From the Department of Molecular and Cell Biology,
Division of Biochemistry and Molecular Biology, University of
California, Berkeley, California 94720-3202, the ¶ Center for
Advanced Research in Biotechnology, University of Maryland
Biotechnology Institute, Rockville, Maryland 20850, and the
Howard Hughes Medical Institute, University of California,
Berkeley, California 94720-3204
Received for publication, August 4, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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Frq1, a 190-residue N-myristoylated
calcium-binding protein, associates tightly with the N terminus of
Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis
of an Frq1-binding fragment, Pik1-(10-192), showed that
residues within 80-192 are necessary and sufficient for Frq1
association in vitro. A synthetic peptide (residues
151-199) competed for binding of [35S]Pik1-(10-192) to
bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199)
did not. Correspondingly, a deletion mutant, Pik1( Recognition that phosphoinositides and inositol phosphates are key
regulators of many processes in eukaryotic cells has brought increased
attention to the enzymes that regulate the synthesis and turnover of
these molecules (reviewed in Refs. 1-3). Of particular interest are
the enzymes responsible for producing the various polyphosphoinositides
situated on the cytosolic face of cellular membranes, which initiate
several different signaling pathways by serving as highly specific
recognition determinants for the selective recruitment of proteins to
membranes (reviewed in Refs. 4-7) and as the precursors for several
intracellular second messengers (reviewed in Refs. 8-10). The first
committed step in the synthesis of the polyphosphoinositide,
phosphatidylinositol 4,5-bisphosphate, is considered to be
ATP-dependent phosphorylation of the hydrophilic myo-inositol head group of phosphatidylinositol
(PtdIns)1 at the
D-4 position by PtdIns 4-kinase
(ATP:1-phosphatidyl-1D-myo-inositol 4-phosphotransferase, EC
2.7.1.67) (reviewed in Refs. 11-13) . The resulting product,
PtdIns(4)P, can be phosphorylated on the D-5 position by
PtdIns(4)P 5-kinase to generate PtdIns(4,5)P2, PtdIns(4,5)P2 can be phosphorylated on the D-3
position by yet other lipid kinases, and the phosphoinositides so
generated can be converted to other species by specific phosphatases
and phospholipases (reviewed in Refs. 14-17).
The first PtdIns 4-kinase to be purified to homogeneity from any
organism (18), and to have the corresponding gene cloned (19, 20), was
Pik1 from the yeast Saccharomyces cerevisiae. Thereafter, a
second isoform, Stt4, which is the product of a discrete gene, was
described (21). Absence of either Pik1 or Stt4 is lethal, and
overproduction of each protein cannot compensate for absence of the
other, indicating that these enzymes participate in distinct cellular
processes and generate discrete pools of PtdIns(4)P that are essential
for yeast cell viability. Indeed, subsequent work has shown that,
together, Pik1 and Stt4 account for all of the PtdIns(4)P generated in
the yeast cell (22) and that Pik1 is required for vesicular trafficking
in the late secretory pathway (23, 24) and perhaps for cytokinesis
(20), whereas Stt4 plays roles in cell wall integrity, maintenance of
vacuole morphology, and aminophospholipid transport from the
endoplasmic reticulum to the Golgi (25-27). The presence of Pik1- and
Stt4-like isoforms is also conserved in metazoans (11, 12, 28).
We have shown previously that Frq1, a small calcium-binding protein,
co-purifies with Pik1 and is required for optimal activity of the
enzyme (29). Frq1 is the yeast ortholog of a protein called frequenin,
first described in Drosophila (30), but referred to as
neuronal-calcium-sensor-1 (NCS-1) in mammalian cells. Members of
a large subfamily of small, EF-hand-containing, calcium-binding proteins that includes frequenin (31-34) are characterized by a consensus signal for N-terminal myristoylation and four
Ca2+-binding sites (of which the first and, in some cases,
the fourth or another contain substitutions that make them
non-functional). We have shown previously that Frq1 binds three
Ca2+ (35). Available evidence indicates that
frequenin/NCS-1 may also modulate PtdIns 4-kinase activity in animal
cells (36, 37).
Frq1, which is itself essential for the viability of yeast cells (29),
associates with membranes in a manner that depends on both the
N-myristoyl group and conformational changes induced upon
Ca2+ binding (35). Thus, in addition to its stimulation of
enzymic activity, Frq1 may contribute to the optimal function of Pik1 by assisting with its membrane recruitment, because Pik1 itself lacks
any obvious membrane-targeting motifs. Indeed prior work indicated that
N-myristoylation of Frq1 is important, but not essential,
for its function (29). In some Ca2+-binding regulatory
proteins, in addition to the N-terminal myristoyl group, palmitoylation
of a cysteine residue near the N terminus is also required for
efficient membrane association (38, 39). Frq1 has only two Cys
residues, one is near its N terminus and the other buried in the
interior (35).
In this study, as a prelude to structural analysis to determine at
atomic resolution how Frq1 recognizes Pik1, we have applied several
independent methods to determine the affinity and stoichiometry of the
Frq1-Pik1 interaction, used different approaches to delineate the
sequences in Pik1 responsible for high affinity binding of Frq1 and
utilized both biochemical and genetic techniques to explore the role,
if any, of S-palmitoylation in the function of Frq1.
Strains and Growth Conditions--
Yeast strains used in this
study are listed below in Table I. Standard rich (YP) and synthetic
complete (SC) media (40) were supplemented with carbon sources
(either 2% Glc, or 2% Raf/0.2% Suc, or 2% Gal/0.2% Suc, as
indicated) and with appropriate nutrients for the selection and
maintenance of plasmids. Yeast was cultivated at 30 °C, unless
otherwise noted. Conventional methods for DNA-mediated transformation
and other genetic manipulations of yeast cells were used (41).
Plasmid Construction--
Plasmids were constructed using
standard methods for the manipulation of recombinant DNA (42).
Escherichia coli strain DH5
Fragments of the N terminus of Pik1 were tagged with a C-terminal
His6 tract and expressed in E. coli, as
follows. pET23d-PIK1(10-192) was constructed by inserting
via blunt-end ligation the HindIII fragment of
PIK1 into pET23d (Novagen, Madison, WI) that had been cleaved with NcoI and EcoRI.
pET23d-PIK1(10-192,
Multicopy (2-µm, DNA-based) URA3-marked plasmids,
YEp352-FRQ1, YEp352-GAL-FRQ1, and
YEp352-FRQ1(G2A) have been described previously (29).
YEp352-GAL-FRQ1-(His)6 was constructed by excising the corresponding fragment from pET23d-FRQ1-(His)6
(29) and inserting it into YEp352-GAL (44).
YEp352-FRQ1(G2A,C15A) was generated by replacing an
HindIII-BglII fragment of
YEp352FRQ1(G2A) with a PCR product, carrying the
appropriate mutations to encode C15A, that was digested with the same
enzymes. The low copy (CEN-based) URA3-marked
plasmid, pRS316-FRQ1(G2A,C15A), was generated by inserting an EcoRI fragment from YEp352-FRQ1(G2A,C15A) into
pRS316-FRQ1 (29). Construction of the TRP1-marked
CEN plasmids, RS314-PIK1, pRS314-GAL1,10, and pRS314-GAL-mycPIK is
described elsewhere (45). The cloning intermediate,
Litmus28-PIK1, was produced by inserting a
BamHI-SacI fragment containing the entire coding
sequence of PIK1, excised from pRS314-PIK1, into
Litmus28. Litmus28PIK1( Production of Radiolabeled Proteins by Coupled in Vitro
Transcription and Translation--
Vectors for in vitro
production of mRNA were constructed, as follows. A fragment
containing the FRQ1 coding sequence, generated by PCR and
containing an NcoI site overlapping the translation initiation codon and a BamHI site introduced 3' to the stop
codon, was inserted into pBAT4 (46), which had been cleaved with
NcoI and BamHI, yielding pBAT4-FRQ1.
A PCR fragment was amplified from pET23d-PIK1(10-192)
using appropriate primers to introduce a SmaI site 5' to the
coding sequence and a HindIII site 3' to codon 192 of the
PIK1 open reading frame, cleaved with SmaI and
HindIII, and ligated into pBAT4 that had been cleaved with
the same enzymes, generating pBAT4-PIK1(10-192).
35S-Labeled proteins were produced by coupled in
vitro transcription and translation in the presence of
[35S]Met and [35S]Cys (PerkinElmer Life
Sciences, Boston, MA) using the TNTTM coupled
reticulocyte lysate system (Promega, Madison, WI), according to the
manufacturer's instructions. Translation mixtures were clarified by
centrifugation (10 min, 4 °C) at maximum speed in a microcentrifuge.
If not used immediately, the resulting supernatant fractions were mixed
with an equal volume of glycerol and stored at Bacterial Expression and Purification of
(His)6-tagged Proteins--
The Pik1 and Frq1 constructs
containing a C-terminal His6 tag were expressed in E. coli strain BL21(DE3) (Novagen, Madison, WI) and purified using
Ni2+-saturated NTA-agarose (Qiagen, Valencia, CA) under
denaturing conditions according to the manufacturer's specifications.
To confirm their identity and purity, proteins recovered by binding to
Ni2+-saturated NTA-agarose were resolved by SDS-PAGE and
visualized by staining with Coomassie Brilliant Blue and by
immunoblotting. Protein concentration was determined by the dye-binding
method of Bradford (47) using a commercial kit (Bio-Rad, Inc.,
Hercules, CA) with bovine serum albumin as the standard.
