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Journal of Bacteriology, April 1999, p. 2527-2534, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Extracellular Domain of the Saccharomyces
cerevisiae Sln1p Membrane Osmolarity Sensor Is Necessary for
Kinase Activity
Darin B.
Ostrander
and
Jessica A.
Gorman*
Department of Leads Discovery, Bristol-Myers
Squibb Pharmaceutical Research Institute, Princeton, New Jersey
08543-4000
Received 26 October 1998/Accepted 8 February 1999
 |
ABSTRACT |
The function of the extracellular domain (ECD) of Sln1p, a plasma
membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast Saccharomyces cerevisiae. Truncations of SLN1
that retain an intact kinase domain are capable of complementing the
lethality of an sln1
strain. By observing levels of
Hog1p phosphorylation as well as the phosphorylation state of Sln1p,
the kinase activities of various SLN1 constructions were
determined. In derivatives that do not contain the first TMD, Sln1p
activity was no longer dependent on medium osmolarity but appeared to
be constitutively active even under conditions of high osmolarity.
Removal of the first TMD (TMD1 construct) gave a protein that was
strongly phosphorylated whereas Hog1p was largely dephosphorylated, as
expected if the active form of Sln1p is phosphorylated. When both TMDs
as well as the ECD were deleted, so that the kinase domain is
cytosolic, Sln1p was not phosphorylated whereas Hog1p became
constitutively hyperphosphorylated. Surprisingly, this hyperactivity of
the HOG mitogen-activated protein kinase signaling pathway was not
sufficient to result in cell lethality. When the ECD of the TMD1
construct was replaced with a leucine zipper motif, Sln1p was
hyperactive, so that Hog1p became mostly unphosphorylated. In contrast,
when the Sln1p/leucine zipper construct was crippled by a mutation of
one of the internal leucines, the Sln1 kinase was inactive. These
experiments are consistent with the hypothesis that the ECD of Sln1p
functions as a dimerization and activation domain but that osmotic
regulation of activity requires the presence of the first TMD.
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INTRODUCTION |
The yeast Saccharomyces
cerevisiae responds to high external osmolar conditions by
increasing the intracellular concentration of glycerol (26).
The high-osmolarity glycerol (HOG) pathway in yeast consists of a
double two-component phosphorelay cascade coupled to a
mitogen-activated protein kinase (MAPK) cascade. The SLN1
gene encodes an enzyme with histidine kinase and aspartate phosphotransferase activities and functions as a plasma membrane sensor. Under normal, low-osmolar conditions, Sln1p actively transfers a phosphate to Ypd1p, which in turn transfers a phosphate to Ssk1p (20). Phosphorylated Ssk1p inhibits the kinase cascade in
the HOG MAPK pathway (13). Under high-osmolarity conditions,
Sln1p is deactivated. The lack of phosphorelay through the
two-component pathway causes inactivation of Ssk1p and activation of
the HOG MAPK pathway. The MAPKKKs Ssk2p and Ssk2p are kinases which
phosphorylate the MAPKK Pbs2p, which phosphorylates the MAPK Hog1p.
Phosphorylated Hog1p has been shown to activate transcription factors
that increase the production of enzymes involved in glycerol synthesis
and stress response (23).
SLN1 was originally identified as an allele that is
synthetically lethal with ubr1, encoding the recognition
component of the N-end-rule ubiquitin system (17). The
predicted protein sequence is 1,220 amino acids in length and possesses
homology to both the sensor histidine autophosphorylation and response regulator proteins of bacterial two-component systems. The histidine and aspartate phosphorylation sites are residues 576 and 1144, respectively (13), encoded by
SLN1H576 and SLN1D1144.
Sln1p is also predicted to possess two transmembrane domains (TMDs)
like some bacterial sensor kinases. The autophosphorylation of
histidine residues in the bacterial sensor kinases is believed to occur
in trans. That is, bacterial histidine kinases are thought to require dimerization of the protein (29). The homology of Sln1p with the bacterial proteins predicts that Sln1p may also require
dimerization for autophosphorylation.
It is known that Sln1p can transfer a phosphate group from the sensor
to the receiver domain in trans (13). Neither
SLN1H576Q nor SLN1D1144N
complements sln1. Complementation is observed, however,
when the mutant proteins are coexpressed, demonstrating that an
intermolecular transfer of the phosphate group can occur. Likewise, the
isolated histidine kinase region (amino acids 450 to 1070) has been
observed to transfer a phosphate group to the isolated receiver domain region (amino acids 1059 to 1220) (20). Phosphotransfer was observed between the two protein fragments but not with fragments possessing mutations in either His576 or Asp1144. Although these experiments demonstrate that Sln1p phosphate transfer can occur in
trans, they do not show whether Sln1p histidine
autophosphorylation and kinase activation normally occur by an
intermolecular or intramolecular interaction.
