From the Section Yeast Genetics, Institute of
Molecular Plant Sciences, Leiden University, Wassenaarseweg 64,
2333 AL Leiden, § Department of Parasitology,
Leiden University Medical Center, Albinusdreef 2, 2333 ZA
Leiden, ¶ Department of Molecular Cell Biology, Leiden University
Medical Center, Wassenaarseweg 72, 2333 AL Leiden, and
Kluyver
Laboratory for Biotechnology, Delft University of Technology,
Julianalaan 67, 2628 BC Delft, The Netherlands
Received for publication, December 9, 2002, and in revised form, January 10, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Fin1 protein of the yeast
Saccharomyces cerevisiae forms filaments between the
spindle pole bodies of dividing cells. In the two-hybrid system it
binds to 14-3-3 proteins, which are highly conserved proteins involved
in many cellular processes and which are capable of binding to more
than 120 different proteins. Here, we describe the interaction of the
Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail.
Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This
interaction is strongly reduced after dephosphorylation of Fin1p.
Surface plasmon resonance analysis showed that Fin1p has a higher
affinity for Bmh2p than for Bmh1p (KD 289 versus 585 nM). Sequences in both the central
and C-terminal part of Fin1p are required for the interaction with
Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the
14-3-3 proteins are not required for this process. Fin1 also interacts
with itself in the two-hybrid system. For this interaction sequences at
the C terminus, containing one of two putative coiled-coil regions, are
sufficient. Fin1p-Fin1p interactions were demonstrated in
vivo by fluorescent resonance energy transfer between cyan
fluorescent protein-labeled Fin1p and yellow fluorescent
protein-labeled Fin1p.
The Saccharomyces cerevisiae FIN1 gene encodes a
protein of 291 amino acids with a predicted pI of 10 that contains two
putative coiled-coil regions at its C terminus. We showed recently (1) that the subcellular localization of the
GFP1-Fin1 protein is highly
cell cycle-dependent (1). In large budded cells the protein
is visible as a filament between the two spindle pole bodies. In
resting cells the protein is undetectable, and in small budded cells it
is localized in the nucleus. During late mitosis it localizes on the
spindle pole bodies. Purified His6-tagged Fin1p
self-assembles into filaments with a diameter of ~10 nm after
dialysis against low salt buffers as is observed for other intermediate
filament-forming proteins. The Fin1 protein is an interaction partner
of the yeast 14-3-3 protein Bmh2 (1).
The 14-3-3 proteins form a family of highly conserved acidic dimeric
proteins that are present, often in multiple isoforms, in all
eukaryotic organisms (for review see Refs. 2-6). They bind to more
than 120 different proteins and play a role in the regulation of many
cellular processes, including signaling, cell cycle control, apoptosis,
exocytosis, cytoskeletal rearrangements, transcription, and regulation
of enzymes. Although the exact function of the 14-3-3 proteins is still
not completely understood, three main mechanisms seem to be important.
First, 14-3-3 proteins positively or negatively regulate the activity
of enzymes; second, 14-3-3 proteins may act as localization anchors,
controlling the subcellular localization of proteins; and third, 14-3-3 proteins can function as adaptor molecules or scaffolds, thus
stimulating protein-protein interactions. Binding motifs have been
identified in a number of proteins that bind to the 14-3-3 proteins.
Many of these binding motifs consist of a phosphorylated serine
residue, flanked by a proline and arginine residue (7-9). However,
other 14-3-3 binding motifs have been identified that are seemingly
unrelated and do not contain a phosphoserine (10, 11).
The yeast S. cerevisiae has two genes,
BMH1 and BMH2, encoding 14-3-3 proteins (12-15).
A bmh1 bmh2 disruption is lethal in most but not all
laboratory strains, and the lethal bmh1 bmh2 disruption can
be complemented by at least four of the Arabidopsis isoforms
and by a human and Dictyostelium isoform (16). As in higher
eukaryotes, the S. cerevisiae 14-3-3 proteins are involved in many cellular processes, and many different binding partners have
been identified (14), including the protein kinases Ste20p (17) and
Yak1p (18) and the transcription factors Rtg3 (19), Msn2, and Msn4
(20). Recently, it has been shown (21) that the yeast 14-3-3 proteins
bind to cruciform DNA.
Here, we describe the two-hybrid screen identifying the Fin1 protein as
an interaction partner of the Bmh2 protein and detailed studies on the
interaction between of the Fin1 protein and the yeast 14-3-3 proteins.
In addition, the self-association of Fin1p was studied using both the
yeast two-hybrid system and fluorescence resonance energy transfer in
intact yeast cells.
Strains, Culture Media, and Plasmids--
S.
cerevisiae HF7c (22) was used as reporter strain in two-hybrid
screens. S. cerevisiae strain GG3100 is CEN-PK113-5D
(MATa ura3-52; P. Kötter, Göttingen, Germany)
containing plasmid pRUL182. The bmh1 bmh2 strain RR1249 and
the isogenic control strain F3 Nucleic Acid Manipulations--
Nucleic acid manipulations were
performed by standard techniques. The QIAprep mini spin kit (Qiagen)
was used to isolate plasmid DNA from E. coli. The same kit
was used to purify plasmid DNA from S. cerevisiae, after
yeast cells were incubated with 1 mg/ml lyticase in buffer P1 (Qiagen)
for 30 min. Subsequently, the isolated plasmid DNA was amplified in
E. coli XLI-blue.
Two-hybrid Screen--
Library Y2HL (22), consisting of
0.5-3-kb fragments of genomic DNA fused to the GAL4ad, was
a gift of Philip James. Strain HF7c was first transformed (23) with
pRUL551 and subsequently with 50 µg of each of the libraries Y2HL-C1,
Y2HL-C2, and Y2HL-C3. Transformants with an activated HIS3
reporter were selected on MY medium supplemented with adenine (20 µg/ml), lysine (30 µg/ml), and 20 mM 3-aminotriazole
and subsequently assayed for Purification of Bmh1p and Bmh2p--
His6-tagged
recombinant Bmh1p and Bmh2p were purified from E. coli XLI
containing pRUL526 and pRUL527, respectively. Cells were grown to an
A620 of 0.25 in TB supplemented with ampicillin. After addition of
isopropyl-1-thio- Purification of His6-Fin1p--
S.