Purification of Frq1-(His)6 from Yeast
Cells--
Protease-deficient strain, BJ2168 (Table I), transformed
with YEp352-GAL-FRQ1-(His)6, was grown in SC-Raf
lacking uracil at 30 °C to mid-exponential phase and induced by
addition of Gal (2% final concentration). After incubation for 6 h at 30 °C, cells were collected by centrifugation and resuspended
in an equal volume of distilled water. The cell suspension was frozen by dripping into liquid nitrogen, and the resulting pellets were crushed with a pestle in a precooled mortar. The resulting frozen cell
powder was dissolved in lysis buffer (5 mM imidazole, 145 mM NaCl, 50 mM Na-PO4 (pH 7.5); 20 ml/liter yeast culture) containing a mixture of protease inhibitors
(CompleteTM, Promega, Madison, WI). The crude lysate was
clarified by centrifugation at maximum rpm in a microcentrifuge at
4 °C for 15 min and then at 72,000 × g for 90 min
in a L8-80M ultracentrifuge (Beckman-Coulter Inc., Fullerton, CA). The
resulting supernatant fraction was then applied to a
Ni2+-saturated NTA-agarose column (1.5-ml bed volume) that
had been pre-equilibrated with three volumes of lysis buffer. After
washing with 10 bed volumes of lysis buffer and 6 volumes of wash
buffer (20 mM imidazole, 145 mM NaCl, 50 mM Na-PO4 (pH 7.5)), the bound Frq1-His6 was eluted with 3 volumes of elution buffer (120 mM imidazole, 145 mM NaCl, 50 mM
Na-PO4 (pH 7.5)). The eluate was concentrated by
ultrafiltration through an anisotropic membrane (3-kDa cut-off,
Centricon YM-3, Amicon, Beverly, MA) until a final concentration of 0.5 µg/ml was reached. To confirm identity and purity, the resulting
fraction was resolved by SDS-PAGE and visualized by staining with
Coomassie Brilliant Blue and by immunoblotting.
In Vitro Protein Binding Assays--
Prior to use,
Ni2+-saturated NTA-agarose beads used in protein binding
and peptide competition experiments were pre-blocked by incubation for
30 min in 8 volumes of buffer A (10 mM imidazole, 100 mM NaCl, 1 µM CaCl2, 1 mM dithiothreitol, 50 mM Tris-HCl (pH 7.4))
containing 5 mg/ml ovalbumin at room temperature. All in vitro binding assays were carried out at 4 °C. Radiolabeled
Frq1, prepared by coupled in vitro transcription and
translation, was mixed with an equal volume of a slurry of pre-blocked
Ni2+-saturated NTA-agarose beads in buffer A and incubated
on a roller drum for 30 min. The beads and any nonspecifically
bound radioactivity were removed by brief sedimentation in a
microcentrifuge, and the resulting pre-cleared supernatant fraction was
collected. Aliquots (400 µl) of the pre-cleared fraction were mixed
either with 40 µl of pre-blocked Ni2+-saturated
NTA-agarose beads, as a control for background binding, or with
Ni2+-saturated NTA-agarose beads on which had been
immobilized Pik1-(10-192)-(His)6 or its deletion
derivatives (30 µg of protein/40 µl of beads) and incubated for
1 h on a rollerdrum. The beads were collected by centrifugation
for 15 s in a microcentrifuge and washed three times with buffer
A. Bound proteins were eluted from the beads in 50 µl of buffer A
containing 300 mM imidazole, and samples of the resulting
eluate were resolved by SDS-PAGE on a 12% gel and visualized by autoradiography.
Synthetic peptides corresponding to Pik1-(174-199), Pik1-(164-199),
and Pik1-(151-199) were prepared by standard solid phase peptide
synthesis (using Fmoc chemistry) on an automated synthesizer (Model ABI
431A, PerkinElmer Life Sciences-Applied Biosystems, Foster City, CA),
purified by high-performance liquid chromatography, and confirmed by
electrospray ionization mass spectrometry. A slurry (30 µl), of
either pre-blocked Ni2+-saturated NTA-agarose beads or the
same beads pre-coated to saturation with purified
Frq1-His6, was mixed with 500 µl of 2×-concentrated buffer A, 290 µl of H2O, and 100 µl of either an
aqueous solution of the indicated peptide in 10% acetonitrile or
H2O containing 10% acetonitrile as a control. After
preincubation of the samples on a roller drum for 30 min, 80 µl of
35S-labeled Pik1-(10-192), produced by coupled in
vitro transcription and translation, was added to each tube, and
the mixture was incubated for a further 1.5 h. The beads were
collected by sedimentation in a microcentrifuge and washed twice
with buffer A. Bead-bound radioactivity was solubilized by boiling in
SDS-PAGE sample buffer (48), and the resulting eluate was subjected to
SDS-PAGE. The species corresponding to
[35S]Pik1-(10-192) was quantitated using a
PhosphorImagerTM (Amersham Biosciences, Sunnyvale, CA). At
each peptide concentration, the amount of radioactivity bound
nonspecifically to empty beads was subtracted from the amount of
radioactivity bound to the beads coated with Frq1-His6.
NMR Spectroscopy--
15N-Labeled and unlabeled
samples of unmyristoylated Frq1 were prepared as described previously
(35). Chemical synthesis of the 49-residue synthetic peptide
corresponding to residues 151-199 of Pik1 was described in the
preceding section. Using PCR with appropriate primers, an 83-residue
segment from the N terminus of Pik1 consisting of residues 110-192,
Pik1-(110-192), was tagged at its C terminus with an His6
tract, inserted as an NcoI-XhoI fragment into the
corresponding sites in the vector, pET23d, and expressed in E. coli strain BL21(DE3), as described above; in this construct, the
Pik1-derived sequence is preceded by a 2-residue leader (Met-Ala-).
Pik1-(110-192)-(His)6 was produced primarily in inclusion
bodies, which were solubilized using 6 M guanidine hydrochloride (49) and purified using Ni2+-saturated
NTA-agarose chromatography, essentially as described above. Samples for
NMR analysis were prepared by dissolving 15N-labeled Frq1
(0.4 mM) with various amounts (0, 1, or 2 molar equivalents) of Pik1-(110-192) or the synthetic peptide,
Pik1-(151-199), in 0.5 ml of a 95% H2O/5%
[2H]H2O solution containing 10 mM
imidazole (pH 6.7), 10 mM
[2H10]dithiothreitol, and either 1 mM EDTA (Ca2+-free) or 5 mM
CaCl2 (Ca2+-bound). All NMR experiments were
performed at 37 °C on a Model DRX-500 or DRX-600 NMR spectrometer
(Bruker Instruments, Billerica, MA) equipped with a four-channel
interface and a triple-resonance probe with triple-axis pulsed field
gradients as described before (35). The NMR spectra were processed and
analyzed as described previously (50).
Fluorescence Spectroscopy--
The effect of Pik1 peptides on
the intrinsic tryptophan fluorescence emission of Frq1, excited at 290 nm, was measured (at 300-420 nm) using a SPEX fluorometer (Jobin Yvon
Inc., Edison, NJ). Neither Pik1-(151-199) nor Pik1-(110-192) contain
any tryptophan, and they do not contribute any fluorescence under these
conditions. Titrations were performed using 5 µM Frq1 in
2 ml of 50 mM HEPES (pH 7.5), 0.1 M KCl, 1 mM CaCl2, 1 mM dithiothreitol at
25 °C. Various amounts (0, 0.5, 1.0, 1.5, 2.0, 3.0, and 4.0 molar
equivalents) of either Pik1-(151-199) or Pik1-(110-192) were injected
into the sample cuvette containing the Frq1 solution, and fluorescence emission spectra were recorded after each addition.
Isothermal Titration Calorimetry--
Binding of
Ca2+-Frq1 to either Pik1-(151-199) or Pik1-(110-192) was
measured by isothermal titration calorimetry (51) using a MicroCal
VP-ITC MicroCalorimeter (MicroCal Inc., Northampton, MA). Frq1
and Pik1-(110-192) protein samples were dialyzed against a buffer
containing 10 mM HEPES (pH 7.4), 5 mM
CaCl2, and 1 mM dithiothreitol. Lyophilized
Pik1-(151-199) was dissolved in the same buffer used for the dialysis
of Frq1. Experiments were performed at 25 °C. Into the sample cell
containing a solution (10 µM) of either Pik1-(151-199)
or Pik1-(110-192) were injected a total of 270 µl of 58 µM Frq1 in 18 aliquots. Heats of dilution, determined by
titrating Frq1 into same buffer alone, were subtracted from the raw
titration data before data reduction and analysis. A single-site model
was used to fit the titration data for Pik1-(151-199) binding to Frq1.
A two-site model was needed to describe the multiphasic interaction of
Pik1-(110-192) with Frq1.
Preparation of Yeast Cell Extracts, Immunoprecipitation, and
Immunoblot Analysis--
Lysis conditions and centrifugation for the
preparation of clarified yeast cells extracts for immunoprecipitation
were as described previously (29). Samples (1 mg of total protein) of extract were diluted into lysis buffer (220-µl final volume) and mixed with 40 µl of a 1:1 (settled beads:buffer) suspension of protein G-/protein A-coupled agarose beads (Oncogene Research Products,
Boston, MA) and 2 µl of an irrelevant mouse antibody (anti-6-His mAb,
BAbCO, Richmond, CA) and incubated at 4 °C on a roller drum for
1 h. Beads were removed by brief centrifugation, and the
precleared supernatant fraction was transferred to a new tube. Samples
of the precleared fraction were then incubated overnight at 4 °C
with either 2 µl of Analytical Size Exclusion Chromatography--
Size exclusion
chromatography was performed on a Superdex 200 column (Amersham
Biosciences, Piscataway, NJ) using an fast protein liquid
chromatography apparatus (650E Advanced Protein Purification System,
Waters, Milford, MA) at a constant flow rate of 0.1 ml/min in 50 mM Tris-HCl (pH 7.6) containing 150 mM NaCl. Protein standards (either High Molecular Weight Gel Filtration Calibration kit from Amersham Biosciences, Piscataway, NJ, or Gel
Filtration Standard from Bio-Rad, Hercules, CA) were prepared according
to the manufacturer's specifications and loaded onto the column (90-ml
bed volume). Fractions (0.75 ml) were collected, and the elution
volumes (Ve) determined by measuring the protein concentration of each fraction by the Bradford method, as described above. The column void volume (V0) was assessed
using blue dextran 2000. Kav values for each
protein were calculated using the equation, Kav = (Ve Mass Spectrometry--
Mass measurements were performed by
electrospray-ionization mass spectrometry using a Model 3000 ion trap
mass spectrometer (Bruker Instruments, Billerica, MA). Prior to
determining its mass spectrum, each peptide or protein was desalted by
microbore reversed-phase high-performance liquid chromatography.