We undertook to address the question of whether Sln1p requires
dimerization for histidine autophosphorylation to occur. We also
attempted to delimit the structural region of Sln1p that may function
as a dimerization domain. These studies also defined regions that are
essential for other functions of the protein such as the osmolarity
response. Together the results suggest some generalized
structure-function relationships for this two-component sensor kinase.
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MATERIALS AND METHODS |
Strains and plasmids.
The SLN1/sln1 heterozygous
strain (his3-200/his3-200 leu2-3,112/leu2-3,112
lys2-801/lys2-801 SLN1/sln1::HIS3 trp1-1/trp1-1 ura3-52/ura3-52) was provided by I. Ota
(17). The SLN1 expression vector was created as
follows. The carboxyl-terminal region of SLN1 was isolated
from a vector provided by I. Ota (17) and inserted into both
pDO105 (high copy) (16) and pDO120 (low copy) (18), using the same restriction sites. The amino-terminal
domain was cloned from total genomic DNA of strain Y294 (5)
by PCR to add a restriction site just upstream of the initiation codon (Fig. 1A, arrow 1).
Site-directed mutagenesis (SDM) was performed with the QuikChange
mutagenesis system (Stratagene). SDM was accomplished with two
oligonucleotides, the first adding a restriction site just after the
first TMD (TMD1) (Fig. 1A, arrow 2) and the second adding a restriction
site just before TMD2 (Fig. 1A, arrow 3). The latter mutation is
silent, while the former creates a conservative amino acid substitution
(N49S). The mutagenized amino-terminal region of SLN1 was
isolated and added to the vector containing the carboxyl-terminal region to create plasmid pDO108. This construct will be referred to as
full length (Table 1).
To create the
TMD1 construct (Table
1), pDO108 was cut with
restriction enzymes deleting the amino-terminal intracellular
region
and TMD1, and annealed oligonucleotides were added. The
new
amino-terminal coding sequence includes the
STE3 signal
sequence,
MSYKS (
7), to direct the protein into the
secretory pathway.
The
TMD1&ECD plasmid was created in the same
manner, adding annealed
oligonucleotides, which again adds the
STE3 signal sequence to
the amino terminus. The kinase
domain expression plasmid (Table
1, KD) apparently uses Met439 as
initiator, as judged by the
apparent molecular weight of the protein
product (not
shown).
To obtain a noncomplementing
SLN1 allele, pDO108 was cut
with a single restriction enzyme just upstream of the kinase domain,
treated with Klenow fragment, and reclosed. The additional four
base
pairs cause premature termination of the reading frame upstream
of the
kinase domain (Table
1,
KD). To add a CAAX box plasma
membrane
localization signal to the carboxyl terminus of the
SLN1 kinase domain (Table
1, KD/CAAX), the carboxyl terminus of
SLN1 was isolated on a plasmid. The vector was then
mutagenized with
oligonucleotides, adding a silent restriction site
near the end
of the open reading frame. This vector was cut, and
annealed oligonucleotides
were inserted. The new sequence adds the
amino acids CIIS of the
RAS2 gene (
21). The
modified carboxyl terminus was subsequently
isolated and reinserted
into
pDO108.
To replace the extracellular domain (ECD) of
SLN1 with a
leucine zipper sequence (Table
1,
ECD/LeuZip), the relevant sequence
from a C/EBP (CCAAT element-binding protein) clone (
12) was
isolated by PCR and inserted into pDO108. To make the construction
single transmembrane (
TMD1&ECD/LeuZip), this vector was cut and
the
same
STE3 signal sequence oligonucleotides described above
for the
TMD1 construct were added. The undimerizable leucine
zipper
derivative was created by SDM of the leucine zipper fragment
described
above. This mutagenesis alters the central leucine to
a proline. The
undimerizable
SLN1TMD1&ECD/LeuZip* construct was
then
created as described
above.
To add the FLAG epitope tag to various
SLN1 constructions,
the mutagenized carboxyl-terminal domain plasmid with the silent
restriction site near the end of the open reading frame was used.
The
plasmid was cut and annealed oligonucleotides were ligated,
adding
sequence containing the DYKDDDDK FLAG sequence (IBI). The
carboxyl-terminal domain of
SLN1 in pDO108 was then replaced
with
the FLAG sequence, and all constructs were recreated as previously
described. Immunoblotting and immunoprecipitation used anti-FLAG
monoclonal antibodies
(Kodak).
To disrupt the
SHO1 gene in this strain, we amplified 350- and 500-bp fragments of the
SHO1 5' and 3' nontranslated
regions,
respectively, and placed them on either side of a
hisG/URA3/hisG cassette (
1). This construct was
used to disrupt the
SHO1 gene
in a
sln1
haploid strain, and selection on 5-fluoorotic acid
isolated those cells
which had recombined at the
hisG repeats
and had recovered
uracil
auxotrophy.