cerevisiae strain GG3100 was grown in 500 ml of MY medium to an
A620 of 0.8. Cells were harvested, washed, and
cultured for 16 h in 500 ml of MYZ medium supplemented with 1%
galactose to induce the expression of the recombinant protein. Cells
were harvested by centrifugation, and the pellet (3.8 g) was vortexed with 4 g of glass beads (600 µm; Sigma) in 40 ml of lysis buffer (8 M urea, 50 mM Tris-HCl, pH 8, 500 mM NaCl, 2 mM 2-mercaptoethanol) containing 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A, 2 µg/ml chymostatin, 1 mM Enzyme-linked Immunosorbent Assay (ELISA)--
Microtiter plates
(Flow Laboratories) were coated overnight at 4 °C with varying
amounts of purified His6-Fin1p or cytochrome c
(Sigma) in 50 µl of PBS (50 mM phosphate buffer, pH 7.5, 150 mM NaCl). After washing with 200 µl of PBS, the wells
were blocked with 200 µl of 1% blocking reagent (Roche Molecular
Biochemicals) in PBS for 90 min at room temperature. When
desired, bound Fin1p was dephosphorylated by incubation with 100 units
of Surface Plasmon Resonance Measurements--
Surface plasmon
resonance analysis was done with a BIAcore-3000. 2000 resonance units
of Fin1p were coupled to a CM5 sensor chip (BIAcore) by the
N-hydroxysuccinimide/N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (NHS/EDC) method (25). Fin1p was dialyzed
against 2.5 mM phosphate buffer, pH 7.5, 25 mM
NaCl. The reference channel was made by activating and deactivating the
surface without coupling a protein. Measurements were done in
HBS buffer (BIAcore) at 25 °C at a flow rate of 30 µl/min.
Each run consisted of an association step of 5 min using either
His6-Bmh1p or His6-Bmh2p dissolved in HBS,
followed by a dissociation step with HBS buffer. Between runs the chip
was regenerated with 10 mM glycine, pH 10. Analysis of
kinetic data was performed with the BIAevaluate 3.0 software (BIAcore)
using the 1:1 (Langmuir) binding fitting model. Simulations with
experimentally found kinetic parameters were done with BIAsim (BIAcore)
and Clamp software (26).
FRET Analysis--
Yeast cells were cultured in MY medium, and
exponentially growing cells were analyzed using a Zeiss Axiovert 135 TV
microscope (Zeiss, Jena, Germany), equipped with a 100-watt mercury
lamp and a ×100/NA 1.30 Plan Neofluar phase objective. YFP was
detected with a filter set consisting of a 500/20-nm bandpass filter, a 515-nm dichroic mirror, and a 535/30-bandpass emission filter. For FRET
images the emission filter of the YFP filter set was used in
combination with the CFP excitation filter and dichroic mirror. CFP was
detected with a filter set consisting of a 436/20-nm bandpass
excitation filter, a 455-nm dichroic mirror, and a 480/20-nm bandpass
emission filter (all from Chroma Technologies). Images were acquired
using a cooled CCD camera (Princeton Instruments), in the sequence
YFP-, FRET-, and CFP-image (27). Typical exposure times were 0.2 s. The set of three images was corrected for background and, when
necessary, for pixel shift. Overlap from the CFP and YFP into the FRET
image was corrected at each pixel. Images were further processed with
in-house software. For calculation of relative FRET values the formula,
FRETC = (IFRET Identification of Fin1p as a Binding Partner for Bmh2p--
To
identify binding partners of the yeast 14-3-3 protein Bmh2 we made use
of the two-hybrid system. To facilitate analysis of positive clones, we
constructed "bait" and "prey" vectors with a different
selection marker. Therefore, we replaced the ampicillin resistance
marker of bait vectors pGBD-C1, pGBD-C2, and pGBD-C3 (22) by the
kanamycin resistance marker. Then, we constructed plasmid pRUL551
encoding a fusion between Bmh2p and Gal4bd. This construct was able to
complement a lethal bmh1 bmh2 disruption, indicating that
the fusion protein has retained (at least) its essential 14-3-3 function (data not shown). pRUL551 alone already activated the
HIS3 reporter of HF7c, but this could be suppressed by
addition of 20 mM 3-aminotriazole. Of 22 × 106 transformants, five had both an active HIS3
and
We isolated plasmids from the His+
In Vitro Interaction between the Yeast 14-3-3 Proteins and
Fin1p--
To confirm the interaction between the 14-3-3 proteins and
Fin1p found in the two-hybrid system, we used a sandwich ELISA with
purified proteins. To this end we isolated recombinant
His6-tagged Bmh1 and Bmh2 proteins from E. coli
and His6-tagged Fin1p from yeast (see "Materials and
Methods"). Various amounts of purified Fin1p (0-200 ng) were
immobilized to the wells of a microtiter plate. After blocking and
washing, these wells were incubated with Bmh1p or Bmh2p, and the amount
of Bmh protein bound was determined immunologically using our
anti-14-3-3 protein antiserum (13). Fig.