Residues 164-192 of Pik1 Are Necessary for the Binding of
Frq1--
We previously demonstrated that Frq1 associates tightly with
Pik1 and that a fragment of the N-terminal domain of Pik1 comprising residues 10-192 is sufficient to mediate this interaction (29). We
also noted before (19) that this segment of Pik1 contains a sequence
element (residues 35-110), distinct from the catalytic domain
per se, that is weakly conserved among PtdIns 3-kinase, PtdIns 4-kinases, and even more distantly related enzymes that appear
to be protein kinases, such as the Tor proteins. This motif has been
referred to subsequently as the "lipid kinase unique domain" (LKU)
(12, 28, 53). Because Frq1 binds to the region of Pik1 that contains
the LKU, we suggested that the motif itself might be the binding site
for this regulatory protein (29).
To test this hypothesis directly and to begin to define the minimal
region in Pik1 responsible for Frq1 binding, we constructed three
deletion derivatives of the Frq1-binding Pik1-(10-192) fragment. One
deletion ( Residues 151-199 of Pik1 Are Sufficient for Binding to
Frq1--
To determine if the region of Pik1 from 164 to 192, or some
sub-domain of it, was sufficient for Frq1 binding, we used an approach
involving competition by synthetic peptides corresponding to different
portions of Pik1 (Fig.
2A). For these
binding experiments, the arrangement was reversed. In this case,
[35S]Pik1-(10-192), prepared by coupled in
vitro transcription and translation, was incubated with either
empty Ni2+-saturated NTA-agarose beads (as a control for
nonspecific binding) or the same beads coated with purified
Frq1-His6, preincubated in the absence or the
presence of increasing concentrations (1 nM to 20 µM) of the competing peptides. We found that the
36-residue peptide corresponding to residues 164-199 was unable to
block the binding of [35S]Pik1-(10-192) to immobilized
Frq1 (Fig. 2B), and, not surprisingly, a shorter 26-residue
peptide, residues 174-199, was also ineffective (data not shown).
Thus, although residues 164-192 of Pik1 are necessary for Frq1
binding, they are not sufficient. Indeed, we found that a longer
49-residue peptide, corresponding to residues 151-199, was able to
prevent the binding of [35S]Pik1-(10-192) to Frq1 in a
dose-dependent manner (Fig. 2B). Hence, we
conclude that the Frq1-binding site includes residues in Pik1 upstream
of position 164.
The results from three independent trials measuring the ability of the
49-residue peptide (151-199) to compete for the binding of
[35S]Pik1-(10-192) to bead-immobilized
Frq1-His6 were quantitated, after correcting for the
minimal background binding of [35S]Pik1-(10-192) to
empty beads at each peptide concentration (Fig. 2C). The
resulting data were then normalized to the amount of radioactivity
bound in the absence of peptide and fitted to the following equation:
[Frq·Pik(+Pep)]/[Frq·Pik( Structural Characterization of Frq1 Target Complexes Using
NMR--
NMR spectroscopy was used to examine whether any
conformational changes occurred in Frq1 upon binding to either the
49-residue synthetic peptide (151-199) or to a somewhat larger
83-residue fragment of Pik1, Pik1-(110-192), prepared by expression in
and purification from bacterial cells (see "Experimental
Procedures"). Two-dimensional NMR experiments
(1H-15N HSQC) were performed on samples of
uniformly 15N-labeled Ca2+-free and
Ca2+-bound forms of Frq1, prepared as described previously
(35), in the presence and absence of saturating amounts of either the 151-199 peptide or the Pik1-(110-192) fragment. Initial attempts to
prepare NMR samples of Frq1 complexed with the 151-199 peptide were
unsuccessful, because simple addition of the peptide to a solution of
Frq1 at a concentration (0.4 mM) sufficient for NMR analysis resulted in irreversible denaturation and aggregation. In
contrast, when a dilute solution of Ca2+-bound Frq1 (20 µM) was added slowly to an equal volume of a dilute solution of the Pik1 peptide or Pik1 fragment (20 µM), a
soluble complex was produced that could then be concentrated more than 20-fold to yield a stably soluble sample adequate for NMR studies. This
approach was only successful in preparing complexes with Ca2+-bound Frq1. Mixtures of Ca2+-free Frq1
with the Pik1 peptide or Pik1 fragment were much less soluble and,
hence, were not characterized further.
Two-dimensional NMR (1H-15N HSQC) spectra for
Ca2+-bound Frq1 in the absence and presence of
Pik1-(110-192) are shown in Fig. 3. The
spectrum of Ca2+-bound Frq1 alone (Fig. 3A) was
analyzed previously (35). Free Ca2+-bound Frq1 contains
fewer peaks than the expected number of amide groups in the protein
(180 versus 220), apparently because some of the amide
resonances have extremely weak intensity and therefore escape
detection. Moreover, the wide range of NMR peak intensities suggested
that Ca2+-bound Frq1 exists as a somewhat heterogeneous
population of species. In agreement with this conclusion, dynamic light
scattering measurements performed on the sample of free
Ca2+-bound Frq1 used for the NMR analysis was indicative of
significant polydispersity, suggesting that Ca2+-bound Frq1
consists of a heterogeneous mixture of monomer, dimer, and higher
oligomeric species under the concentration conditions required for
NMR.
In marked contrast, the NMR spectrum of Ca2+-bound Frq1 in
the presence of a saturating amount of Pik1-(110-192) (Fig.
3B) looked quite different from that of free
Ca2+-bound Frq1, suggesting that Pik1 binding induces
conformational changes in Frq1. The NMR spectrum of
Ca2+-bound Frq1 in the presence of the 151-199 peptide
looked essentially identical to that of the Frq1·Pik1-(110-192)
complex (data not shown). The spectrum of each complex exhibits
significantly sharper peaks compared with those in the spectrum of
Ca2+-bound Frq1 alone, suggesting, first, that Frq1 is
monomeric in each complex. Second, in the spectrum of the complex, some
new peaks are observed that are not seen in the spectrum of free
Ca2+-bound Frq1 (compare Fig. 3, A and
B). The total number of observable peaks in the spectra of
the complexes of Ca2+-bound Frq1 with either the 151-199
peptide or the Pik1-(110-192) fragment are now very close to the
expected number of amide resonances (218 versus 220). Third,
the intensities of all peaks in the spectra of the complexes are much
more uniform than those of free Ca2+-bound Frq1, suggesting
that both complexes are stable and homogeneous. In agreement with this
conclusion, dynamic light scattering measurements performed on the
sample of Ca2+-bound Frq1 at a saturating concentration of
Pik1-(110-192) that was used for NMR analysis indicated a monodisperse
scattering profile with a molecular mass of ~35 kDa, consistent with
a monomer of Ca2+-saturated Frq1 (22.1 kDa) bound to one
molecule of the His6-tagged 85-residue Pik1-(110-192)
fragment (10.0 kDa). Finally, the changes in the NMR spectrum of
Ca2+-bound Frq1 induced in the presence of Pik1-(110-192)
reached saturation upon the addition of 1 molar equivalent of
Pik1-(110-192), an observation also consistent with formation of a
high affinity 1:1 complex.
The sample of Ca2+-bound Frq1 complexed with the 151-199
peptide that was used for NMR analysis also exhibited a monodisperse light scattering profile indicative of a homogeneous species. However,
unexpectedly, despite the smaller size of the 49-residue peptide (5.6 kDa), the molecular mass of this complex was somewhat larger (~36
kDa) than that observed for the complex of Ca2+-bound Frq1
with the Pik1-(110-192) fragment, suggesting the species present was
monomeric Ca2+-saturated Frq1 bound to two molecules of the
151-199 peptide. In agreement with this interpretation, the changes in
the NMR spectrum of Ca2+-bound Frq1 induced by the presence
of the 151-199 peptide saturated upon addition of 2 molar equivalents
(data not shown), consistent with formation of a high affinity
peptide·Frq1 complex with a stoichiometry of 2:1. Moreover, the NMR
spectrum of Ca2+-bound Frq1 in the presence of 1 molar
equivalent of the 151-199 peptide (data not shown) resembled the
composite that would be expected from equal parts of the spectra (Fig.
3, A and B) for free and fully complexed
Ca2+-bound Frq1. Thus, it seems that in the presence of 1 molar equivalent of the 151-199 peptide, half of the Frq1 molecules
are peptide-bound and half are unbound. Furthermore, these observations
indicate that the population of peptide-bound Frq1 molecules exchanges with the unbound species only very slowly, such that the dissociation rate must be slower than the time scale of NMR chemical shift, as
expected for a high affinity complex. On this basis, we estimate that
the dissociation constant for binding of the 151-199 peptide to
Ca2+-bound Frq1 in solution must be in the nanomolar range.