Antisera.
To obtain antisera to the carboxyl-terminal region
of Sln1p, a DNA fragment encoding amino acids 494 to 1220 was excised
from pDO108. The fragment was ligated into vector pMAL-C2 (New England Biolabs) to create a malE/SLN1 fusion.
Isopropyl-
-D-thiogalactopyranoside (IPTG) induction of
this plasmid in Escherichia coli produced large amounts of a
124-kDa fusion protein. A cell extract was prepared, and the fusion
protein was isolated on an amylose column and eluted with maltose. The
Sln1p fusion protein was purified by preparative electrophoresis,
electroeluted, dialyzed, and concentrated. The protein was injected
into rabbits, and antisera were isolated as described previously
(15). Protein gel analysis of various SLN1
constructions used 7.5 to 15% gradient acrylamide gels with a vertical
slab gel unit (Hoefer).
The
HOG1 open reading frame was cloned from total genomic
Y294 (
5) DNA by PCR. The product was ligated into pET23B
(Novagen)
to create a His
6-tagged Hog1p. IPTG induction of
this plasmid
in
E. coli produced a 45-kDa product. The
protein was isolated
on a Ni-nitrilotriacetic acid agarose column
(Qiagen) and used
to create antisera as described
above.
Hog1p phosphorylation assay.
Strains were grown in YPD
(rich) or SD (defined) medium (10) at 28°C. For
high-osmolarity conditions, sorbitol or NaCl was added to the cultures
to a final concentration of 1 or 0.3 M, respectively. The cultures were
incubated in high-osmolarity medium for 10 min for low-copy-number
constructs or 12 h for high-copy-number constructs unless
otherwise indicated. Subsequent steps were performed at 5°C.
Mid-log-phase cultures (2 × 107 to 3 × 107 cells/ml) were harvested by centrifugation. Cells were
broken in protein isolation buffer (25 mM HEPES [pH 7.4], 10%
glycerol, 1 mM Na2EDTA) with protease inhibitors (87 µg
of phenylmethylsulfonyl fluoride, 2.5 µg of
N
-p-tosyl-L-lysine chloromethyl ketone, 0.75 µg of pepstatin A, 0.5 µg of leupeptin, and 0.5 µg of aprotinin per ml) with 500 µm-diameter acid-washed glass beads in a Mini-Bead Beater (BioSpec Products). In some cases, a phosphatase inhibitor cocktail (10 mM NaF, 5 mM -glycerol phosphate, 1 mM sodium vanadate) was added to the breakage buffer. The extracts were subjected to
centrifugation for 1 h at 100,000 × g in a TL-100
ultracentrifuge (Beckman), and the supernatant was collected in order
to isolate the cytosolic fraction. Aliquots containing 100 µg of
cytosolic protein were used for immunoprecipitation (22)
with 10 µl of anti-Hog1p antisera and 5 mg of protein A-Sepharose
CL-4B (Pharmacia). To remove the immunoglobulin G from the
immunoprecipitate, the final washed pellets were resuspended in 1%
sodium dodecyl sulfate (SDS) and centrifuged (8).
Supernatants were collected and placed in SDS-polyacrylamide gel
electrophoresis (PAGE) loading buffer.
Hog1p immunoblot analyses used 10% Ready Gels in the Mini-Protean cell
system (Bio-Rad) which were blotted to polyvinylidene
difluoride
membranes in a Mini-V8 apparatus (Life Technologies).
Immunoblot
analyses used the PhotoBlot chemiluminescence detection
system
(Gibco-BRL). Rabbit polyclonal antiphosphotyrosine antisera
was
obtained from Transduction Laboratories. The relative levels
of Hog1p
phosphorylation were normalized to amounts of Hog1p immunoprecipitated
by scanning densitometry of a duplicate immunoblot that used anti-Hog1p
antisera. A comparison of extracts prepared with or without addition
of
phosphatase inhibitors demonstrated that there was no significant
difference in the levels of Hog1p phosphorylation
found.
Cell fractionation and membrane localization.
Cells were
grown and harvested as described above. Cells were broken with glass
beads in membrane isolation buffer (300 mM sucrose, 50 mM Tris-HCl [pH
7.5], 10 mM -mercaptoethanol, 1 mM Na2EDTA) with
protease inhibitors at 5°C. Unbroken cells and glass beads were
removed by centrifugation at 1,000 × g for 5 min.
Membrane and cytosolic fractions were separated as before. Pellets
(membrane fractions) were directly resuspended in SDS-PAGE loading
buffer or were used for additional fractionation as described below, while supernatants (cytosol) were first concentrated with a
Centricon-10 concentrator (Amicon) at 4,500 × g for
4 h at 5°C.
