Brookdale Department of Molecular, Cell, and Developmental Biology, Mount
Sinai School of Medicine, New York, NY 10029, USA
* Present address: Servei de Cardiologia, Escala 1, planta 6, Hospital
Clínic de Barcelona, Villaroel 170, Barcelona 08036, Spain
Present address: Applied Biosystems, 35 Wiggins Avenue, Bedford, MA 01730,
USA
Author for correspondence (e-mail:
jeanne.hirsch{at}mssm.edu)
Accepted 11 November 2002
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Summary |
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Key words: GPA2, KRH1, KRH2, Kelch repeat
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Introduction |
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One component of the Gpa2p pathway that has been identified is the G
protein-coupled receptor that interacts with the Gpa2p -subunit. This
receptor, called Gpr1p, was identified by a two-hybrid screen using Gpa2p as
the bait (Kraakman et al.,
1999
; Xue et al.,
1998
; Yun et al.,
1997
). The presence of this receptor on the cell surface suggests
that it recognizes an extracellular ligand
(Xue et al., 1998
). However,
the ligand that activates Gpr1p has not been identified as yet. Like GPA2,
GPR1 is required for the switch to pseudohyphal and invasive forms of
growth (Lorenz et al., 2000
;
Tamaki et al., 2000
). A
constitutively active allele of GPA2 suppresses the filamentous
growth defect conferred by a gpr1
mutation, in agreement with
the idea that Gpr1p couples to Gpa2p and initiates signaling through the Gpa2p
pathway (Lorenz et al.,
2000
).
The signaling pathway that functions downstream of Gpr1p and Gpa2p is not
well understood. One possibility that has been proposed is that Gpa2p acts in
an analogous manner to the mammalian -subunit G
s,
which directly activates adenylyl cyclase. This potential mechanism would
constitute a second way of activating adenylyl cyclase in yeast, in addition
to the known mechanism of direct activation of adenylyl cyclase by Ras
proteins (Broach, 1991
;
Thevelein and de Winde, 1999
).
Production of cAMP by adenylyl cyclase activates the cAMP-dependent kinases
Tpk1p, Tpk2p and Tpk3p. These kinases phosphorylate substrates that regulate
metabolism, growth and filament formation
(Borges-Walmsley and Walmsley,
2000
; Madhani and Fink,
1998
).
cAMP-dependent kinase is involved in many cellular processes, and the three
different forms of this kinase that are present in yeast are not equivalent
for all functions. Although TPK1, TPK2 or TPK3 can provide
the essential function that is revealed when all three genes are mutated
(Toda et al., 1987b), they
appear to play different roles with respect to filamentous growth. Whereas
Tpk2p is required for filamentous growth, Tpk1p and Tpk3p have either no
effect or a small inhibitory effect on this process
(Pan and Heitman, 1999
;
Robertson and Fink, 1998
).
Activation of the cAMP-dependent kinases occurs by binding of cAMP to a
regulatory subunit, Bcy1p, which is presumed to bind to all three forms of the
kinase. It is therefore likely that the different cAMP-dependent kinases are
subject to additional types of regulation that affect their involvement in
different cellular processes.
Several observations suggest that there is a relationship between
Gpa2p-mediated signaling and cAMP-dependent signaling. First, overexpression
of GPA2 augments the rapid increase in cAMP levels that occurs when
glucose is added to glucose-starved cells
(Nakafuku et al., 1988).
Second, the defect in pseudohyphal growth conferred by a gpa2
or gpr1
mutation is reversed by the addition of cAMP
(Kübler et al., 1997
;
Lorenz and Heitman, 1997
;
Lorenz et al., 2000
;
Tamaki et al., 2000
). Finally,
a gpa2
or gpr1
mutation eliminates the
glucose-induced increase in cAMP levels under certain conditions
(Colombo et al., 1998
;
Kraakman et al., 1999
;
Lorenz et al., 2000
;
Yun et al., 1998
). Gpa2p has
been shown to function independently of Ras, which would be consistent with
the possibility that Gpa2p directly activates adenylyl cyclase
(Colombo et al., 1998
;
Xue et al., 1998
). However,
direct activation of adenylyl cyclase by Gpa2p has not been demonstrated
experimentally as yet.