Pik1-Frq1 Interaction Monitored by Fluorescence
Spectroscopy--
The 190-residue Frq1 polypeptide contains just
two tryptophan residues (Trp-30 and Trp-103). The fluorescence
emission of Trp is very sensitive to its surrounding chemical
environment and, hence, provides a well-documented method for probing
structural changes in a protein (54). Hence, we used the change in the intrinsic Trp fluorescence of Frq1 as an independent means both to
monitor the effects of and to quantitate the binding of Frq1 to the
Pik1-derived fragment and the synthetic peptide (see Fig. S1 in the
Supplemental Material). Neither the Pik1-(110-192) fragment nor
the 151-199 peptide contain any Trp; as expected, the fluorescence spectra of neither the Pik1-(110-192) fragment alone nor the 151-199 peptide alone exhibited any significant emission over the range of
wavelengths examined. The fluorescence emission spectrum of free
Ca2+-bound Frq1 exhibited a maximum at 340 nm, as observed
before (35). Addition of a saturating concentration of either the
Pik1-(110-192) fragment or the 151-199 peptide increased the emission
intensity by 30% and caused the emission maximum to shift very
slightly toward the blue. These results indicate that, upon binding of the Pik1 sequences, conformational changes in Frq1 occur that cause one
or both of its Trp residues to become more constrained and/or to enter
a less solvent accessible and more non-polar environment. The emission
intensity of Frq1 increased linearly with the addition of the
Pik1-(110-192) fragment in the range from 0 to 1 molar equivalents,
and no further increase was observed when more than 1 equivalent was
added. Thus, in agreement with the conclusions drawn from the NMR and
light scattering analysis, the stoichiometry of the complex of
Pik1-(110-192) with Ca2+-bound Frq1 is 1:1. In contrast,
the emission intensity of Frq1 increased linearly with the addition of
the 151-199 peptide in the range from 0 to 2 molar equivalents, and no
further increase of intensity was observed when more than 2 molar
equivalents of peptide was added. Again, this result agrees with the
conclusions drawn from the NMR and light scattering analysis, which
indicated that the stoichiometry of the complex of the 151-199 peptide
with Ca2+-bound Frq1 is 2:1.
Energetics of Pik1-Frq1 Interaction--
As yet another
independent means to examine the association of Frq1 with its apparent
binding site in Pik1, we used isothermal titration calorimetry, which
also permitted assessment of the energetics of the binding interaction
between Ca2+-bound Frq1 and either the Pik1-(110-192)
fragment (Fig. 4, A and
B) or the 151-199 peptide (Fig. 4, C and
D). These calorimetric titrations were conducted at 25 °C
in 10 mM HEPES (pH 7.4). In both cases, the heat change was
endothermic, indicating that the binding reaction is largely
entropically driven, most consistent with desolvation of one or both
partners and a hydrophobic interaction between them. After correction
for the heats of dilution (see "Experimental Procedures") and
normalization, the concentration dependence of the absorbed heats were
plotted for the interaction of Ca2+-bound Frq1 with the
Pik1-(110-192) fragment (Fig.
5B) and with the 151-199
peptide (Fig. 5D). Once the ratio of Ca2+-bound
Frq1 to the Pik1-(110-192) fragment exceeded 1:1, the heat no longer
changed, again consistent with a one-to-one complex. However, the
binding of Frq1 to Pik1-(110-192) was multiphasic and was fit to a
two-site model, yielding dissociation constants (KD1 = 62 nM and
KD2 = 200 nM) and positive (non-favorable) enthalpies of binding ( Size Determination of Native Pik1·Frq1 Complexes by Size
Exclusion Chromatography--
Despite the finding that the
Pik1-(110-192) fragment formed a 1:1 complex with
Ca2+-bound Frq1, the fact that the smaller 151-199 peptide
could form 2:1 complexes with Ca2+-bound Frq1, raised the
possibility, albeit remote, that in vivo Frq1 might serve to
bridge two Pik1 molecules and thereby promote formation of enzyme
dimers. To determine if Pik1 and Frq1 form stable complexes in cell
extracts and to estimate their apparent molecular mass, analytical size
exclusion chromatography was performed on extracts of yeast cells
(strain YPH499) overproducing an epitope-tagged derivative of Pik1,
which is otherwise quite an inabundant protein (18). For this purpose,
the yeast cells carried a low copy number (CEN) plasmid
expressing from the Gal-inducible GAL1 promoter full-length
Pik1 containing an in-frame N-terminal c-Myc epitope. This construct is
able to fully complement the inviability of a pik1 Site-directed Mutagenesis of the Frq1 Binding Region in
Pik1--
Comparison of Pik1 to the sequences of type III PtdIns
4-kinases from other organisms indicated that the region of Pik1 that is necessary and sufficient for Frq1 binding (residues 151-192) shows
weak, but detectable, conservation. To determine whether any of the
most conserved residues are critical for Frq1 binding, we generated
derivatives of the Frq1-binding fragment,
Pik1-(10-192)-(His)6, carrying a variety of site-directed
mutations. These mutants included two double mutants, Pik1(10-192;
P181A,V183A) and Pik1(10-192; R188A,R189A), a triple mutant,
Pik1(10-192; L175A,P181A,V183A), and a quadruple mutant
Pik1(10-192; E154A,N155A,V156A,P158A). The resulting constructs
were expressed in and purified from E. coli, immobilized on
Ni2+-saturated NTA-agarose bead, and their ability to bind
[35S]Frq1 in vitro was examined, as described
above (see Fig. 1). None of the four mutants proteins showed any
significant reduction in their capacity to bind radiolabeled Frq1 under
these conditions (data not shown). In two of the mutants, charged or
polar side chains (Glu-154 and Asn-155, and Arg-188 and Arg-189,
respectively) were replaced with the hydrophobic residue, Ala.
Likewise, all of the other substitutions replaced more bulky
hydrophobic side chains with the less bulky, but nevertheless
non-polar, Ala residue. Thus, all of the mutations, although perturbing
individual resides, did not dramatically change the overall hydrophobic
character of this region. The fact that Frq1 binding was not affected
by these alterations provides a further indication that the association of Frq1 with Pik1 is largely a hydrophobic interaction, fully consistent with the data from calorimetry (Fig. 4) and fluorescence spectroscopy (see Fig. S1 in the Supplemental Material). Moreover, preliminary NMR analysis of the NOE patterns for the complex of 15N-labeled Pik1-(110-192) with Ca2+-bound
Frq1 indicates that a 13-residue segment in this region (Ala-157 to
Ala-169) that contains no charged residues (and was not altered
significantly in any of the site-directed mutants we generated) makes
intimate contact with Frq1 (see "Discussion").
Deletion of the Frq1-binding Site Compromises Pik1
Function in Vivo--
In contrast to the substitution mutations,
truncations of Pik1-(10-192) did greatly impair Frq1 binding (see Fig.
1). Therefore, to determine whether the region of Pik1 found to be
necessary and sufficient for Frq1 binding in vitro is also
required for association of Frq1 with Pik1 in vivo and plays
a role in the physiological function of this enzyme, we generated a
mutant allele, pik1(
Before testing its phenotype, we wanted to confirm that the
pik1(
To examine the consequences of defective Frq1 binding on the function
of Pik1 in vivo, a heterozygous
pik1
We have demonstrated before that, when PIK1 is highly
overexpressed, it is able to support the growth of frq1 Cys-15 Is Neither S-Palmitoylated in Vivo nor Required for Frq1
Function--
One apparent role for the association of Frq1 with Pik1
is to promote its association with membranes, and we have shown
previously that the N-terminal myristoyl group of Frq1 is important,
but not essential, for this function (29, 35). First, we found that,
like wild-type Frq1, a mutant, Frq1(G2A), that cannot be and is not
myristoylated is capable of rescuing the inviability of
frq1
First, we generated a Frq1(C15A) single mutant and found that its
properties were indistinguishable from those of wild-type Frq1.3 Next, we generated a
Frq1(G2A,C15A) double mutant and examined its ability to rescue the
temperature-sensitive lethality of frq1-1ts cells
(strain YKBH4, Table I) when expressed from the native FRQ1
promoter in a multicopy vector. The empty vector was unable to permit
cell growth at the non-permissive temperature, as expected, whereas
both normal FRQ1 and frq1(G2A,C15A) expressed
from the same vector suppressed the temperature-sensitive phenotype of the frq1-1ts cells (Fig.
7A). Likewise, when expressed
from the same vector, expression of both normal Frq1 and Frq1(G2A,C15A)
was able to restore viability to otherwise inviable spores carrying a
frq1
Second, to determine whether Frq1 is S-palmitoylated at
either Cys-15 or Cys-38 in vivo, we tagged the 3'-end of the
FRQ1 gene with a sequence encoding an in-frame
His6 tag, expressed the protein product in yeast cells,
purified the protein to apparent homogeneity by chromatography on
Ni2+-saturated NTA-agarose, and performed electrospray
ionization mass spectrometry on the purified protein. The calculated
molecular mass for Frq1-His6 with an N-terminal myristoyl
group (but lacking any palmitoyl moiety) is 23,839.90 Da. After
deconvolution of the spectrum of mass species observed (see Fig.
S4B in the Supplemental Material), we found a single
homogenous component with a molecular mass of 23,838.18 Da. We
conclude, therefore, that N-myristoylation at Gly-2 is the
only lipophilic modification present in native Frq1 and,
correspondingly, that neither Cys-15 nor Cys-38 is
S-palmitoylated. Thus, S-palmitoylation is not
required for the function of Frq1, as also confirmed by our mutagenesis
and phenotypic tests.
We showed previously using primarily genetic means that the sole
essential target of the small Ca2+-binding protein, Frq1,
in the yeast S. cerevisiae is the PtdIns 4-kinase, Pik1
(29). At the biochemical level, Frq1 is present in a stoichiometric
amount in preparations of Pik1 (18) that were purified more than
25,000-fold by ammonium sulfate fractionation followed by
chromatography on five different columns in buffers lacking
Ca2+ and containing chelator (EDTA). Thus, Frq1 is
constitutively bound to Pik1, even in the absence of Ca2+,
and should be considered a non-catalytic subunit of the enzyme. Indeed,
we demonstrated, using an assay in which the substrate (PtdIns) was
displayed in detergent micelles, rather than in authentic biological
membranes, that the presence of Frq1 is required for optimal activity
of the enzyme (29). As judged by cell fractionation experiments and
other methods, Frq1 associates with the membrane-containing particulate
fraction in a manner that depends on both its N-terminal myristoyl
group and Ca2+-induced changes in the protein (35). Thus,
in vivo, Frq1 associated with Pik1 presumably also assists
in targeting Pik1 to membranes in a
Ca2+-dependent manner. In our prior work, based
on both genetic and biochemical approaches, we showed that the region
of Pik1 responsible for Frq1 binding seemed to reside within the first
~200 residues of the protein. In this present study, we sought to
further define the Frq1-binding site and to characterize the nature of
this interaction in greater detail.