Plasma membranes were isolated as described previously (
24),
with modifications (
14). Measurement of plasma membrane
H
+-ATPase activity (
11) was used to assess the
purity of this
fraction. Vanadate-sensitive ATPase activity accounted
for greater
than 95% of the total ATPase activity in the plasma
membrane fraction.
Mitochondrial fractions were isolated from cell
extracts by differential
centrifugation (1,000 to 20,000 ×
g) (
2). Measurement of mitochondrial
ATPase activity
was used to assess the purity of this fraction.
Sodium azide-sensitive
ATPase activity accounted for greater than
98% of the total ATPase
activity in the mitochondrial membrane
fraction. A crude microsomal
membrane fraction was also isolated
from cell extracts by differential
centrifugation as previously
described (27,000 to 100,000 ×
g) (
6). All membrane fractions
were suspended in
membrane isolation buffer to a volume equivalent
to that of the
cytosolic
fraction.
Sln1p phosphorylation assay.
Strains containing different
FLAG-tagged SLN1 plasmids were labeled with
32Pi (375 µCi/ml) in low-phosphate medium
(27) with or without 1 M sorbitol. Cells were harvested,
broken in radioimmunoprecipitation assay buffer (150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris [pH 8.0])
(8) with protease inhibitors (see above) and phosphatase
inhibitors (10 mM NaF, 5 mM -glycerophosphate, and 1 mM sodium
vanadate), and total membrane and cytosolic fractions were separated as
described above. Sln1p was isolated from both fractions by
immunoprecipitation with FLAG monoclonal antibodies (Kodak). The
immunoprecipitates were separated on polyacrylamide gels in duplicate.
One gel was transferred to membranes for immunoblot analysis with
anti-Sln1p antisera to assess total immunoprecipitated protein levels,
while the second was exposed to X-ray film at 
80°C for 2 days with
an intensifying screen to assess phosphorylation levels.
| |
RESULTS AND DISCUSSION |
SLN1 mutagenesis.
To test the in vivo effects of
different structural alterations in Sln1p, three restriction enzyme
sites were introduced into SLN1 by SDM (Fig.
1A): one immediately upstream of the
initiator ATG (arrow 1), a second immediately downstream of TMD1 (arrow 2), and a third immediately upstream of TMD2 (arrow 3). This mutated copy of SLN1 was cloned behind the ADH1 promoter
on both low- and high-copy-number shuttle plasmids. We used the
ADH1 promoter to obviate any potential expression regulatory
effects that might be caused by the nascent SLN1 promoter in
these constructs.
A
SLN1/sln1 heterozygous strain was used to test the
ability of the mutagenized
SLN1 plasmid to complement
sln1. The strain
containing this plasmid was sporulated,
and tetrads were dissected.
Because an
SLN1 null mutation is
lethal in this strain, complementation
could be tested by growth of the
spores. Four viable colonies
were obtained with the mutagenized
SLN1 plasmid with every tetrad
(Fig.
2A), while only two were found with the
parent vector (Fig.
2B). The restriction site mutations incorporated
into
SLN1 therefore
do not affect complementation of
sln1 (Table
1). No difference
was detected in the growth
rates of strains containing mutagenized
or wild-type
SLN1 in
either low- or high-osmolarity medium. These
data were observed with
both low- and high-copy-number
SLN1-containing
vectors.
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FIG. 2.
SLN1/sln1 tetrad dissections with various
SLN1 expression constructs. (A) Full-length SLN1
with mutations incorporating restriction enzyme sites; (B) parental
vector control; (C) SLN1KD (1-438); (D)
SLN1KD (543-1220). These results were obtained with
both low- and high-copy-number SLN1-containing vectors.
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SLN1 deletion studies.
As a first step to
understanding the relationship between the structure of Sln1p and its
function in cells, various structural deletion constructs were tested
for the ability to complement the lethal sln1 phenotype.
The restriction sites that were introduced into the gene were used to
remove sequence corresponding to the short amino-terminal intracellular
region and TMD1. This construct, TMD1, possesses a deletion of the
first 49 amino acids of Sln1p (Table 1). In hopes of correctly
orienting the truncated protein in membranes, the STE3
signal sequence (7) was added to the amino terminus of the
construction. The amino terminus should therefore be extracellular,
with only a single TMD (Fig. 1C). This plasmid was used to transform
the SLN1/sln1 strain, which was subsequently sporulated
and subjected to tetrad dissection to test the ability of the deletion
to complement sln1. The construct was able to complement
the mutation (Table 1). These data were observed with both low- and
high-copy-number SLN1-containing vectors.
Continuing the deletion of the structural domains of Sln1p, the ECD was
deleted from the
TMD1 construct (Fig.