1 shows that increasing the amount of
immobilized Fin1p resulted in increased binding of Bmh1 and Bmh2p ( Surface Plasmon Resonance Analysis--
The dissociation constants
of the Fin1p-Bmh1p and Fin1p-Bmh2p interactions were determined by
surface plasmon resonance analysis. To this end 2000 resonance units of
Fin1p were covalently immobilized to the surface of a CM5 sensor chip,
and solutions with various concentrations of Bmh1p or Bmh2p were
injected over the chip. A relatively fast binding of Bmh1p or Bmh2p to
Fin1p was observed, whereas dissociation was relatively slow (Fig.
2). Dissociation was not affected by
addition of 2 M NaCl, 2 M KCl, or 2 M LiCl to the buffer, indicating that the Bmh-Fin1p
interaction is resistant to high salt concentrations (data not shown).
Using the 1:1 Langmuir binding model, we calculated for Bmh1p an
association rate constant (ka) of 1.85 × 103 M Effect of Dephosphorylation on the Bmh2p-Fin1p
Interaction--
Western blot analysis with an antibody recognizing
phosphorylated serine, threonine, and tyrosine residues suggested that Fin1p is a (partly) phosphorylated protein (data not shown). To study
the role of Fin1p phosphorylation in the interaction with the 14-3-3 proteins, increasing amounts of Fin1p (0-250 ng) were immobilized to
the wells of a microtiter plate, and the immobilized Fin1p in one
series of wells was treated with the nonspecific Fin1p Filament Formation in Cells Deficient in 14-3-3 Proteins--
The Fin1 protein forms cell cycle-specific filaments
between the spindle pole bodies of dividing yeast cells. To study the role of the 14-3-3 proteins in Fin1p filament formation we made use of
the observation that in Self-association of Fin1p--
Transformation of reporter strain
HF7c with plasmids pRUL348 and pRUL558 encoding fusions between of
Fin1p (amino acids 11-291) with Gal4ad and Gal4bd, respectively,
yielded transformants that were able to grow on medium without
histidine and that displayed Mapping of the Fin1p Domains Responsible for Self-association and
Bmh2p Binding--
To map the regions of Fin1p that are involved in
self-association and in the binding to the 14-3-3 proteins, plasmids
were constructed that expressed in-frame fusions of GAL4ad
with various parts of FIN1 (Fig.
5). The N terminus of Fin1p up to amino
acid 253 could be deleted without affecting the interaction with
Gal4bd-Fin1p (Fig. 5). On the other hand, deletion of the C-terminal
part of Fin1p completely abolished the interaction with Gal4bd-Fin1p
(Fig. 5). A fusion containing amino acids 266-282 was unable to
interact with Gal4bd-Fin1p. In contrast, a Gal4ad fusion protein
containing amino acids 254-280 of Fin1p (pRUL369) was able to interact
with both Gal4bd-Fin1p and with Gal4bd fused to amino acids 254-280 of
Fin1p. These data indicate that amino acids 254-280 of the C-terminal
part of Fin1p are required for self-association and that this region
alone is sufficient for this self-association. This region contains one
of the two putative coiled-coil regions present in the Fin1 protein,
suggesting that this coiled-coil region is involved in the
self-association. The interaction with Bmh2p was also completely
abolished after deletion of the C-terminal sequences of Fin1p. The
interaction was not disturbed after deletion of the first 56 amino
acids from the N terminus of Fin1p but was abolished after deletion of
the first 58 amino acids from the N terminus, indicating that sequences
in this part of the Fin1 protein are involved in 14-3-3 binding. This
part of the protein does not contain sequences resembling known 14-3-3 binding sequences. As 14-3-3 binding to Fin1p was affected by
dephosphorylation, we replaced the threonine residue at position 58 by
a valine residue. However, binding to 14-3-3 still occurred (data not
shown). Our results suggest that both sequences around residue 58 and
the C-terminal part of Fin1p are required for association with Bmh2p. Alternatively, association with Bmh2p might require a dimeric Fin1p. In
that case, deletion of the C terminus disrupts the Fin1p self-association, and deletion of sequences around residue 58 might
remove the Bmh2p-binding domain.
FRET between CFP- and YFP-tagged Fin1 Proteins--
FRET
measurements were used to study the self-association of Fin1p in the
intact yeast cell. To this end strain GG3066 was constructed expressing
both CFP-tagged Fin1p and YFP-tagged Fin1p. Both labeled Fin1 proteins
behaved similarly as we reported for GFP-Fin1p (1). In the dividing
cell shown in Fig. 6A, both CFP-Fin1p as YFP-Fin1p were present in the spindle pole bodies. After
correction for the spectral overlap of CFP and YFP, a clear energy
transfer from the CFP-tagged Fin1p to the YFP-tagged Fin1p could be
observed in the spindle pole bodies. In the dividing cell shown in Fig.
6B, both CFP-Fin1p and YFP-Fin1p were present in a filament
between the spindle bole bodies. Again energy transfer was observed,
although less than in the spindle pole bodies. No significant energy
transfer was observed in cells expressing both free CFP and YFP (Fig.
6C).