We generated deletions in an His6-tagged 183-residue
fragment of Pik1, Pik1-(10-192)-(His)6, that, when
immobilized on Ni2+-saturated NTA-agarose beads, is able to
capture [35S]Frq1 from solution with high affinity. Using
this in vitro binding method, we found that the region from
164 to 192 was essential for the observed interaction, whereas the
segment from residue 31 to 79 was totally dispensable. However, using
competition assays with synthetic peptides, we found that the 164-192
region of Pik1 was not sufficient for Frq1 binding. A peptide
corresponding to residues 164-199 was not able to prevent the binding
of [35S]Pik1-(10-192) to bead-immobilized
Frq1-His6, whereas a longer peptide corresponding to
residues 151-199 did compete. Thus, residues N-terminal to the
164-192 region are also important for high affinity binding of Frq1 to
Pik1. It also appears that the segment corresponding to residues
80-109 is dispensable for the interaction of Frq1 with Pik1, because a
smaller 83-residue fragment of Pik1, Pik1-(110-192), bound to Frq1
with high affinity and a 1:1 stoichiometry, as judged by NMR, intrinsic
Trp fluorescence, titration calorimetry, and light scattering.
Revealingly, the 151-199 peptide also bound to Frq1 avidly, however,
as judged by the same criteria (NMR, intrinsic Trp fluorescence, titration calorimetry, and light scattering), the stoichiometry of
peptide:Frq1 binding was 2:1, unlike that of the Pik1-(110-192) fragment. Moreover, as judged by calorimetric measurements in solution,
the association of Frq1 with the 83-residue Pik1-(110-192) fragment
was tighter (apparent Kd = 62 nM) than
its association with the 49-residue 151-199 peptide (apparent
Kd = 140 nM). When measured by
competition for the binding of [35S]Pik1-(10-192) to
bead-bound Frq1-His6, the Ki for the 151-199 peptide was 1 µM. However, in this assay format
and given that a tracer level of the radioactive probe was used,
competition was only observed when the amount of peptide added
approached that of the large excess of immobilized Frq1 present.
Nevertheless, the 151-199 peptide may be missing residues upstream of
151, which are present in Pik1-(110-192), that also contribute to
maximal binding affinity.
The intriguing 2:1 stoichiometry observed for binding of the 151-199
peptide suggests that the Pik1-binding site in Frq1 may be, in some
sense, bipartite. Indeed, preliminary HSQC spectra for
[15N]Pik1-(110-192) bound to Ca2+-saturated
Frq1 indicate that contact is made with two short segments in the Pik1
fragment of 12 residues (Phe-125 to Gln-136) and 13 residues (Ala-157
to Ala-169), respectively, both of which assume an We derived a model for the three-dimensional structure of
Ca2+-bound yeast Frq1 based on NMR analysis in solution
(35). A very similar structure for the Ca2+-bound form of
its human ortholog, NCS-1, was determined by x-ray analysis and refined
to 1.9-Å resolution (36). Both structures revealed that frequenin has
two tightly folded domains that pack against each other to form an
accessible crevice lined primarily with hydrophobic side chains. This
hydrophobic cleft would seem the mostly likely site for binding of the
13-residue hydrophobic segment in the Frq1-binding region of Pik1. By
contrast, the 12-residue segment in the Pik1-(110-192) fragment
(-FQVARRVLNNLQ-) that also appears to associate with Frq1 contains both
polar and charged residues, the most striking feature of which is a
tandem pair of Arg residues (Arg-129 and Arg-130). We note that,
although the 151-199 peptide lacks this basic hydrophilic sequence
found in Pik1-(110-192), there is a 12-residue segment at the
C-terminal end of the 151-199 peptide that has some similarity
(-ESQGRRQKAFVF-), including a tandem pair of Arg residues (Arg-188 and
Arg-189). Thus, it is possible that the 151-199 peptide binds to Frq1
with a 2:1 stoichiometry because one copy of the peptide uses the
13-residue hydrophobic motif (Ala-157 to Ala-169) near its N-terminal
to correctly occupy the corresponding hydrophobic pocket in Frq1, and
another copy of the peptide uses the basic segment near its C-terminal
end to artifactually occupy the site in Frq1 that would normally
recognize the 12-residue hydrophilic motif (Phe-125 to Gln-136).
Indeed, our site-directed mutagenesis experiments indicated that the
Arg-188/Arg-189 pair is not required for the binding of Frq1 to
Pik1-(10-192), consistent with the view that normally it is the
upstream Arg-129/Arg-130 pair that fulfills this function.
The fact that a single Frq1 was able to bind two 151-199 peptides
raised the possibility that one role of Frq1 in vivo might be to promote dimerization of Pik1. However, the fact that the Pik1-(110-192) fragment bound to Pik1 with a 1:1 stoichiometry made
such a dimerization scenario unlikely. Nonetheless, there is precedent
for protein dimerization being induced by the binding of small, EF-hand
type, Ca2+-binding proteins, most notably the
oligomerization of the SK class of potassium channels promoted
by calmodulin binding (57). Hence, we examined the nature of the native
Frq1·Pik1 complexes found in yeast cell extracts by size-exclusion
chromatography. Consistent with the binding of Pik1-(110-192) to Frq1
in vitro, we found that the bulk of the Pik1·Frq1
complexes in yeast cell extracts (Peak II) had an apparent molecular
mass most consistent with a 1:1 complex. However, we did observe a
fraction (Peak I) that contained both Pik1 and Frq1 that had a much
larger apparent size. Because the extracts were prepared in the absence
of detergent, and Frq1 has a propensity to interact with membranes,
this fraction most likely represents the 1:1 Pik1·Frq1 complex
associated with small vesicles or membrane fragments. On the other
hand, we cannot rule out the possibility that this fraction indeed
represents a higher order Pik1·Frq1 oligomer or a novel complex of
Pik1·Frq1 that contains other tightly associated polypeptides.
We demonstrated previously that Frq1 lacking its N-terminal myristoyl
group retains a residual capacity to associate with membranes in a
Ca2+-dependent manner (35). This behavior could
be due to greater exposure of previously buried hydrophobic side chains
upon Ca2+-induced conformational change. Alternatively,
however, it was possible that some other lipophilic substituent is also
present in Frq1 that becomes more solvent exposed when Ca2+
binds to the protein. In other small, EF-hand type,
Ca2+-binding proteins, such as a flagellar regulator in
Trypanosoma cruzi (38) and the novel mammalian potassium
channel regulator, KChIP2 (39), Cys residues located near the N
terminus are S-palmitoylated and are important for the
ability of those proteins to associate with membranes. However, using
mass spectrometry we found that Frq1 purified from yeast is fully
N-myristoylated but not palmitoylated on either of its two
Cys residues (Cys-15 and Cys-38). Moreover, site-directed mutagenesis
of the most N-terminal Cys (Cys-15) did not compromise the function of
Frq1 in vivo, nor did it exacerbate the effects of a
mutation (G2A) that prevents N-myristoylation of Frq1.
Therefore, S-palmitoylation is not involved in the
physiological function of Frq1.
Taken together, our data support the conclusion that a single molecule
of Frq1 docks onto a single molecule of Pik1 and does so by binding to
a site that includes as its core a 13-residue hydrophobic sequence
(Ala-157 to Ala-169). As expected if this region is critical for high
affinity binding of Frq1 to Pik1, we found that a deletion mutation,
pik1(152-191), did not
co-immunoprecipitate efficiently with Frq1 and did not support growth
at elevated temperature. Site-directed mutagenesis of Pik1-(10-192)
suggested that recognition determinants lie over an extended region.
Titration calorimetry demonstrated that binding of an 83-residue
fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high
affinity (Kd ~100 nM) and is largely
entropic, consistent with hydrophobic interaction. Stoichiometry of
Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration
calorimetry, by changes in NMR spectrum and intrinsic tryptophan
fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1
exist mainly in a heterodimeric complex, as shown by size exclusion
chromatography. Cys-15 in Frq1 is not
S-palmitoylated, as assessed by mass spectrometry; a
Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A)
double mutant rescued the inviability of frq1
cells.
This study defines the segment of Pik1 required for high affinity
binding of Frq1.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(43) was used for routine
manipulation and propagation of plasmids. Unless otherwise indicated,
all PCR reactions were performed using Pfu DNA polymerase
(Stratagene, La Jolla, CA). All recombinant plasmids were verified by
dideoxy chain termination sequencing.
