control in ®ssion yeast. It is possible that this
phenotype is caused by perturbation of the function
of a Bni1p-like protein in S. pombe.
Another intriguing hypothesis for the physiological
signi®cance of the Bni1p-EF1a interactions is that Bni1p
localizes EF1a to regulate translation of speci®c
mRNAs in speci®c areas of cells. In this respect, it
should be noted that the bni1 mutation has been isolated
as a mutation, she5, which is de®cient in daughter cellspeci®c localization of Ash1p (Jansen et al., 1996).
Ash1p is a repressor of the HO gene, which is speci®cally
transcribed in mother cells. Recently, the ASH1 mRNA
has been shown to be speci®cally localized in daughter
cells (Long et al., 1997). Therefore, it is possible that
EF1a bound to Bni1p is responsible for the translation
of the ASH1 mRNA in daughter cells. The localization
of speci®c mRNAs has been shown to play an important
role in generation of cell polarity (Bassell and Singer,
1997). The Rho1p-Bni1p system may regulate reorganization of the actin cytoskeleton and/or translation of
speci®c mRNAs by interacting with EF1a.
Materials and methods
Yeast and E. coli strains and growth conditions for yeast cells
Yeast strains, BJ5457 (MATa ura3-52 leu2D1 trp1 his3D200
lys2-801 pep4::HIS3 prb1D1.6R can1) and HIY11 (MATa
ura3 leu2 trp1 his3 ade2 bni1::HIS3 bnr1::URA3), were
used for isolation and puri®cation of a Bni1p-binding
protein and functional study of the bni1 mutant gene,
respectively. An E. coli strain DH5a was used for
construction and propagation of plasmids and purification
of recombinant proteins. Yeast strains were grown in rich
medium that contained 2% Bacto-peptone (Difco Laboratories, Detroit, MI), 1% Bacto-yeast extract (Difco),
0.04% adenine sulfate, and 0.02% uracil and 2% glucose
(YPDAU) or 3% galactose and 0.2% sucrose (YPGalAU).
Yeast transformations were performed by the lithium
acetate methods (Gietz et al., 1992). Transformants were
selected on SD medium that contained 2% glucose and
0.7% yeast nitrogen base without amino acids (Difco) and
amino acids were supplemented to SD medium when
required as described (Sherman et al., 1986).
Isolation and puri®cation of a Bni1p-binding protein
All manipulations were carried out at 0 ± 48C. Yeast cells of
BJ5457 (30 g wet weight) were suspended in 60 ml of Bu€er
A (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM
dithiothreitol (DTT), 10 mg/ml of leupeptin, 20 mg/ml of
aprotinin, 10 mM (p-amidinophenyl) methanesulfonyl fluoride and 1 mg/ml of pepstatin A). The cell suspension was
homogenized with 30 ml of glass beads (0.45-mm diameter).
The homogenate was centrifuged at 1700 g for 10 min and
the supernatant was further centrifuged at 100 000 g for 1 h.
The resulting supernatant (60 ml, 700 mg of protein) was
subjected to ammonium sulfate fractionation. The 50 ± 80%
precipitate was dissolved in 10 ml of Bu€er A and the
sample was dialyzed against Bu€er A. One ml of the
dialyzed sample was applied onto an amylose resin column
which was prebound to 30 mg of MBP-Bni1p(1229 ± 1650).
After the column was washed three times with 20 ml of
Bu€er B (20 mM Tris/HCl at pH 7.5, 1 mM EDTA and
1 mM DTT), the bound proteins were eluted with 300 ml of
Bu€er B containing 10 mM maltose. The eluted proteins
were subjected to SDS ± PAGE, followed by protein staining
with Coomassie brilliant blue.
Bni1p-EF1a interactions
M Umikawa et al
Peptides sequencing of a Bni1p-binding protein
The protein band of Mr of 47 kDa (about 100 mg) of the
Bni1p-binding proteins was cut out from the gel and
digested with Achromobacter protease I, and the digested
peptides
were
separated
by
TSKgel
ODS-80Ts
(0.46615 cm, Tosoh) reverse phase high-performance
liquid column chromatography as described (Imazumi et
al., 1994). The amino acid sequences of the two peptides
were determined with a peptide sequencer (HP G1005A
protein sequencing system, HP). The peptide sequences
determined
were
GWEK
and
SVEMHHEQLEQGVPGDNVGF.
Materials and chemicals for biochemical assays
Globular actin (G-actin) was prepared from rabbit muscle
as described (Pardee and Spudich, 1982). Recombinant
proteins used in this study were puri®ed from overexpressing E. coli. His10-EF1a was puri®ed as described
(Shiina et al., 1994). Bni1p(1229 ± 1650) was puri®ed as a
MBP fusion protein using an amylose resin column (New
England BioLabs, Inc., Beverly, MA) as described (Guan
et al., 1987). Human pro®lin (Imamura et al., 1997),
Bni1p(1229 ± 1650), Bni1p(1229 ± 1513), Bni1p(1328 ± 1513),
Bni1p(1229 ± 1328), and Bni1p(1416 ± 1953) were puri®ed as
GST fusion proteins using a glutathione Sepharose 4B
column (Pharmacia P-L Biochemicals Inc., Milwaukee,
WI) as described (Kikuchi et al., 1992). The anti-poly-Lhistidine antibody was purchased from Santa Cruz
Biotechnology, Inc., Santa Cruz, CA.