1D). This construct,
TMD1&ECD, possesses a deletion of the first 330 amino acids of
Sln1p
(Table
1). Subsequently, the second TMD was deleted to
yield a kinase
domain construct (KD) possessing a deletion of
the first 438 amino
acids of Sln1p (Fig.
1E). The KD construct
did not contain the
secretion signal sequence used with the other
truncation mutants.
Surprisingly, both of these deletion protein
constructions complemented
sln1 (Fig.
2C; Table
1).
As a negative control, a mutation that retained all the membrane
structural elements but deleted the kinase domain (
KD; amino
acids
543 to 1120) (Fig.
2D) was used. Complementation was not
observed
(Table
1).
Subcellular localization.
To observe the localization of the
different Sln1p truncations, rabbit polyclonal antibodies were prepared
against the carboxyl-terminal region of Sln1p that contains the kinase
domain. The antisera were used to show that the constitutively
expressed full-length mutant SLN1p was present in approximately
fivefold excess over wild-type levels (not shown). No difference in
expression level was observed between the full-length construct and any
of the deletion constructs. This result suggests that the deletion
constructs are overexpressed to the same extent and are as stable as
the full-length construct in cells. The sizes of the different
truncated proteins as determined by gel migration were as predicted
(not shown).
Crude membrane preparations were separated from cytosol by
centrifugation and analyzed for the presence of the
SLN1
constructs
by immunoblot analysis (not shown). The full-length,
TMD1,
TMD1&ECD,
and
KD constructs were all found by immunoblot
analysis to be
exclusively associated with the membrane fraction. The
KD construct,
which possesses no membrane structural components or
localization
signals, was found to be completely
cytosolic.
Membrane localization experiments were used to determine the organelles
in which the truncated proteins were present. Crude
membrane
preparations were separated by differential centrifugation
and analyzed
for purity by ATPase activity specific for each organellar
membrane
(see Materials and Methods). With the exception of the
KD construct
mentioned above, each showed a similar pattern of
localization in that
between 30 and 50% of the protein was specifically
associated with the
plasma membrane, with the remainder associated
with microsomes
(endoplasmic reticulum, Golgi complex, and vesicles).
Wild-type cells
and strains expressing the truncation constructs
from low-copy-number
vectors show a higher percentage (~80%) of
Sln1p in the plasma
membrane fraction (Fig.
3). No
localization
to mitochondrial membranes was observed (not shown).
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FIG. 3.
Sln1TMD1p subcellular localization. The
SLN1TMD1-expressing strain was examined for Sln1p
subcellular distribution by immunoblot analysis using anti-Sln1p
antisera. Cells were grown to the mid-logarithmic phase of growth,
harvested, and lysed, and membrane-associated and cytosolic proteins
separated. The total membrane fraction was used to make a crude plasma
membrane preparation by differential centrifugation. The purity of the
plasma membrane fraction was tested by the ability of plasma membrane
H+-ATPase activity to be specifically inhibited by vanadate
(see Materials and Methods). The remaining membranes were termed the
microsomal fraction. Lanes 1 to 3, high-copy-number vector; lanes 4 to
6, low-copy-number vector. Lanes 1 and 6, total cytosolic protein;
lanes 2 and 5, plasma membrane fraction; lanes 3 and 4, microsomal
fraction. Sizes here and in Fig. 5 to 7 are in kilodaltons.
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Hog1p phosphorylation assay.
To observe the effect of the
different Sln1p truncation mutants on activation of the HOG pathway,
the relative levels of Hog1p phosphorylation were determined. To
accomplish this, cytosolic protein extracts were used for
immunoprecipitation of the Hog1 protein with anti-Hog1p antisera,
separated by SDS-PAGE, and subjected to immunoblot analysis with
antiphosphotyrosine antisera.
It was found that the maximal induction of the pathway with
high-copy-number
SLN1-containing plasmids was obtained when
the
cells were grown in high-osmolarity medium for approximately eight
generations (Fig.
4). These data for the
high-copy-number constructs
contrast with reports that HOG pathway
signaling peaks within
5 min and returns to undetectable
phosphorylation levels within
20 min (
3,
4,
19,
28). The
overexpression of
SLN1 in
our system lengthened the time of
HOG pathway induction, as evidenced
by Hog1p phosphorylation levels.
Results in agreement with the
literature were obtained when a
wild-type, nonoverexpressed
SLN1 strain was used or when
SLN1 was expressed from a low-copy-number
plasmid in an
sln1 strain. Under these conditions, Hog1p
phosphorylation
peaked within 30 min and fell to basal levels within 75 min (Fig.
4). Interestingly, Maeda and coworkers found that
overexpression
of
SSK1, the second aspartate phosphoacceptor
of the pathway,
leads to an increased duration of Hog1p phosphorylation
in response
to high-osmolarity growth (
13).