Another candidate for a downstream component of the Gpa2p pathway is the
kinase Sch9p. The SCH9 gene was isolated based on the fact that its
overexpression suppresses the growth defect conferred by mutations in genes
that encode components of the cAMP signaling pathway
(Toda et al., 1988). Sch9p
appears to function in a separate pathway from Ras, adenylyl cyclase and the
cAMP-dependent kinases (Hartley et al.,
1994
; Toda et al.,
1988
). The possibility that Sch9p acts downstream of Gpa2p was
raised by the finding that an sch9
mutation eliminates the
sensitivity to heat shock conferred by a constitutively active version of
Gpa2p (Xue et al., 1998
).
However, Sch9p and Gpa2p do not appear to function in a linear signaling
pathway because sch9
mutant cells have a different phenotype
from gpa2
mutant cells. Cells containing an
sch9
mutation display a significant growth defect
(Toda et al., 1988
) and are
defective for induction of trehalase activity in response to nitrogen addition
(Crauwels et al., 1997
). Cells
containing a gpa2
mutation do not have a growth defect
(Nakafuku et al., 1988
) and
respond normally to nitrogen addition
(Kraakman et al., 1999
).
Moreover, double mutant sch9
gpa2
cells
display a much more severe growth defect than single mutant
sch9
cells, indicating that the functions of these two genes
cannot be completely overlapping (Kraakman
et al., 1999
; Lorenz et al.,
2000
).
In summary, the Gpa2p -subunit cannot be placed in the context of a
simple, linear signaling pathway because all potential downstream components
of the Gpa2p pathway respond to multiple inputs. Here, we report the
identification of two novel genes, KRH1 and KRH2, encoding
proteins that play a role in the Gpa2p pathway. The KRH1 and
KRH2 gene products act downstream of Gpa2p and function by exerting a
negative regulatory effect on haploid invasive growth.
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Materials and Methods |
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Construction of plasmids YEpSCH9 and YEpADH-SCH9 was described previously
(Toda et al., 1988). Plasmid
pS9CLON.111 was made by cloning a 7.6 kb BamHI fragment from YEpSCH9
into the BamHI site of YEplac111. Plasmid pS9-7K.Bs was made by
cloning a 7.0 kb BamHI-KpnI fragment from pS9CLON.111 into
the BamHI-KpnI sites of pBluescript. Plasmid pURA3.Bs was
made by cloning a 1.2 kb XbaI fragment containing the URA3
gene from pAC100-2 into the SpeI site of pBluescript. To construct a
URA3 disruption of SCH9, a 1.0 kb
EcoRI-NsiI fragment from pURA3.Bs was cloned into the
EcoRI-PstI sites of pS9-7K.Bs to produce
pS9-7K::URA3.Bs.
Construction of plasmid YEpTPK2 was described previously
(Toda et al., 1987b). Plasmid
YEpTPK2.2 was made by cloning a 2.0 kb BglII fragment from YEpTPK2
into the BamHI site of YEp351.
The KRH1 gene was cloned by amplifying a 1.2 kb fragment from yeast genomic DNA by polymerase chain reaction (PCR) using primers 5TH14-1 [5'-CGCTGCAGTGATTCATTGGCAGGTCC-3' (genomic sequences are underlined in all primers)], which contains a flanking PstI site (shown in bold), and 3TH14-1 (5'-CGGTCCGTTAATTTGGATCC-3'), which contains an internal BamHI site present in the genomic DNA site (shown in bold). This fragment was cloned into the PstI/BamHI sites of pUC19 to create pUC19.Th14. To construct a HIS3 disruption of KRH1, a 1.8 kb HincII-SmaI fragment from pUC18-HIS3 was cloned into the HincII sites of KRH1 to produce Th14::HIS3. Plasmid YEp181-FLKRH1 was cloned by PCR using yeast genomic DNA as a template and contains sequences from the EcoRI site at nucleotide -755 (where +1 is the A of the start codon) to nucleotide +2778.