In this study we performed a two-hybrid screen to identify
interaction partners of the Bmh2 protein. One of the binding partners was the Fin1 protein. While the manuscript was in preparation interaction between Fin1p and the yeast 14-3-3 proteins has also been
reported by others (29). Fin1p is a 291-amino acid protein of unknown
function with a pI of 10 that contains two putative coiled-coil regions
in the C terminus. The protein is ~45% similar to myosins and
tropomyosins from various organisms. In addition, it shares ~45%
similar residues with parts of the kinesin-like proteins Kar3p from
S. cerevisiae and human CENP-E and CENP-F. We showed
recently (1) that Fin1p form cell cycle-specific filaments between the
spindle poles of dividing cells. In vitro, the protein can
form 10-nm filamentous structures resembling the filaments formed by
other intermediate filament proteins. Here, we showed that in the
two-hybrid system sequences at the C terminus of the Fin1 protein
containing one of the two putative coiled-coil regions are sufficient
for self-association (Fig. 5). In addition, we showed FRET between
CFP-Fin1p and YFP-Fin1p in intact in yeast cells (Fig. 6). The function
of the Fin1p filaments is still unknown. However, in the two-hybrid
assay Fin1p interacts not only with the 14-3-3 proteins and Fin1p
itself but also with Reb1p, Fir1p, and Wss1p (1). Both the Reb1 and
Fir1 proteins are involved in RNA metabolism. The Reb1 protein is a
DNA-binding protein required for termination of RNA polymerase I
transcription (30, 31), and the Fir1 protein (32) has been identified
as a factor interacting with the Ref2 protein, which is involved
in 3' mRNA processing (33). Wss1p is a weak suppressor of a
SMT3 mutation, a gene that may be involved in the function
and/or structure of the kinetochore (34). In a large scale search for
protein-protein interactions the Fin1 protein was found to interact
with the Glc7 protein phosphatase (35).
The yeast S. cerevisiae is widely used as model organism for
studies on cellular processes. We use this yeast to elucidate the
function of the 14-3-3 proteins. 14-3-3 proteins are present in all
eukaryotic organisms investigated, and more than 120 interaction partners have been identified (for review see Refs. 2-6). These interaction partners belong to many different classes of proteins, such
as protein kinases, protein phosphatases, receptors, transcription factors, filament-forming proteins, and enzymes. In this study we
identified the filament-forming Fin1 protein as an interaction partner
of the yeast 14-3-3 proteins. Previously, the protein kinase Ste20p and
Yak1p and the transcription factors Msn2p, Msn4p, and Rtg3p have been
identified as interaction partners for the yeast 14-3-3 proteins,
whereas a large scale analysis of yeast protein-protein interactions
(35) revealed interactions with the extracellular membrane protein
Ecm13p, with the inositolhexaphosphate kinase Kcs1p and with Bop3p,
overexpression of which can bypass mutations in the phosphatase
suppressor gene PAM1. Thus also in the relatively simple
yeast cell the 14-3-3 proteins have different interaction partners,
belonging to different classes of proteins.
In a large number of proteins interacting with 14-3-3 proteins specific
binding motifs have been identified. Many of these motifs contain a
phosphorylated serine residue flanked by a proline and arginine residue
(7-9). The Fin1 protein does not contain any of these
phosphoserine-containing motifs, nor does it contain any of the
previously defined 14-3-3 binding motifs lacking a phosphorylated
serine residue (10, 11). However, phosphorylation of the Fin1 protein
is involved in 14-3-3 binding. Deletion studies suggested that both
sequences around residue 58 and in the C-terminal part of Fin1p are
required for association with Bmh2p. These results may imply that
multiple sites are responsible for 14-3-3 binding, that a dimeric Fin1
protein is required, or that a truncated Fin1 protein does not fold correctly.
Using surface plasmon resonance analysis we have determined the
dissociation constants of the Fin1p-Bmh1p and Fin1p-Bmh2 interactions. These dissociation constants are in the same range as those published for other 14-3-3 binding partners (Table
III). We showed that Bmh2p has a higher
affinity than Bmh1p for Fin1p. However, Bmh1p and Bmh2p are for 97%
identical over the first 250 amino acids. Therefore, any isoform
specificity probably resides in the divergent C termini. The
physiological significance of this difference in affinity remains to be
elucidated. In the yeast cell the difference in affinities might be
counterbalanced by an ~5-fold higher Bmh1p concentration than Bmh2p
concentration.2 In
literature mainly qualitative examples of isoform specificity can be found. Muslin et al. (7) found little differences in the affinity constants for the interaction between a number of 14-3-3 isoforms (
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
were obtained from Dr. G. R. Fink (MIT, Cambridge, MA) (17). GG3066 expressing both CFP-Fin1 and
YFP-Fin1, was constructed by transformation of CENPK113-3B
(MAT
ura3-52 his3; P. Kötter, Göttingen, Germany) with the plasmids pRUL1005 (1) and pRUL1008. The control strain GG3087 expressing free CFP and free YFP was constructed by transformation of CENPK113-3B with the plasmids pRUL1001 (1) and pRUL1004. Escherichia coli
(strain XL1-blue) and yeast were cultured as described before
(13). The plasmids constructions are listed in Table
I. The sequences of the oligonucleotides used are shown in Table II.
Plasmid constructions
Oligonucleotide sequences
-galactosidase activity. Library
plasmids were isolated from His+
-galactosidase+ transformants by amplification in
E. coli after selection for ampicillin resistance.
-D-galactopyranoside to a final
concentration of 1 mM, the cells were grown for an
additional 2.5 h to an A620 of 0.82. Two
grams (wet weight) of cells were harvested by centrifugation,
resuspended in 15 ml of 50 mM sodium phosphate buffer, pH
7.8, containing 300 mM NaCl, 1 mg/ml lysozyme, 10 µg/ml
RNase A, and 10 µg/ml DNase I and incubated for 30 min at 0 °C.