31-79) was produced by
cleaving pET23d-PIK1(10-192) with EcoRI and
NcoI and religating the plasmid after filling in the
recessed 3'-ends using Klenow fragment of E. coli DNA
polymerase I. pET23d-PIK1(10-163) and pET23d-PIK1(10-125) were generated by PCR amplification of
pET23d-PIK1(10-192) using as primers an oligonucleotide
spanning the NcoI site of PIK1 and
oligonucleotides introducing a NotI site 3' to the sequences encoding, respectively, either codon 163 or codon 125 of the
PIK1 open-reading-frame. The resulting PCR fragments were
ligated into pET23d-PIK1(10-192) that had been cleaved
with NcoI and NotI. An
XbaI-XhoI fragment of
pET23d-PIK1(10-192) was inserted into Litmus28 (New
England BioLabs, Beverly, MA) cleaved with the same enzymes, to
generate the cloning intermediate, Litmus28-PIK1(10-192). Litmus28-pik1(10-192; P181A,V183A) and
Litmus28-pik1(10-192; L175A,P181A,V183) were generated by
exchanging the TfiI fragment in
Litmus28-PIK1(10-192) with TfiI-digested PCR
products carrying the appropriate substitutions, which were introduced
by site-directed mutagenesis using a commercial kit (QuikChange,
Stratagene, La Jolla, CA), according to the manufacturer's instructions. The XbaI-XhoI fragments from
Litmus28-pik1(10-192; P181A,V183A) and
Litmus28-pik1(10-192; L175A,P181A,V183) were then
transferred back into pET23d that had been cleaved with XbaI and XhoI, to generate pET23d-pik1(10-192;
P181A,V183A) and pET23d-pik1(10-192; L175A,P181A,V183). A PCR product carrying the appropriate
substitutions and a XhoI site 3' to sequences encoding
residue 192 of Pik1, all introduced by site-directed mutagenesis, was
cleaved with EcoRI and XhoI and inserted into the
corresponding sites of pET23d-PIK1(10-192), yielding
pET23d-pik1(10-192; R188A,R189A).
pET23d-pik1(10-192; E154A,N155A,V156A,P158A) was
constructed using a three-way ligation strategy, as follows.
pET23d-PIK1(10-192) was cleaved with EcoRI and
PstI and ligated with a PCR product (carrying the
appropriate substitutions to produce E154A, N155A, and V156A)
that had been cleaved with EcoRI and PvuII and a
second PCR product (carrying the appropriate substitutions to produce
N155A, V156A, and P158A) cleaved with PvuII and
PstI. pET23d-FRQ1 has been described previously (29, 35).
152-191) was generated
by insertion of a PCR-derived HindIII fragment, encoding
residues 10-151 of Pik1, into Litmus28-PIK1, that had been
cleaved with HindIII.
pRS314-PIK1(
152-191) was produced by
subcloning the EcoRI fragment of
Litmus28-PIK1(
152-191) into pRS314-PIK1. Inserting the BamHI-SacI
fragment of pRS314-PIK1(
152-191) into
pRS314-GAL1,10 generated
pRS314-GAL-PIK1(
152-191).
pRS314-GAL-mycPIK1(
152-191) was
constructed by replacing the NcoI-SacI fragment
of pRS314-GAL-mycPIK1 with the corresponding fragment from
pRS314-GAL-PIK1(
152-191).
20 °C.
-c-Myc mAb 9E10 (52), 1.5 µl of
-Frq1, or
1.5 µl of pre-immune serum from the same rabbit, which have been
described before (29). To capture the soluble immune complexes, a fresh
aliquot (30 µl) of the suspension of protein G-/protein A-coupled
agarose beads was added and the mixture was incubated on the roller
drum for 45 min at 4 °C. Bead-bound immune complexes were collected
by brief centrifugation in a microcentrifuge, washed four times
with lysis buffer (1 ml each), resuspended in SDS-PAGE sample buffer,
and solubilized by boiling in a water bath for 5 min. After removal of
the beads by centrifugation, equal volumes of the resulting supernatant
fractions were resolved by SDS-PAGE and analyzed by immunoblotting with
appropriate antibodies.
V0)/(Vt
V0), where V0 = 36 ml and
Vt = 90 ml, and plotted semilogarithmically
against the corresponding molecular weight. To examine the apparent
molecular weight of native Pik1·Frq1 complexes in yeast cell
extracts, strain YPH499, transformed with pRS314-GAL-mycPIK1, was grown to
A600 nm = 0.6 in SCRaf-Trp at 30 °C and
induced by addition of Gal (2% final concentration) and incubated for
1.5 h. Induced cells were collected by centrifugation, washed once
with distilled water, resuspended in ice-cold 50 mM Tris-HCl (pH 7.6) containing 150 mM NaCl and protease
inhibitors (CompleteTM, Promega, Madison, WI). The washed
cells were broken by 10 pulses (1 min each) of vigorous vortex mixing
with glass beads. The resulting crude extract was clarified by
centrifugation in a TL-100 tabletop ultracentrifuge (Beckman Coulter
Inc., Fullerton, CA) at 49,000 × g for 30 min at
4 °C. The protein concentration of the clarified extract was
determined, and 250 µl (~2.5 mg of total protein) was loaded onto
the gel filtration column. Fractions (0.75 ml) were collected, and
protein was concentrated by precipitation with 10% trichloroacetic
acid in the presence of 0.15% deoxycholate for 10 min at room
temperature. The precipitates were dissolved in 30 µl of SDS-PAGE
sample buffer, and the remaining acid was neutralized by addition of 5 µl of an unbuffered saturated solution of Tris. The resulting
fractions were split into two, resolved by SDS-PAGE, and analyzed
separately by immunoblotting with either anti-c-Myc or anti-Frq1
antibodies, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
31-79) removed the most conserved core of the LKU motif;
the other two deletions were truncations that removed 29 residues
(
164-192) and 67 residues (
126-192), respectively, from the
C-terminal end (Fig. 1A).
Full-length Pik1-(10-192) and each of the three deletions were tagged
with a C-terminal His6 tract, expressed in and purified
from E. coli, and immobilized on Ni2+-saturated
NTA-agarose beads. Solubilization and analysis by SDS-PAGE followed by
staining with Coomassie Brilliant Blue demonstrated that equivalent
amounts of each construct were affixed to the beads (Fig.
1B, lower panel). The beads were then incubated
with [35S]Frq1, prepared by coupled in vitro
transcription and translation, washed, and subjected to SDS-PAGE, and
the amount of radiolabeled Frq1 bound was analyzed by autoradiography.
As observed previously using unlabeled Frq1 (29), Pik1-(10-192) bound
radiolabeled Frq1 avidly, and there was little or no nonspecific
binding to empty beads (Fig. 1B, upper panel).
Strikingly, the 49-residue deletion lacking the heart of the LKU motif
showed no detectable diminution in its ability to bind
[35S]Frq1. In marked contrast, even the shortest
(29-residue) C-terminal truncation completely ablated Frq1 binding.
This result suggested that residues in the region 164-192 of Pik1 are
necessary for the high affinity binding of Frq1. Moreover, these data
indicated that the LKU motif does not mediate the interaction between
Pik1 and Frq1.
View larger version (27K):
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Fig. 1.
In vitro binding of
[35S]Frq1 to immobilized
Pik1-(10-192)-(His)6 and derived mutants. A,
schematic diagram of Pik1 (top bar) and, in an expanded
view, the N-terminal Pik1-(10-192) fragment (second bar)
and an internal deletion mutant and two C-terminal truncation mutants
derived from Pik1-(10-192). The LKU domain (residues 35-110) and the
C-terminal catalytic domain (residues 792-1066) are also indicated.
B, Pik1-(10-192)-(His)6 and the three derived
mutants shown in A were expressed in bacteria, purified,
bound to Ni2+-saturated NTA-agarose, and incubated with
[35S]Frq1 prepared by in vitro translation.
Bead-bound radioactivity was solubilized by boiling in SDS-gel sample
buffer, resolved by SDS-PAGE, and detected by autoradiography
(upper panel). To verify purity and equivalent loading of
Pik1-(10-192) and the three derived mutants, bead-bound proteins were
subjected to SDS-PAGE and visualized by staining with Coomassie
Brilliant Blue dye (lower panel).
View larger version (14K):
[in a new window]
Fig. 2.
Competition by synthetic peptides for binding
of [35S]Pik1-(10-192) to immobilized
Frq1-(His)6. A, schematic representation of
three synthetic peptides spanning the Frq1-binding segment of the
N-terminal region of Pik1. B,
[35S]Pik1-(10-192), prepared by in vitro
translation (input, lane 1), was incubated with
either empty Ni2+-saturated NTA-agarose beads, in the
absence (lane 2) or the presence (lane 3) of
synthetic peptide, or with the same beads coated with
Frq1-His6, in the absence (lane 4) and at the
indicated concentrations of synthetic peptide (lanes 5-9).
Bead-bound radioactivity was solubilized by boiling in SDS-gel sample
buffer, resolved by SDS-PAGE, and detected by autoradiography
(upper panel). Competition by the 151-199 peptide
(upper panel) and the 164-199 peptide (lower
panel) are shown. C, results from three independent
trials of competition by the 151-199 peptide for binding of
[35S]Pik1-(10-192) to Frq1-His6 were
quantitated using a PhosphorImager (Amersham Biosciences,
Sunnyvale, CA) and corrected for the low nonspecific binding of the
probe to ovalbumin-blocked Ni2+-saturated NTA-agarose
beads at each peptide concentration used. The values obtained were then normalized to
the signal observed in the absence of any competing peptide and fit,
assuming a stoichiometry of [35S]Pik1-(10-192) binding
to Frq1-His6 of 1:1, to the following equation:
[Frq1·Pik1(+Pep)]/[Frq1·Pik1( Pep)] = a/b + c(1 + [Pep]/KI), where Frq1·Pik1(+Pep) is the
concentration of the complex of [35S]Pik1-(10-192) bound
to Frq1-His6 at a given peptide concentration and
Frq1·Pik1(
Pep) is the concentration of the complex of
[35S]Pik1-(10-192) bound to Frq1-His6 in the
absence of peptide, a, b, and c are
arbitrary constants, [Pep] is the concentration of the competing
peptide, and KI is the dissociation constant for
the binding of the peptide to Frq1-His6.
Pep)] = a/b + c(1 + [Pep]/KI), where
KI is the apparent dissociation constant for peptide binding, Frq·Pik(+Pep) is the amount of
radiolabeled Pik1-(10-192) bound to Frq1 at a given peptide
concentration and Frq·Pik(
Pep) is the amount of radiolabeled
Pik1-(10-192) bound to Frq1 in the absence of peptide and
a, b, and c are arbitrary constants.