The KRH2 gene was cloned by amplifying a 0.8 kb fragment from yeast genomic DNA by PCR using primers oYOR1.5 (5'-CCGAGCTCGTATGGTATGGTGCCCATCAC-3'), which contains a flanking SacI site (shown in bold), and oYOR4 (5'-CCTTAGGTCTACCGTCAAAAGC-3'). This fragment was cloned into the SacI/HindIII sites of pUC19, using the introduced SacI site and an internal HindIII site, to produce pYOR37.1. A 1.1 kb fragment was amplified from yeast genomic DNA by PCR using primers oYOR5 (5'-CGAGTGTAATGCCAAGTGCCA-3') and oYOR6 (5'-CCAAGCTTAATTGCATCATCCTCTAAATA-3'), which contains a flanking HindIII site (shown in bold). This fragment was cloned into the HindIII site of pYOR37.1, using the introduced HindIII site and an internal HindIII site, to produce pYOR37.12. To construct a URA3 disruption of KRH2, a 1.2 kb XbaI fragment from pAC100-2 was cloned into the XbaI sites of pYOR37.12 to produce pYOR37.12U. Plasmid YEp112-KRH2 was cloned by PCR using yeast genomic DNA as a template and contains sequences from the EcoRI site at nucleotide -766 (where +1 is the A of the start codon) to nucleotide +2701.
The plasmid used for isolating the FLO11 probe was made by
amplifying a 0.5 kb fragment from pYSL12
(Lo and Dranginis, 1998) by
PCR using primers oFLO3,
5'-GGGGATCCGTAACTCCTGCCACTAATGC-3', which
contains a flanking BamHI site (shown in bold), and oFLO4,
5'-CCACATAAAGTTTCCAAGAACCTTG-3'. The fragment was
cloned into the BamHI/XhoI sites of pBluescript, using the
introduced BamHI site and an internal XhoI site, to produce
pFLO11CT.Bs. The plasmid used for isolating the ACT1 probe was made
by amplifying a 0.9 kb fragment from yeast genomic DNA by PCR using primers
GMS288 (5'-CCTCGTGCTGTCTTCCCATCTATC-3') and GMS289
(5'-GCATTCTTTCGGCAATACCTGG-3'). The fragment was cloned
into plasmid PCR2.1-TOPO to produce pActin.21.
Strain construction and media
Strains used in this study are listed in
Table 1. The
gpa2::TRP1 allele was made by transformation of cells with the 1.4 kb
BamHI fragment from pgpa2-1::TRP1
(Xue et al., 1998). The
sch9::URA3 allele was made by transformation of cells with a 3.8 kb
HindIII fragment from plasmid pS9-7K::URA3.Bs. The
tpk2::HIS3 allele was made by transformation of cells with a 3.6 kb
EcoRI fragment from plasmid ptpk2::HIS3
(Toda et al., 1987b
). The
tpk2::TRP1 allele was made by transformation of a tpk2::HIS3
strain with a 3.8 kb SmaI/XhoI fragment from marker swap
plasmid pHT6 (Cross, 1997
). The
krh2::URA3 allele was made by transformation of cells with the 1.6 kb
SacI/HindIII fragment from pYOR37.12U. The
krh2::TRP1 allele was made by transformation of a krh2::URA3
strain with a 3.6 kb SmaI fragment from marker swap plasmid pUT11
(Cross, 1997
). The
krh1::HIS3 allele was made by transformation of cells with the 2.3 kb
SacI fragment from Th14::HIS3. The flo11::lacZ-HIS3 allele
was made by transformation of cells with BstEII-digested plasmid
pMUC1-lacZ. All strain constructions involving transformations were confirmed
by Southern blot.
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Strains were grown on YEPD (2% glucose), and strains under selection were
grown on synthetic dropout media, as described
(Guthrie and Fink, 1991).
Two-hybrid screen and yeast methods
pG2CT-T9.2 was transformed into reporter strain HF7c (Clontech) and the
resulting strain was individually transformed with each of three yeast genomic
DNA fusion libraries, Y2HL-C1, Y2HL-C2 and Y2HL-C3
(James et al., 1996).