Then Triton X-100 was added to a final concentration of 0.1%, and the
cells were incubated on ice for an additional 10 min. The lysate was
cleared by two centrifugation steps at 13,000 × g for
10 min at 4 °C. One ml of nickel-nitrilotriacetic acid-agarose
suspension (Qiagen) was added to the lysate, and after a 90-min
incubation at 4 °C under continuous stirring the mixture was poured
into a column. The column was washed with 100 ml of 50 mM
phosphate buffer, pH 7.8, 300 mM NaCl, at 20 ml/h, followed
by washing with 55 ml of Buffer B (50 mM phosphate buffer, pH 6.5, 300 mM NaCl, 10% glycerol). The
His6-tagged proteins were eluted with a 40-ml 0-500
mM gradient of imidazole in Buffer B. Fractions containing
the purified His6-Bmh1p or His6-Bmh2p were dialyzed against 50 mM phosphate buffer, pH 7.5, 50%
glycerol, 300 mM NaCl. Protein concentrations were
determined by Bradford analysis (24), and purity was estimated by
SDS-PAGE followed by Coomassie and silver staining. The purified
proteins were stored at
80 °C.
-aminocaproic acid, 1 µg/ml
E-64, 1 µg/ml leupeptin, 2 µg/ml aprotonin, and 1 mM
Na3VO4. Cell debris was removed by
centrifugation for 10 min at 13,000 × g, and the pH of
the clear supernatant was adjusted to 8.0 with NaOH. The extract was
incubated with 500 µl of nickel-nitrilotriacetic acid-agarose (Qiagen) for 16 h at room temperature. All washing and elution steps were performed in spin columns. The nickel-nitrilotriacetic acid-agarose was washed six times with 0.5 ml of lysis buffer, 10 times
with 0.5 ml of lysis buffer containing 10 mM imidazole, and
single times with 0.5 ml of lysis buffer containing 20, 30, and 40 mM imidazole. His6-Fin1p was eluted with 0.5 ml
of lysis buffer containing 400 mM imidazole. The eluted
protein was dialyzed against 50 mM phosphate buffer, pH
7.5, 500 mM NaCl, 50% glycerol at 4 °C. Alternatively,
the protein was stored at
20 °C in the 8 M
urea-containing elution buffer. The protein concentration was
determined with the Bradford assay, and purity was estimated by
SDS-PAGE and Coomassie staining.
protein phosphatase (New England Biolabs) in 100 µl of
protein phosphatase buffer for 60 min at 30 °C. Controls
contained heat-inactivated protein phosphatase (10 min at 100 °C in
the presence of 100 mM EDTA, pH 8.0). After five washes
with 200 µl of PBST (PBS, 0.5% blocking reagent, 0.05% Tween 20),
the wells were incubated with 50 µl of PBS containing the indicated
amounts of purified His6-Bmh1p or His6-Bmh2p
for 1 h at room temperature. The wells were washed five times with
200 µl of PBST, and 100 µl of anti-Bmh1 antiserum (13) (1:2000) in
PBST was added followed by a 1-h incubation at room temperature. After
washing with PBST, goat anti-rabbit IgG alkaline phosphatase
conjugate (Promega) and nitrophenylphosphate were used to detect bound
antiserum. Bound Fin1p was detected by a similar procedure except that
anti-RGSH4 antibody (Qiagen) and anti-mouse/rabbit
IgG-peroxidase (Roche Molecular Biochemicals) were used.
a *
ICFP
b *
IYFP)/ICFP, essentially
as described (28), was used, where FRETC is the corrected
FRET value, and IFRET,
ICFP, and IYFP are
intensities measured through FRET, CFP, and YFP filter sets,
respectively. a and b are bleed-through
percentages measured in cells only expressing CFP or YFP. Calculated
FRETC values are displayed in pseudocolor.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase reporter.
-galactosidase+ transformants, and after transformation
of E. coli, colonies were selected on medium
supplemented with ampicillin. None of these E. coli colonies
contained the bait plasmid pRUL551. In two other two-hybrid screens using bait plasmids with a kanamycin marker we did not isolate
the bait plasmid either (data not shown), indicating the power of the
use of different selection markers in the bait and prey plasmids.
Cotransformation of HF7c with each of the five isolated library
plasmids and pRUL551 resulted again in His+ transformants
with
-galactosidase activity. Cotransformation of HF7c with the
isolated library plasmids and the empty vector pGBDK-C1 yielded
His
transformants without
-galactosidase activity.
This demonstrates that the activation of the reporter genes of HF7c in
these five positives is dependent on the presence of both pRUL551 and
the library plasmid. Sequencing of the yeast DNA insert of one of the
isolated plasmids revealed an in-frame fusion between the GAL4ad and the 3'-end of GCR2, encoding amino
acids 223 to 534 of the Gcr2 protein. GCR2 encodes a
transcription factor required for full activation of glycolytic genes.
Three other plasmids contain SPT21. However, for
SPT21 there is not an in-frame fusion with the
GAL4 activating domain. The last plasmid contains an in-frame fusion between the GAL4ad and YDR130c,
lacking the first 30 base pairs. As the protein encoded by this open
reading frame forms cell cycle-specific filaments, we named this open
reading frame FIN1 (filaments in
between nuclei 1) (1). The reverse combination,
BMH2 fused to the GAL4ad and FIN1
fused to GAL4bd, also reacts positively in the two-hybrid
system (data not shown). We studied the interaction between Fin1p and
the yeast 14-3-3 proteins in more detail.
and
, respectively). In the absence of a Bmh protein, no signal was
observed (Fig. 1,
). The apparent stronger binding of Bmh1p in
comparison to Bmh2p is not caused by a higher affinity of Bmh1p for
Fin1p (as will be discussed later) but is because of the higher
affinity of the anti-14-3-3 antiserum for Bmh1p. As Fin1p is a highly
basic protein, whereas the 14-3-3 proteins are acidic, we also
performed an ELISA experiment in which we used another basic protein,
cytochrome c, instead of Fin1p. As shown in Fig. 1 (
),
Bmh2p does not bind to cytochrome c. Similar results were
obtained for Bmh1p (data not shown), demonstrating that there is a
specific in vitro interaction between the yeast 14-3-3 proteins and Fin1p.