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FIG. 4.
Hog1p phosphorylation time course. The relative levels
of Hog1p phosphorylation were determined at different times of
incubation with high-osmolarity medium with different SLN1
constructs. Total cytosolic protein was subjected to
immunoprecipitation with anti-Hog1p antisera, separated by SDS-PAGE,
and immunoblotted with antiphosphotyrosine antisera, and the
intensities of the bands corresponding to Hog1p were quantified by
scanning densitometry. All values were corrected for the amount of
Hog1p loaded (as judged with duplicate anti-Hog1p immunoblots) and
normalized to the average value observed with the low-copy-number
wild-type SLN1 construct grown in low-osmolarity medium.
Data represent averages of three independent experiments; standard
deviations are less than 5%. Data for wild-type SLN1
strains are not substantially different from those for
sln1 strains containing SLN1 on a
low-copy-number plasmid; data for the SLN1 KD-containing
strains are not substantially different when grown in high- or
low-osmolarity medium.
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With full-length
SLN1, Hog1p phosphorylation increases when
cells are grown in high- as opposed to low-osmolarity medium (Fig.
5, lanes 1 and 2). (The identities of the
100- and 105-kDa bands
are unknown). Therefore, as in wild-type cells,
Sln1p kinase activity
appears to be high in low-osmolarity medium and
the HOG pathway
is inactive (Table
1). This situation is reversed in
high-osmolarity
medium.
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FIG. 5.
Hog1p phosphorylation with various SLN1
truncation mutations. Antiphosphotyrosine immunoblot analysis was
performed on immunoprecipitated Hog1p separated by SDS-PAGE. (A) Rich
medium; (B) defined medium. Odd-numbered lanes, low-osmolarity medium;
even-numbered lanes, high-osmolarity medium. Lanes 1 and 2, full-length
SLN1 with restriction sites (Fig. 1A); lanes 3 and 4, TMD1 construction (Fig. 1C); lanes 5 and 6, KD construction (Fig.
1E). Duplicate anti-Hog1p immunoblots demonstrated that the same
amounts of Hog1p were immunoprecipitated in all cases.
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Sln1p with a single TMD (
TMD1) fails to show any response to the
osmolarity of the medium, and Hog1p appears to be primarily
unphosphorylated (Fig.
5, lanes 3 and 4). Therefore, without TMD1,
Sln1p appears to be constitutively active, and the HOG pathway
is
inactive as in low-osmolarity medium (Table
1). When the
SLN1 kinase domain alone (KD) is expressed, again no
response to changes
in osmolarity is observed. However, Hog1p appears
to possess a
relatively high level of phosphorylation (Fig.
5, lanes 5 and
6). Therefore, the soluble kinase domain peptide appears to lack
activity, as the HOG pathway is as active as it is with the wild-type
SLN1 construct in high-osmolarity medium (Table
1). These
experiments
were conducted in both rich (Fig.
5A) and defined (Fig.
5B)
media.
The
TMD1&ECD construct also resulted in constitutive
activation
of the HOG pathway (Table
1), showing no change when grown
in
media of different osmolarities (not shown). The use of
low-copy-number
constructs produced results that are substantially the
same (not
shown).
To control for the effect of Sho1p in these experiments, the gene
encoding the protein was disrupted in these strains. Sho1p
is another
plasma membrane osmosensor that directly activates
the MAPKK Pbs2p,
which in turn phosphorylates Hog1p (
20). No
substantive
differences in the relative levels of Hog1p were observed
with these
constructs in an
sln1 sho1 strain (not
shown).
These results demonstrate that only full-length Sln1p is capable of
altering kinase activity in response to differences in
medium
osmolarity. Further, the results suggest that Sln1p anchoring
to the
membrane is insufficient for activation of kinase
activity.
Plasma membrane localization.
The observation that the
TMD1&ECD construct appeared to be mostly inactive suggested that
plasma membrane localization alone is insufficient to fully activate
Sln1p kinase. As further evidence, a CAAX farnesylation sequence was
added to the carboxyl terminus of the KD construct (Fig. 1F, KD/CAAX)
to tether it to the plasma membrane (25). (This effort was
assisted by the fact that the carboxyl-terminal region of Sln1p has
numerous basic amino acids, a necessary feature of the CAAX motif
[21].) This construct was shown to complement
sln1 lethality (Table 1). By immunoblot analysis,
approximately 80% of this protein was found associated with the
membrane fraction. However, 100% of the membrane-associated protein
was found specifically associated with the plasma membrane (not shown).
As with TMD1&ECD, this construct caused constitutive Hog1p
phosphorylation (Table 1). These experiments demonstrate that membrane
localization alone is not responsible for the full activation of the
kinase activity of Sln1p.