Transformation mixtures were plated on medium lacking histidine, and positive
transformants were retested for ß-galactosidase expression by incubation
in the presence of 0.3 mg/ml X-gal. Plasmid TH14, which encodes a fusion to
KRH1 at codon 531, was isolated in this screen.
Heat shock assays, sporulation assays, and yeast RNA extraction were
performed as described previously (Xue et
al., 1998). Invasive growth assays were performed according to
Kuchin et al. (Kuchin et al.,
2002
). Yeast transformations were performed by the lithium acetate
method using standard procedures (Guthrie
and Fink, 1991
).
Northern blots
Total RNA was isolated from cells grown to logarithmic phase. RNA was
electrophoresed in a 0.9% agarose formaldehyde-containing gel. Following
electrophoresis, the gel was incubated in 0.05 M NaOH, 0.15 M NaCl for 20
minutes and neutralized in 0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl for 30
minutes. RNA was transferred to a nylon GeneScreen membrane (NEN) by applying
a pressure of 80 mmHG with the Posiblot Pressure Blotter (Stratagene) for 2.5
hours using 1.5 M NaCl, 0.15 M sodium citrate as the transfer buffer. The RNA
was UV crosslinked to the membrane using a Stratalinker UV box.
Prehybridization was carried out at 42°C in a buffer containing 50%
formamide, 0.75 M NaCl, 75 mM sodium citrate, 50 mM sodium phosphate (pH 6.5),
0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.1% SDS,
and 0.1 mg/ml of denatured salmon testes DNA (Sigma). Hybridization was
carried out at 42°C in a buffer containing 50% formamide, 0.75 M NaCl, 75
mM sodium citrate, 50 mM sodium phosphate (pH 6.5), 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, 0.1% bovine serum albumin, 12.5% dextran sulfate, and
0.1 mg/ml of denatured salmon testes DNA. Blots were washed four times for 15
minutes at 65°C with 0.3 M NaCl, 30 mM sodium citrate, 0.5% SDS and
exposed to a phosphor storage screen and scanned with a PhosphorImager
(Molecular Dynamics) or exposed to film. The probes used were gel-purified DNA
restriction fragments 32P-labeled by random primer labeling using a
Prime-It kit (Stratagene). The fragments used were a 0.5 kb
BamHI/XhoI fragment from plasmid pFLO11CT.Bs for the
FLO11 probe and a 0.9 kb EcoRI fragment from plasmid
pActin.21 for the ACT1 probe.
Microscopy
Cells were viewed using Nomarski optics for differential interference
contrast microscopy on a Zeiss Axiophot microscope. They were photographed
with a 100x objective. Plates were viewed using a Zeiss Axioplan 2
microscope and photographed with a 2.5x objective.
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Results |
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KRH1 and KRH2 encode negative regulators of
FLO11 expression and invasive growth
A search of the Saccharomyces Genome Database revealed that a gene
with high homology to KRH1 is present in the yeast genome. The
protein encoded by this gene, which is called KRH2, is 35% identical
to Krh1p. If Krh1p and Krh2p function in the Gpa2p signaling pathway, then
deletion of the KRH1 and KRH2 genes would be expected to
affect cellular processes that require GPA2. Such processes include
the ability of cells to undergo the transition to pseudohyphal or invasive
growth (Kübler et al.,
1997; Lorenz and Heitman,
1997
). The involvement of the KRH1 and KRH2 gene
products in invasive growth was investigated in a strain of the
1278b
background, which contains an intact signaling pathway for this process
(Kron, 1997
).