View larger version (19K):
[in a new window]
Fig. 1.
ELISA to study the binding of Bmh1p and Bmh2p
to Fin1p. Increasing amounts of purified His6-Fin1p or
cytochrome c ( ) were coated to the wells of a microtiter
plate. After blocking and washing, the wells were incubated with a
solution containing 2 µg Bmh2p (
and
), 2 µg Bmh1p (
), or
no protein (
). The amount of 14-3-3 protein retained in the wells
after washing was determined immunologically (A415).
1 s
1 and a
dissociation rate constant (kd) of 1.08 × 10
3 s
1 and for Bmh2p a
ka of 2.57 × 103
M
1 s
1 and a
kd of 7.44 × 10
4
s
1. In these calculations we included our observation
that the recombinant Bmh proteins form dimers (data not shown). These
data resulted in a dissociation constant (KD)
of 585 nM for the Fin1p-Bmh1p interaction and of 289 nM for the Fin1p-Bmh2p interaction, indicating that Bmh2p
has a higher affinity than Bmh1p for Fin1p. Simulated curves obtained
by using the found dissociation constants matched with the experimental
curves.
View larger version (24K):
[in a new window]
Fig. 2.
Overlay plots of sensograms of Bmh1p
(A) or Bmh2p (B) binding to
immobilized Fin1p during surface plasmon resonance analysis. Two
thousand resonance units of purified Fin1p were immobilized to a CM5
chip. Solutions containing the indicated concentrations of Bmh1p or
Bmh2p were applied to allow binding (association). For dissociation
buffer without protein was used.
protein
phosphatase. As a control, the other series of wells was incubated with
inactivated protein phosphatase. As shown in Fig.
3 (
), Bmh2p binds to Fin1p treated
with inactive phosphatase in a similar way as untreated Fin1p. However,
treatment of Fin1p with protein phosphatase resulted in a strong
reduction in Bmh2p binding (Fig. 3,
). The reduced binding of Bmh2p
to Fin1p after dephosphorylation was not caused by a decrease in the
amount of immobilized Fin1p, as the amount of immobilized Fin1p was not affected by the protein phosphatase treatment (Fig. 3,
and
). Similar results were obtained with Bmh1p (data not shown). In addition,
during surface plasmon resonance analysis dephosphorylation of Fin1p
resulted in a strong reduction (~60%) of the Bmh2p binding (data not
shown).
View larger version (20K):
[in a new window]
Fig. 3.
ELISA to study the effect of phosphatase
treatment of Fin1p on the interaction with Bmh2p. Increasing
amounts of Fin1p were immobilized to the wells of a microtiter plate.
Two series of wells were treated with either active ( and
) or
inactivated (
and
)
protein phosphatase. After blocking and
washing, both series of wells were incubated with a solution containing
1 µg of Bmh2p. The amounts of Bmh2p bound to Fin1p (
) and to
dephosphorylated Fin1p (
) were determined immunologically using
anti-14-3-3 protein antiserum (A415). To determine the
effect of phosphatase treatment on the amount of Fin1p present in the
well, two other series of wells containing immobilized Fin1p were
treated with inactivated (
) and active (
) protein phosphatase.
Subsequently the amount of Fin1p was determined immunologically using
anti-RGSH4 antibody (A492).
1288-derived strains the 14-3-3 proteins are
not essential (17). To this end the bmh1 bmh2 strain RR1249
and the isogenic control strain F3
were transformed with plasmid
pUG36[FIN1] encoding an N-terminal GFP fusion of Fin1p. As
shown in Fig. 4, in both strains
fluorescent filaments can be seen. In theory it is possible that in the
1288-derived strains the loss of 14-3-3 proteins is compensated by
an unknown activity that replaces the 14-3-3 proteins during Fin1p
filament formation. Therefore, we also looked at Fin1p filament
formation in a strain in which BMH1 and BMH2 are
replaced by a temperature-sensitive bmh2 allele (19). Both
at the permissive and restrictive temperature, filaments formed by
GFP-Fin1 were observed (data not shown). Also in a strain in which both
BMH1 and BMH2 are replaced by cDNA encoding a
plant 14-3-3 protein isoform under control of the GAL1
promoter (13), GFP-Fin1 filaments were observed after growth on
medium with glucose as sole carbon source resulting in
undetectable levels of 14-3-3 proteins (data not shown). These results
indicate that the 14-3-3 proteins are not essential for Fin1p filament
formation in vivo, in agreement with our previous
observation that purified Fin1p forms filaments in vitro in
the absence of other proteins.
View larger version (55K):
[in a new window]
Fig. 4.
Fluorescence microscopy of
F3 (BMH1 BMH2)
(A) and RR1249 (bmh1 bmh2)
(B) cells containing plasmid
pUG36[FIN1] expressing GFP-Fin1p.
-galactosidase activity (data not
shown). In addition, during a two-hybrid screen for interaction
partners of Fin1p, we isolated several clones containing a
GAL4ad-FIN1 fusion (1). Therefore, Fin1p is able
to interact with itself in the two-hybrid system. This observation is
in agreement with our previous observation (1) that purified
His6-Fin1p self-assembles into 10-nm filaments after
dialysis against low salt buffers.
View larger version (28K):
[in a new window]
Fig. 5.