Sln1p ECD dimerization.
Sln1p seems to be more active with an
untethered ECD, while it is nearly inactive in the absence of the ECD.
Therefore, it was hypothesized that the ECD of Sln1p is involved in
dimerization of the protein and that this is essential for kinase
activity. To test this hypothesis, the restriction enzyme sites
incorporated into SLN1 were used to replace the Sln1p ECD
with the rat C/EBP leucine zipper region (Table 1, ECD/LeuZip). This
construction retains the first intracellular and transmembrane domains.
Immunoblot analysis revealed that this construct is, like the previous
Sln1p truncation constructions, predominantly associated with the
plasma membrane. This construct complemented sln1 but
resulted in high levels of Hog1p phosphorylation (Table 1), which
suggests that the Sln1p kinase activity is minimal with this construct.
The leucine zipper motif is much shorter than the nascent Sln1p ECD (50 as opposed to 280 amino acids) and is known to adopt an alpha-helical motif in its native conformation. Therefore, it was hypothesized that
the physical constraint of having both sides of the zipper tethered to
the membrane might prevent dimerization, resulting in a less active
form of Sln1p kinase. To test this hypothesis, the first intracellular
and transmembrane domains of SLN1 were removed in this
construct, allowing the amino-terminal leucine zipper motif to freely
adopt its native conformation (Fig. 1G; Table 1). The resulting protein
was again observed to be membrane associated, primarily with the plasma
membrane. This construct resulted in only basal levels of Hog1p
phosphorylation (Fig. 6A, lane 1; Table
1), suggesting that the unconstrained zipper motif was free to dimerize
and activate the Sln1p kinase domains.
View larger version (45K):
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|
FIG. 6.
Hog1p phosphorylation with Sln1p/leucine zipper
constructions. (A) Antiphosphotyrosine immunoblot analysis of
immunoprecipitated Hog1p. Lane 1, cells containing the
TMD1&ECD/LeuZip construct; lane 2, cells containing the
TMD1&ECD/LeuZip* (mutated, undimerizable leucine zipper) construct.
(B) Duplicate immunoblot analysis of panel A with anti-Hog1p antisera
showing similar amounts of Hog1p immunoprecipitated in all cases. The
cells were grown in low-osmolarity medium.
|
|
The same construct was mutagenized to convert the central leucine codon
of the zipper motif to a proline, a mutation that
is reported to
abolish dimerization of a related leucine zipper
(Fig.
1H)
(
9). This construct demonstrated constitutive high-level
Hog1p phosphorylation (Table
1; Fig.
6A, lane 2). These observations
support the hypothesis that Sln1p must be dimerized in order to
achieve
full kinase
activity.
Sln1p phosphorylation.
The model for osmotically induced
signal transduction implies that Hog1p phosphorylation levels are
opposite those of Sln1p (13). To directly confirm this, the
phosphorylation state of Sln1p was observed. A restriction enzyme site
was introduced into the carboxyl-terminal domain of SLN1 by
SDM and used to introduce DNA sequence encoding the FLAG epitope. This
tag was inserted into the full-length, TMD1, and KD, as well as both
wild-type and undimerizable mutant forms, of TMD1&ECD/LeuZip
expression constructs. Immunoblot analysis both with anti-FLAG and
anti-Sln1p antibodies demonstrated all three SLN1 constructs
were expressed in similar amounts (not shown).
The FLAG-tagged constructs were used to test the phosphorylation state
of Sln1p under different growth conditions. Cells were
labeled with
32P
i in both high- and low-osmolarity media.
The cells were harvested
and broken, and cytosol was separated from the
membrane fraction.
The Sln1p in each fraction was immunoprecipitated
with anti-FLAG
antibodies, separated by SDS-PAGE, and visualized by
autoradiography.
Duplicate immunoblots with anti-Sln1p antisera
demonstrated approximately
equal amounts of Sln1p immunoprecipitated in
each case (Fig.
7B
and D).
View larger version (45K):
[in this window]
[in a new window]
|
FIG. 7.
Phosphorylation of various Sln1p constructions. (A)
Autoradiogram of a polyacrylamide gel of FLAG-tagged Sln1p proteins
immunoprecipitated from the membrane fraction of
32Pi-labeled cells. Lanes 1 and 3, low-osmolarity medium; lanes 2 and 4, high-osmolarity medium; lanes 1 and 2, full-length Sln1p; lanes 3 and 4, TMD1. (B) Duplicate
immunoblot analysis of panel A with anti-Sln1p antisera. (C)
Autoradiogram of a polyacrylamide gel of FLAG-tagged Sln1p
immunoprecipitated from the membrane fraction of
32Pi-labeled cells. Lane 1, TMD1/LeuZip;
lane 2, TMD1/LeuZip* (mutated, undimerizable leucine zipper). (D)
Duplicate immunoblot analysis of panel C with anti-Sln1p antisera.
|
|
Full-length Sln1p was associated only with the membrane fraction and
was highly labeled when cells were grown in low-osmolarity
medium (Fig.