Assuming a stoichiometry for binding of
[35S]Pik1-(10-192) to immobilized Frq1-His6
of 1:1, KI for binding of the 49-residue peptide
(151-199) to Frq1 was 1.1 ± 0.5 µM.
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Fig. 3.
Conformational changes in
Ca2+-bound Frq1 upon binding of the Pik1-(110-192)
fragment assessed by NMR. Two-dimensional
[15N]-[1H] HSQC NMR spectra of purified
unmyristoylated Ca2+-bound
[15N]Frq1-His6 in the absence (A)
and in the presence (B) of two molar equivalents of purified
unlabeled Pik1-(110-192) were recorded at 600-MHz
[1H] frequency. Peaks correspond to the proton
resonances of the backbone and side-chain amides, and the indicated
sequence-specific assignments for unmyristoylated
Ca2+-bound [15N]Frq1-His6 are as
reported before (35).
H1 = +5.4 kcal/mol and
H2 = +30 kcal/mol) for the
two sites, respectively. The multiphasic binding of
Ca2+-bound Frq1 to the Pik1-(110-192) fragment might be
explained, in part, by the fact that we observed, using dynamic light
scattering, that the starting solution of the Pik1-(110-192) fragment
alone displayed some polydispersity. This analysis indicated that more than 25% of the free Pik1-(110-192) fragment existed in a
pre-aggregated form, which might account for the highly endothermic and
poorer affinity phase of binding we observed
(KD2 = 200 nM and
H2 = +30 kcal/mol). By contrast, the bulk of
the population of the Pik1-(110-192) molecules was monomeric and
presumably accounts for the major, higher affinity phase of binding
(KD1 = 62 nM and
H1 = +5.4 kcal/mol). The binding of
Ca2+-bound Frq1 to the 151-199 peptide appeared
homogeneous and was fitted to a one-site model, yielding a
KD = 140 nM, and a
H for
binding = +9.5 kcal/mol. In these titrations, the heat no longer
changed when the ratio of Ca2+-bound Frq1 to the 151-199
peptide reached 0.5, again consistent with a complex containing two
peptides bound per Frq1 molecule.
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Fig. 4.
Isothermal titration calorimetry of the
binding of Ca2+-bound Frq1 to Pik1-(110-192) fragment or
151-199 synthetic peptide. A, trace of heat changes
observed upon addition of 19 aliquots (16 µl each) of purified
Ca2+-bound Frq1 to a calorimeter cell containing purified
Pik1-(110-192) fragment. B, integrated multiphasic binding
isotherm obtained from the experiment shown in A was
experimentally fit to a two-site model; parameters were obtained from
the best fit of the data (solid line). C, trace
of heat changes observed upon addition of 18 aliquots (16 µl each) of
purified Ca2+-bound Frq1 to a calorimeter cell containing
the 151-199 peptide. D, integrated monotonic binding
isotherm obtained from the experiment shown in C was
experimentally fit to a one-site model; parameters were obtained from
the best fit of the data (solid line).
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Fig. 5.
Assessment of Frq1-Pik1 interaction by
co-immunoprecipitation. Yeast cells (strain BJ2168; Table I)
overexpressing either full-length myc-Pik1 (lanes 1-3),
myc-Pik1( 152-191) (lanes 4-6), or untagged
Pik1(
152-191) (lanes 7 and 8) were grown and
lysed as described under "Experimental Procedures." Samples of the
extracts were subjected to immunoprecipitation with either rabbit
polyclonal anti-Frq1 antiserum (lanes 3, 6, and
8) or, as a control, pre-immune serum from the same rabbit
(lanes 1 and 4), or with mouse
-c-Myc mAb 9E10
(lanes 2, 5, and 7). The resulting
immune complexes were washed, solubilized, resolved by SDS-PAGE, and
visualized by immunoblotting with the anti-c-Myc mAb.
mutant, even when cells are propagated under conditions (Glc as the
carbon source) that repress the GAL1
promoter.2 Expression of
myc-Pik1 was induced by shifting the cells to Gal-containing medium for
a brief period and lysates were prepared under non-denaturing conditions in a buffer lacking detergent. The proteins in the resulting
lysates were resolved by size exclusion chromatography on a Superdex
200 column that had been pre-calibrated several times with a void
volume (Vo) marker and protein standards of known molecular mass (see "Experimental Procedures"), which did not
differ significantly in their elution behavior from run to run. The
protein content of the fractions obtained from chromatography of the
cell extracts were examined by subjecting samples to SDS-PAGE followed
by immunoblotting with appropriate antibodies (mouse anti-c-Myc mAb
9E10 to detect myc-Pik1 and rabbit polyclonal antibodies to detect
Frq1). Frq1 eluted in three peaks with Kav
values of 0.06 (Peak I, Fraction 44), 0.28 (Peak II, Fraction 60), and
0.61 (Peak III, Fraction 84) (see Fig. S2 in the Supplemental
Material). When plotted on the calibration curve that was prepared from
the Kav values calculated from the
experimentally determined elution volumes (Ve)
of the standards, these three peaks correspond to molecular masses of
724 (Peak I), 162 (Peak II), and 18 kDa (Peak III), respectively. Pik1
eluted in two peaks, both of which were virtually superimposable with
the two largest Frq1-containing peaks (I and II). Given the calculated
molecular masses of Frq1 (22.1 kDa), Pik1 (119.9 kDa), and myc-Pik1
(122.5 kDa), our results are most consistent with the view that Peak
III represents free monomeric Frq1, that Peak II represents a 1:1
complex of myc-Pik1 and Frq1 (calculated molecular mass of 144.6 kDa),
and that Peak I represents the myc-Pik1·Frq1 complex associated with
membrane fragments, because no detergent was included in the buffers
and native N-myristoylated Frq1 tends to associate with
membranes (35). The distribution and apparent stoichiometry of the
myc-Pik1·Frq1 complexes observed in these experiments undoubtedly
reflect the situation in cells in vivo for two reasons.
First, the same elution profile was observed when extracts were
prepared from untransformed YPH499 cells and the endogenous Pik1 was
detected using polyclonal anti-Pik1 antisera (29, 45), although the
signal was weaker (data not shown). Second, the abundance of endogenous
Frq1 exceeds that of endogenous Pik1 in all yeast strains examined to
date2 and, clearly, even when myc-Pik1 was overexpressed.
152-191), in which a
small (40-residue) internal deletion removes the apparent Frq1-binding site.
152-191) mutation does not adversely
affect the catalytic activity of the enzyme per se but does
compromise the ability of Frq1 to bind to Pik1. For this purpose, we
prepared extracts from yeast cells expressing from the GAL
promoter on CEN plasmids versions of either wild-type Pik1
or Pik1-(152-191) tagged at the N terminus with the c-Myc epitope, or,
as a negative control, untagged Pik1-(152-191). These extracts were
then subjected to immunoprecipitation with either anti-Frq1 antibodies
or, as a control, with pre-immune serum from the same rabbit. The
resulting immune complexes were resolved by SDS-PAGE, transferred to
filters, and the amount of Pik1 that co-immunoprecipitated determined
by immunoblotting with anti-Myc mAb. Samples of the same extracts were
also directly immunoprecipitated with the anti-Myc mAb to determine the
level of production of the tagged Pik1 proteins, and to examine their
activity. First, the c-Myc epitope tag allowed for highly sensitive,
specific, and background-free detection, because no signal was observed
after immunoprecipitation of the extract expressing untagged
Pik1(
152-191) with either the anti-Myc mAb or the anti-Frq1
antibodies (Fig. 5, lanes 7 and 8). Second, as
expected, wild-type myc-Pik1 was co-immunoprecipitated by the anti-Frq1
antibodies, but not by the preimmune serum (Fig. 5, lanes 1 versus 3). Moreover, the amount of myc-Pik1
co-immunoprecipitated with Frq1 was similar to the amount of myc-Pik1
that could be captured by direct immunoprecipitation with the anti-Myc
mAb (Fig. 5, lanes 2 versus 3). In
marked contrast, even though copious amounts of myc-Pik1(
152-191)
could be immunoprecipitated directly with the anti-Myc mAb (Fig. 5,
lane 5), only a trace of myc-Pik1(
152-191) was
co-immunoprecipitated by the anti-Frq1 antibodies (Fig. 5, lane
6), although this level was above that seen in the pre-immune serum control (Fig. 5, lane 4). Nonetheless,
Pik1(
152-192) clearly interacted with Frq1 much less efficiently
than wild-type Pik1. In contrast, when immune complexes obtained with
anti-Myc mAb 9E10 were assayed for PtdIns 4-kinase catalytic activity,
by methods that will be described in greater detail elsewhere, the
specific activity of myc-Pik1(
152-191) was quite comparable to that
for myc-Pik1.2
::LEU2/PIK1
diploid strain (YES10; Table I)
was transformed with a CEN vector,
pRS314-myc-pik1(
152-191), expressing
Pik1(
152-191) under the control of the GAL1 promoter, or
with the same vector expressing wild-type myc-PIK1, or the same empty vector (as a control). The transformants then were induced
to sporulate, and the resulting tetrads were dissected on medium
containing Gal as the carbon source and grown at 30 °C. As expected,
the diploids transformed with the empty vector only yielded tetrads in
which two spores (PIK1) were viable and two spores
(pik1
::LEU2) were inviable (see Fig.
S3 in the Supplemental Material), indicating that the absence of Pik1
function is lethal, as shown before (19). In contrast, the diploids
transformed with the vector expressing normal Pik1 yielded many tetrads
in which three or all four spores were viable, indicating that the plasmid-borne copy of PIK1 was able to rescue the
inviability of the pik1
::LEU2
spores. Under the same conditions, the diploids transformed with the
vector expressing pik1(
152-191) also yielded tetrads in which three or four spores were viable. Thus, when overexpressed from the strong inducible GAL1 promoter,
Pik1(
152-191), was able to complement the
pik1
::LEU2 null mutation.