Mapping of the Fin1p domains responsible for
self-association and interaction with Bmh2p using the two-hybrid
system. Various parts of Fin1p were expressed as Gal4ad
fusion proteins. These were tested for interaction with Gal4bd fusions
containing amino acids 11-291 of Fin1p (plasmid pRUL558), amino acids
254-280 of Fin1p (pRUL372), or full-length Bmh2p (pRUL551). A positive
interaction, i.e. when a combination of bait and prey
plasmid activated both the HIS3 and -galactosidase
reporter genes in strain HF7c, is indicated with a plus sign (+).
Double transformants negative for both reporter genes are indicated
with a minus sign (
). Dark boxes in Fin1p indicate
predicted coiled-coil regions.
View larger version (14K):
[in a new window]
Fig. 6.
FRET between CFP-Fin1p and YFP-Fin1p in
intact yeast cells. Corrected FRET values (as described under
"Materials and Methods") are displayed in pseudocolor.
A, GG3036 cells, expressing CFP-Fin1p and YFP-Fin1p, both
localized in the spindle pole bodies; B, GG3036 cells,
expressing CFP-Fin1p and YFP-Fin1p, both localized in a filament
between the spindle pole bodies; C, GG3087 cells, expressing
free CFP and free YFP, both localized mainly in the cytoplasm;
D, relationship between color and pixel value.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
,
, and
) and a Raf-based peptide. The latter
study was performed with short peptides, and we used a complete
protein. Besides a 14-3-3 binding motif, other parts of the protein
might be involved in modulating the interaction (36).
Dissociation constants (KD) for the interactions between 14-3-3 proteins and a number of binding partners
In this study we showed that the yeast 14-3-3 proteins interact with
the intermediate filament protein Fin1p. Interactions between the
14-3-3 proteins and other intermediate filament or cytoskeleton
proteins have been reported (37-39). In keratinocytes the keratin
K8/K18 forms intermediate filaments in a cell cycle-regulated way (40,
41). The 14-3-3 proteins keep the keratin in a soluble form. The yeast
14-3-3 proteins may have a similar function in Fin1p filament
formation. As in 1288-derived strains filament formation was not
affected by the absence of 14-3-3 proteins, and the 14-3-3 proteins are
not essential for filament formation (Fig. 4).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Philip James (Department of Biomolecular Chemistry, University of Wisconsin) for generously supplying the two-hybrid libraries, plasmids, and reporter strains, Dr. G. R. Fink (MIT, Cambridge, MA) for the generous gift of yeast strains, Dr. D. Hailey (Yeast Resource Center, Washington University, Seattle, WA) for plasmids pDH3 and pDH5, and G. E. M. Lamers, K. E. van Straaten, A. Werner, A. van Zon, and M. Hannaart for excellent technical assistance.
![]() |
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.
** To whom correspondence should be addressed. Tel.: 31-71-5274996; Fax: 31-71-5274999; E-mail: Heusden@RULBIM.Leidenuniv.nl.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212495200
2 M. J. van Hemert, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GFP, green fluorescent protein; ad, activating domain; bd, binding domain; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ELISA, enzyme-linked immunosorbent assay.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
van Hemert, M. J.,
Lamers, G. E. M.,
Klein, D. C. G.,
Oosterkamp, T. H.,
Steensma, H. Y.,
and van Heusden, G. P. H.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
5390-5393 |
2. | Aitken, A. (1996) Trends Cell Biol. 6, 341-347[CrossRef] |
3. | Finnie, C., Borch, J., and Collinge, D. B. (1999) Plant Mol. Biol. 40, 545-554[CrossRef][Medline] [Order article via Infotrieve] |
4. | Chung, H. J., Sehnke, P. C., and Ferl, R. J. (1999) Trends Plant Sci. 4, 367-371[CrossRef][Medline] [Order article via Infotrieve] |
5. | Fu, H., Subramanian, R. R., and Masters, S. C. (2001) Annu. Rev. Pharmacol. Toxicol. 40, 617-647[CrossRef] |
6. | Van Hemert, M. J., Steensma, H. Y., and van Heusden, G. P. H. (2001) Bioessays 23, 936-946[CrossRef][Medline] [Order article via Infotrieve] |
7. | Muslin, A. J., Tanner, J. W., Allen, P. M., and Shaw, A. S. (1996) Cell 84, 889-897[Medline] [Order article via Infotrieve] |
8. | Yaffe, M. B., Rittinger, K., Volinia, S., Caron, P. R., Aitken, A., Leffers, H., Gamblin, S. J., Smerdon, S. J., and Cantley, L. C. (1997) Cell 91, 961-971[Medline] [Order article via Infotrieve] |
9. |
Liu, Y. C.,
Liu, Y.,
Elly, C.,
Yoshida, H.,
Lipkowitz, S.,
and Altman, A.
(1997)
J. Biol. Chem.
272,
9979-9985 |
10. | Andrews, R. K., Harris, S. J., McNally, T., and Berndt, M. C. (1998) Biochemistry 37, 638-647[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Petosa, C.,
Masters, S. C.,
Bankston, L. A.,
Pohl, J.,
Wang, B. C.,
Fu, H. I.,
and Liddington, R. C.