Assay for the binding of recombinant EF1a to recombinant
Bni1ps
Puri®ed His10-EF1a (500 pmol) in 500 ml of Bu€er B
containing 150 mM NaCl was loaded onto an amylose
resin column which was prebound to MBP or MBPBni1p(1229 ± 1650) (600 pmol) or a glutathione Sepharose
4B column which was prebound to GST, GST-Bni1p(1229 ±
1650), GST-Bni1p(1229 ± 1513), GST-Bni1p(1328 ± 1513),
GST-Bni1p(1229 ± 1328), or GST-Bni1p(1426 ± 1953) (600
pmol). Each column was then washed with 20 column
volumes of Bu€er B containing 150 mM NaCl. MBPBni1p(1239 ± 1650) or MBP was eluted with 300 ml of
Bu€er B containing 10 mM maltose, whereas the GSTBni1p samples were eluted with 300 ml of Bu€er B
containing 10 mM reduced glutathione. An aliquot (30 ml)
of each eluate was subjected to SDS ± PAGE, followed by
protein staining with Coomassie brilliant blue or Western
blotting with the anti-poly-L-histidine antibody as described
(Imamura et al., 1997) with the ECL Western blotting
detection system (Amersham Corp., Arlington Heights, IL).
Assay for the formation of the Bni1p-EF1a-pro®lin ternary
complex
Puri®ed His10-EF1a (500 pmol) in 500 ml of Bu€er B
containing 150 mM NaCl was loaded onto a glutathione
Sepharose 4B column which was prebound to GST-pro®lin
(500 pmol) or the GST-pro®lin-MBP-Bni1p(1229 ± 1650)
complex (100 pmol), which was prepared as described
(Imamura et al., 1997). Each column was then washed
with 20 column volumes of Bu€er B containing 150 m M
NaCl. GST-Pro®lin was eluted with 300 ml of Bu€er B
containing 10 mM reduced glutathione. An aliquot (50 ml)
of each eluate was subjected to SDS ± PAGE, followed by
protein staining with Coomassie brilliant blue.
Low shear viscometry
Low shear viscosity was measured as described (Pollard and
Cooper, 1982; Kato et al., 1996). Brie¯y, His10-EF1a
(0.5 mM) was mixed with G-actin (0.3 mg/ml) in the
presence of GST-Bni1p(1328 ± 1513) or GST (0 ± 1 mM) in
100 ml of a bu€er containing 20 mM Tris/HCl at pH 7.2,
56 mM piperazine-N,N'-bis(2-ethanesulfonic acid)/NaOH at
pH 6.8, 2.0 mM MgCl2, 1.0 mM EGTA, and 0.1 mM ATP,
and the mixture was sucked into a 0.1-ml micropipette. After
the incubation for 30 min at 258C, the time for a stainless
steel ball to fall a ®xed distance in the pipette was measured.
Assay for sedimentation of F-actin
G-Actin was polymerized by incubation for 30 min at
room temperature in a polymerization bu€er containing
20 mM imidazole/HCl at pH 7.0, 2 mM MgCl2, and 1 mM
ATP. Puri®ed recombinant His10-EF1a (1 mM) and/or
Bni1p(1328 ± 1513) (1 mM) was mixed with F-actin
(0.3 mg/ml) and incubated for 60 min at 48C in 300 ml of
a bu€er containing 56 mM piperazine-N,N'-bis(2-ethanesulfonic acid)/NaOH at pH 6.8, 6 mM imidazole/HCl at
pH 7.0, 1.3 mM MgCl2, 0.7 mM EGTA, and 0.3 mM ATP.
The mixture was then centrifuged at 20 000 g for 30 min at
48C. The supernatant (20 000 g supernatant) was removed,
and the precipitate (20 000 g precipitate) was carefully
washed with the same bu€er. The 20 000 g supernatant was
then centrifuged at 100 000 g for 30 min at 48C. The
100 000 g supernatant and the solubilized samples of the
20 000 g and 100 000 g precipitates were subjected to SDS ±
PAGE, followed by protein staining with Coomassie
brilliant blue.
Molecular biological techniques and construction of plasmids
Standard molecular biological techniques were used for
construction of plasmids, DNA sequencing and polymerase
chain reaction (PCR) (Sambrook et al., 1989). DNA
sequences were determined using ALFred DNA sequencer
(Pharmacia Biotech, Inc., Uppsala, Sweden) and PCRs were
performed using GeneAmp PCR System 2400 (PerkinElmer, Norwalk, CT). Various DNA fragments encoding
truncated Bnilps were generated by PCR and inserted into
pGEX-4T-2-HA (Imamura et al., 1997) and pMAL-c2 (New
England BioLabs, Inc.) for the expression of proteins fused
to GST and MBP, respectively. pET16b-EF1a (Shiina et al.,
1994) was used for the expression of His10-EF1a. pRS316GAL1-BNI1D(1328 ± 1513) was constructed as follows: a
BNI1 DNA fragment deleted in the region from 3985 ± 4540
(codon 1328 to codon 1513) of the BNI1 open reading frame
was generated by the PCR mutagenesis methods (Higuchi,
1989). This fragment was cloned into a yeast vector,
pRS316-GAL1, to express the BNI1 deletion mutant gene
under the control of the GAL1 promoter.
Other procedures
SDS ± PAGE was performed as described (Laemmli, 1970).
Protein concentrations were determined as described with
bovine serum albumin as a reference protein (Bradford,
1976).
Acknowledgements
We thank E Nishida (Kyoto University) for valuable
discussions and pET16b-EF1a, S Tsukita (Kyoto University) for valuable discussions, and D Hirata (Hiroshima
University) for valuable discussions and personal communication. This investigation was supported by grants-in-aid
for Scienti®c Research and for Cancer Research from the
Ministry of Education, Science, Sports, and Culture, Japan
(1996, 1997), by grants-in-aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from
the Ministry of Health and Welfare, Japan (1996, 1997) and
by grants from the Human Frontier Science Program (1996,
1997) and the Uehara Memorial Foundation (1996).
2015
Bni1p-EF1a interactions
M Umikawa et al
2016
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