Nevertheless, one way to determine if a gene product is not operating
optimally is to test its ability to function under conditions of
stress. Indeed, we found that
pik1
::LEU2 spores expressing
Pik1(
152-191) as the sole source of the enzyme were compromised in
their growth at 36 °C and unable to grow at all at 37 °C, whereas
pik1
::LEU2 spores expressing normal
Pik1 from the same vector grew robustly at both temperatures (data not
shown). Thus, even when highly overexpressed, Pik1(
152-191) is not
fully functional under the stressful condition of elevated temperature,
presumably because its efficient association with Frq1 is required for
its optimal function.
S. cerevisiae strains
cells, which are otherwise inviable when Pik1 is present at its normal
level (29). In other words, Frq1 becomes completely dispensable when Pik1 is highly abundant. Therefore, we tested whether Pik1(
152-191) was still able to rescue the inviability of
pik1
::LEU2 cells when it was
expressed at a lower level rather than at the high level that results
from expression from the GAL1 promoter. For this purpose, we
constructed a CEN plasmid,
pRS314-pik1(
152-191), that expressed
Pik1(
152-191) from the native PIK1 promoter and introduced it into the
pik1
::LEU2/PIK1
heterozygous diploid. Even at this lower level of expression, diploids
expressing Pik1(
152-191) yielded tetrads with three and four viable
spores at essentially the same frequency as diploids expressing normal
Pik1 (Fig. 6A). However, as
observed before, pik1
::LEU2 cells
expressing Pik1(
152-191) were unable to grow above 35 °C (Fig.
6B). Thus, under the moderate stress of elevated
temperature, efficient association of Frq1 with Pik1 becomes essential
for the function of the enzyme, regardless of its level of
expression.
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Fig. 6.
Efficient association of Frq1 with Pik1 is
required for growth at elevated temperature. A, a
complementation test was used to assess the function of overexpressed
Pik1( 152-191). Heterozygous
pik1
::LEU2/PIK1 diploid
strain YES10 (Table I) was transformed with either the empty
TRP1-marked CEN vector (left panel),
or the same vector expressing wild-type Pik1 from the native
PIK1 promoter (middle panel), or the same vector
expressing Pik1(
152-191) from the native PIK1 promoter
(right panel) and subjected to conditions that induce
sporulation, and samples of the resulting tetrads (six to ten shown)
were dissected. Viability of the four spores (A-D) was
assessed at 30 °C on medium containing Gal as the carbon source.
B, a representative Leu+ Trp+ spore expressing
myc-Pik1(
152-191) (left) and a representative Leu+ Trp+
spore expressing normal Pik1 (right), obtained as in
A, were tested for growth on SCGal-Leu-Trp at the indicated
temperatures.
cells; however, unlike overexpression of wild-type
Frq1, overexpression of Frq1(G2A) is unable to rescue the
temperature-sensitive lethality of pik1-11ts cells
(29). Second, we found that, like normal Frq1, Frq1(G2A) still
associates with membranes, but does so much less efficiently than
normal Frq1 in either the absence or presence of Ca2+ (35).
One explanation for both of these observations is that Frq1 might carry
a second lipophilic modification that partially compensates for the
absence of the N-myristoyl group. Moreover, there are
precedents for S-palmitoylation of Cys residues situated near the N terminus of other small Ca2+-binding regulatory
proteins (38, 39). Indeed, Frq1 has only two Cys residues, and although
one (Cys-38) appears to be buried, based on our NMR-derived structural
model (35), the other (Cys-15) is located near the N terminus. We took
two independent approaches to address whether
S-palmitoylation has any role in the function of Frq1.
null mutation, whereas the empty vector did not (see
Fig. S4A in the Supplemental Material). Even when expressed
at a lower level from the native FRQ1 promoter on a
CEN vector, Frq1(G2A,C15A) was able to rescue the
inviability of frq1
spores at essentially the same
frequency as wild-type Frq1 expressed from the same plasmid, whereas
the empty vector did not (Fig. 7B). Thus, as judged by these
criteria, Frq1 that is unmyristoylated and non-palmitoylatable (at
Cys-15) showed no impairment in its physiological function in
vivo.
View larger version (55K):
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Fig. 7.
S-Palmitoylation of Frq1 is not
required for its function in vivo. A,
mutant frq1-1ts cells (strain YKBH4; Table I)
carrying either an empty URA3-marked multicopy vector
(YEp352) (upper sector), or the same vector expressing
wild-type FRQ1 from the native FRQ1 promoter
(lower right sector), or the same vector expressing a
non-myristoylated and unpalmitoylatable mutant, frq1(G2A
C15A) (lower left sector), were incubated on SCGlc-Ura
plates at the indicated temperatures. B, heterozygous
frq1 ::HIS3/ FRQ1
diploid strain YKBH1 (Table I) was transformed with either an empty
URA3-marked low copy number (CEN) vector (pRS316), or the
same vector expressing normal FRQ1 from the native
FRQ1 promoter, or the same vector expressing frq1(G2A
C15A) from the native FRQ1 promoter and subjected to
conditions that induce sporulation, and samples of the resulting
tetrads (five to seven shown) were dissected. Viability of the four
spores (A-D) was assessed at 30 °C on medium containing
Glc as the carbon source.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical
conformation in the complex.4
The intervening 20 residues (Thr-137 to Val-156) appear to be unstructured4 and presumably form a loop that does not
interact with Frq1. The 13-residue segment is comparable in length to
the short helices and other sequence elements (e.g.
IQ motif) recognized by the well-characterized
Ca2+-binding regulatory protein, calmodulin (55).
Furthermore, this 13-residue segment (-APALVLSSMIMSA-) is comprised
exclusively of non-polar and uncharged residues, in agreement with the
evidence we obtained from titration calorimetry and intrinsic Trp
fluorescence that the association between Pik1-(110-192) is primarily
a hydrophobic interaction. An interaction between another myristoylated
Ca2+-binding regulatory protein and a largely hydrophobic
element in its target has recently been described. The "CIB"
(calcium- and integrin-binding) protein binds tightly to a 15-residue
-helical sequence (-LVLAMWKVGFFKRNR-) in the cytoplasmic tail of the
integrin
IIb chain (56). This sequence bears some resemblance to the 13-residue Frq1-binding segment of Pik1.
152-191), which removed the 13-residue
hydrophobic motif, produces a protein that, in contrast to normal Pik1,
co-immunoprecipitates very inefficiently with Frq1, yet retains
catalytic activity. The lack of efficient Frq1 binding to Pik1 appears
to compromise the function of the enzyme because, again in contrast to
wild-type Pik1, Pik1(
152-191) was unable to support the growth of
pik1
yeast cells at elevated temperature. We do not know
if the binding modality we have defined for the Pik1-Frq1 interaction
is conserved in the interaction of metazoan frequenins with PtdIns
4-kinase-
and/or any other targets. However, in collaborative
studies, which will be described in greater detail elsewhere, we have
delineated the site in yeast Pik1 bound by human NCS-1 using in
vitro pull-down assays of the sort presented here and using the
two-hybrid method in vivo. As observed for the interaction
of Frq1 with Pik1, the most essential sequences to support the
interaction of human NCS-1 with Pik1 fall between residues 145 and 172, which includes the 13-residue hydrophobic sequence (Ala-157 to
Ala-169).5 Furthermore, as
also observed for Frq1 association with Pik1, residues in the region
100-144, which includes the 11-residue hydrophilic element (Phe-125 to
Gln-136), contribute to the strength of the interaction between human
NCS-1 and Pik1.5
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ACKNOWLEDGEMENTS |
---|
We thank present and former members of the Thorner Laboratory for material support and helpful advice during the course of these studies, especially Nathan C. Rockwell, Claudio Sette, Kristin B. Hendricks, and Bo Qing Wang.
![]() |
FOOTNOTES |
---|
* This work was supported by a Traveling Research Fellowship from the Gottlieb Daimler and Karl Benz Foundation (to I. G. H.); by National Institutes of Health (NIH) Research Grant EY12347, by a Beckman Young Investigator Award, and funds from a center grant provided by the Keck Foundation (to J. B. A.); and by NIH Research Grant GM21841, a grant from the Lowe Syndrome Association, and by facilities provided by the Berkeley campus Cancer Research Laboratory (to J. T.).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.
The online version of this article (available at
http://www.jbc.org) contains Figs. S1-S4.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 510-642-2558; Fax: 510-643-6791; E-mail: jeremy@socrates.berkeley.edu.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M207920200
2 T. Strahl, unpublished observations.
3 I. G. Huttner, unpublished results.
4 J. B. Ames, unpublished results.
5 T. Strahl, B. Grafelmann, J. Dannenberg, O. Pongs, and J. Thorner, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PtdIns, phosphatidylinositol;
PtdIns 4-kinase, phosphatidylinositol 4-kinase;
PI4K, phosphatidylinositol 4-kinase beta isoform;
PtdIns(4)P, phosphatidylinositol-4-phosphate;
PI(4)P 5-kinase, phosphatidylinositol-4-phosphate 5-kinase;
CEN, centromeric;
Gal, galactose;
GAP, GTPase-activating protein;
GEF, guanine nucleotide
exchange factor;
Glc, glucose;
HSQC, heteronuclear single-quantum
coherence spectroscopy;
Kav, average retention
coefficient;
LKU, lipid kinase unique domain;
mAb, monoclonal antibody;
NCS, neuronal calcium sensor;
NOE, nuclear Overhauser effect;
NTA, nitrilotriacetate;
Raf, raffinose;
SC, synthetic complete medium;
Suc, sucrose;
ts, temperature-sensitive;
Fmoc, N-(9-fluorenyl)methoxycarbonyl.
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