(1998)
J. Biol. Chem.
273,
16305-16310 |
12. | van Heusden, G. P. H., Wenzel, T. J., Lagendijk, E. L., Steensma, H. Y., and van den Berg, J. A. (1992) FEBS Lett. 302, 145-150[CrossRef][Medline] [Order article via Infotrieve] |
13. | van Heusden, G. P. H., Griffiths, D. J., Ford, J. C., Chin, A. W.-T., Schrader, P. A., Carr, A. M., and Steensma, H. Y. (1995) Eur. J. Biochem. 229, 45-53[Abstract] |
14. | Van Hemert, M. J., van Heusden, G. P. H., and Steensma, H. Y. (2001) Yeast 18, 889-895[CrossRef][Medline] [Order article via Infotrieve] |
15. | Gelperin, D., Weigle, J., Nelson, K., Roseboom, P., Irie, K., Matsumoto, K., and Lemmon, S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11539-11543[Abstract] |
16. | Knetsch, M. L. W., van Heusden, G. P. H., Ennis, H. L., Shaw, D. R., Epskamp, S. J. P., and Snaar-Jagalska, B. E. (1997) Biochim. Biophys. Acta 1357, 243-248[Medline] [Order article via Infotrieve] |
17. | Roberts, R. L., Mosch, H. U., and Fink, G. R. (1997) Cell 89, 1055-1065[Medline] [Order article via Infotrieve] |
18. |
Moriya, H.,
Shimizu-Yoshida, Y.,
Omori, A.,
Iwashita, S.,
Katoh, M.,
and Sakai, A.
(2001)
Genes Dev.
15,
1217-1228 |
19. | van Heusden, G. P. H., and Steensma, H. Y. (2001) Yeast 18, 1479-1491[CrossRef][Medline] [Order article via Infotrieve] |
20. | Beck, T., and Hall, M. N. (1999) Nature 402, 689-692[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Callejo, M.,
Alvarez, D.,
Price, G. B.,
and Zannis-Hadjopoulos, M.
(2002)
J. Biol. Chem.
277,
38416-38423 |
22. |
James, P.,
Halladay, J.,
and Craig, E. A.
(1996)
Genetics
144,
1425-1436 |
23. | Gietz, R. D., Schiestl, R. H., Willems, A. R., and Woods, R. A. (1995) Yeast 11, 355-360[Medline] [Order article via Infotrieve] |
24. | Bradford, M. M. (1976) Anal. Biochem. 7, 72248-72254 |
25. | Johnsson, B., Lofas, S., and Lindquist, G. (1991) Anal. Biochem. 198, 268-277[Medline] [Order article via Infotrieve] |
26. | Myszka, D. G., and Morton, T. A. (1998) Trends Biochem. Sci 23, 149-150[CrossRef][Medline] [Order article via Infotrieve] |
27. | Hailey, D. W., Davis, T. N., and Muller, E. G. D. (2003) Methods Enzymol., in press |
28. |
Gordon, G. W.,
Berry, G.,
Liang, X. H.,
Levine, B.,
and Herman, B.
(1998)
Biophys. J.
74,
2702-2713 |
29. | Mayordomo, I., and Sanz, P. (2002) Biochem. J. 365, 51-56[CrossRef][Medline] [Order article via Infotrieve] |
30. | Ju, Q. D., Morrow, B. E., and Warner, J. R. (1990) Mol. Cell. Biol. 10, 5226-5234[Medline] [Order article via Infotrieve] |
31. | Lang, W. H., and Reeder, R. H. (1993) Mol. Cell. Biol. 13, 649-658[Abstract] |
32. | Russnak, R., Pereira, S., and Platt, T. (1996) Gene Expr. 6, 241-258[Medline] [Order article via Infotrieve] |
33. | Russnak, R., Nehrke, K. W., and Platt, T. (1995) Mol. Cell. Biol. 15, 1689-1697[Abstract] |
34. |
Biggins, S.,
Bhalla, N.,
Chang, A.,
Smith, D.,
and Murray, A. W.
(2001)
Genetics
159,
453-470 |
35. | Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi-Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J. M. (2000) Nature 403, 623-627[CrossRef][Medline] [Order article via Infotrieve] |
36. | Obsil, T., Ghirlando, R., Klein, D. C., Ganguly, S., and Dyda, F. (2001) Cell 105, 257-267[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Zhang, S. H.,
Kobayashi, R.,
Graves, P. R.,
Piwnica-Worms, H.,
and Tonks, N. K.
(1997)
J. Biol. Chem.
272,
27281-27287 |
38. |
Dorner, C.,
Ullrich, A.,
Haring, H. U.,
and Lammers, R.
(1999)
J. Biol. Chem.
274,
33654-33660 |
39. | Roth, D., Morgan, A., and Burgoyne, R. D. (1993) FEBS Lett. 320, 207-210[CrossRef][Medline] [Order article via Infotrieve] |
40. | Liao, J., and Omary, M. B. (1996) J. Cell Biol. 133, 345-357[Abstract] |
41. |
Ku, N. O.,
Liao, J.,
and Omary, M. B.
(1998)
EMBO J.
17,
1892-1906 |
42. | Itagaki, C., Isobe, T., Taoka, M., Natsume, T., Nomura, N., Horigome, T., Omata, S., Ichinose, H., Nagatsu, T., Greene, L. A., and Ichimura, T. (1999) Biochemistry 38, 15673-15680[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Fuglsang, A. T.,
Visconti, S.,
Drumm, K.,
Jahn, T.,
Stensballe, A.,
Mattei, B.,
Jensen, O. N.,
Aducci, P.,
and Palmgren, M. G.
(1999)
J. Biol. Chem.
274,
36774-36780 |
44. |
Fanger, G. R.,
Widmann, C.,
Porter, A. C.,
Sather, S.,
Johnson, G. L.,
and Vaillancourt, R. R.
(1998)
J. Biol. Chem.
273,
3476-3483 |
45. |
Stomski, F. C.,
Dottore, M.,
Winnall, W.,
Guthridge, M. A.,
Woodcock, J.,
Bagley, C. J.,
Thomas, D. T.,
Andrews, R. K.,
Berndt, M. C.,
and Lopez, A. F.
(1999)
Blood
94,
1933-1942 |
46. |
Banik, U.,
Wang, G. A.,
Wagner, P. D.,
and Kaufman, S.
(1997)
J. Biol. Chem.
272,
26219-26225 |