7A, lane 1). When the cells were grown in high-osmolarity
medium,
full-length Sln1p was unlabeled (Fig.
7A, lane 2). This
result is
consistent with the Hog1p phosphorylation experiments
described above
(Table
1).
TMD1 was also associated exclusively
with the membrane
fraction but was highly labeled in both low-
and high-osmolarity media
(Fig.
7A, lanes 3 and 4), consistent
with the hypothesis that the ECD
of this construct results in
a high percentage of dimerization and
kinase activity (Table
1).
The KD construct was present only in the
cytosolic fraction (not
shown) and was found to be unlabeled under all
conditions tested
(Table
1). The protein was present in cells, however,
as observed
with a duplicate anti-Sln1p immunoblot (not
shown).
The
TMD1&ECD/LeuZip construct showed high levels of Sln1p
phosphorylation, while the undimerizable mutant did not (Fig.
7C).
The
interpretation of the data assumes that the phosphorylation
being
observed with these
SLN1 alleles is due to
autophosphorylation
of the protein through the function of its
histidine kinase activity.
However, the possibility that the observed
phosphorylation is
due to the activity of another kinase cannot be
completely ruled
out. Nevertheless, these results are consistent with
the hypothesis
that dimerization of Sln1p is essential for
autophosphorylation.
Summary.
The results suggest that the function of Sln1p is
dependent on several structural aspects of the protein. First,
regulation of kinase activity in response to osmolarity requires the
presence of TMD1. All truncated forms of the protein that lacked this
domain failed to respond to changes in the osmolarity of the growth
medium, regardless of the apparent level of histidine kinase activity. No changes in either Sln1p autophosphorylation or Hog1p phosphorylation levels were detected with any of the TMD1 truncations upon addition of
high concentrations of sorbitol or salt to the growth medium.
Second, the ECD appears to play an important role in regulating the
level of activity of the kinase domain. Deletion of the
ECD resulted in
a marked reduction of the amount of phosphorylated
Sln1p and, as judged
by the high level of phosphorylated Hog1p,
an attenuation of kinase
activity. Substitution of the Sln1 ECD
with a known dimerization motif
led to a high level of Sln1p autophosphorylation,
reflected by
inactivation of the HOG MAPK pathway. Mutations shown
to prevent
dimerization led to the opposite condition, high-level
constitutive
signaling. Although we have not explicitly shown
that Sln1p kinase
activation is dependent on dimerization of the
protein, these results
are consistent with the hypothesis that
dimerization is essential for
activity of the kinase and that
dimerization is effected by the
ECD.
Third, in agreement with previous studies (
20), it is clear
that the cytoplasmic histidine kinase and receiver domains of
Sln1p are
essential for viability. Mutant proteins in which the
kinase domain is
disrupted or eliminated are unable to support
growth of the
sln1 strain. The toxicity of the
sln1 null
allele
has been attributed to hyperactivation of the HOG MAPK cascade
(
13). Therefore, growth of the
sln1 strain
should reflect the
level of activity of the SLN1p kinase. In several of
our constructs
(
sln1 strains expressing
TMD1&ECD, KD,
KD/CAAX, and
TMD1&ECD/LeuZip*
[Table
1]), the level of Hog1
phosphorylation appeared to be
high regardless of the osmolarity. In
these strains, phosphorylation
of Sln1p was undetectable. While this
suggests a lack of activity,
sln1 strains expressing
these mutant proteins grew normally.
As we also observed this
complementation with low-copy-number
derivatives of the
high-copy-number constructs, it would appear
that the truncated Sln1p
derivatives possess sufficient kinase
activity to relieve the toxicity
associated with constitutive
HOG pathway
signaling.
| |
ACKNOWLEDGMENTS |
We thank David Lach, Pramathesh Patel, and Ramakrishna Seethala
for assistance with E. coli protein expression, Beverly
Remsburg and Michael Kornacker for assistance with the C/EBP leucine
zipper, and Irene Ota for providing the SLN1/sln1
heterozygous strain and the SLN1 plasmid.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Mail Stop
H24-02, Bristol-Myers Squibb Pharmaceutical Research Institute, Rt. 206 and Province Line Rd., Princeton, NJ 08543-4000. Phone: (609) 252-4456. Fax: (609) 252-6813. E-mail: gormanj{at}bms.com.
Present address: Department of Biochemistry and Molecular Biology,
University of Texas Houston Medical School, Houston, TX 77225.
| |
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Journal of Bacteriology, April 1999, p. 2527-2534, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