Haploid invasive growth correlates with induction of the FLO11
gene, which encodes a flocculin that is required for both pseudohyphal and
invasive growth (Lo and Dranginis,
1998). To test whether the KRH1 and KRH2 genes
are involved in the signaling pathway that results in invasive growth, the
expression of FLO11 was determined in strains containing deletions of
these genes. Under conditions of log phase growth, the abundance of
FLO11 RNA was low but detectable in wild-type cells
(Fig. 1A, lane 1). In cells
containing a gpa2
mutation, the level of FLO11 RNA
was greatly decreased relative to wild-type cells (lane 5), as described
previously (Lorenz et al.,
2000
; Tamaki et al.,
2000
). In contrast, individual deletions of KRH1 and
KRH2, or double deletion of both KRH1 and KRH2
resulted in an increase in FLO11 RNA abundance over the level seen in
wild-type cells (lanes 2-4). Quantification of the normalized results from
several experiments showed that the level of FLO11 RNA in
krh1
krh2
cells is three- to fourfold higher
than in wild-type cells. Because deleting the genes causes activation of the
signaling pathway, these results indicate that KRH1 and KRH2
act as negative regulators of signaling. One possible interpretation of these
findings is that Krh1p and Krh2p inhibit signaling of a component that acts
downstream of Gpa2p under conditions that promote turning the pathway off.
Alternatively, Krh1p and Krh2p could be required for modification or
localization of the Gpa2p protein. To distinguish between these possibilities,
the effect of the krh1
and krh2
mutations was
determined in a strain lacking Gpa2p. In triple mutant krh1
krh2
gpa2
cells, the level of FLO11
RNA was substantially higher than that seen in gpa2
cells
(lanes 5 and 6). A comparison of krh1
krh2
cells with krh1
krh2
gpa2
cells revealed that the presence of the gpa2
mutation in a
krh1
krh2
background causes an approximately
two- to threefold decrease in FLO11 RNA abundance (lanes 4 and 6).
However, the level of FLO11 RNA was more than tenfold higher in
krh1
krh2
gpa2
cells than in
gpa2
cells, indicating that the predominant effect of the
krh1
krh2
mutations is to suppress the
phenotype of the gpa2
mutation.
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Induction of the FLO11 gene generally correlates with an increase
in invasive growth in haploid cells and pseudohyphal growth in diploid cells.
To test whether the increased level of FLO11 expression in
krh1 krh2
cells has physiological
consequences, the ability of wild-type and krh1
krh2
strains to undergo haploid invasive growth was
determined. krh1
, krh2
and
krh1
krh2
cells showed a significant increase
in their ability to invade solid medium when compared with wild-type cells
(Fig. 1B). The most pronounced
effect was seen with krh1
krh2
double mutant
cells. Whereas gpa2
cells did not invade the medium to a
significant degree, krh1
krh2
gpa2
cells displayed substantial invasive growth. Therefore,
the krh1
krh2
mutations suppress the defect in
invasive growth conferred by a gpa2
mutation. Deletion of the
FLO11 gene eliminated the increase in invasiveness conferred by the
krh1
krh2
mutations, consistent with
previously identified characteristics of invasive growth
(Fig. 1B).
In addition to the phenotype displayed by krh1
krh2
cells on solid medium, krh1
krh2
cells grown in rich liquid medium for two days displayed
an altered morphology. In liquid medium, krh1
krh2
cells formed chains of elongated cells that appear
similar to cells undergoing the early stages of pseudohyphal growth
(Fig. 1C). Depletion of glucose
from the medium, which occurs under these conditions, is known to promote
invasive growth in haploid cells (Cullen
and Sprague, 2000
). Introduction of a gpa2
mutation into a krh1
krh2
strain had no effect
on the altered morphology of cells grown to saturation in liquid medium (data
not shown). Given that pseudohyphal formation normally requires growth on
solid medium, and that krh1
krh2
cells
overcome this requirement, it can be concluded that the KRH1 and
KRH2 gene products exert a strong negative effect on filamentous
forms of growth in wild-type cells.
Another morphological phenotype conferred by the krh1
krh2
mutations involves the macroscopic appearance of patches
of cells. Deletion of the KRH1 and KRH2 genes causes cell
cultures incubated on solid medium to grow up off the plate in extended sheets
(Fig. 1D). This macroscopic
phenotype is reminiscent of that seen when S. cerevisiae is grown
under conditions of fungal biofilm formation
(Reynolds and Fink, 2001
).
Biofilm formation in S. cerevisiae requires FLO11
(Reynolds and Fink, 2001
),
suggesting that the phenotype of krh1
krh2
mutants may be the result of increased cell adhesion due to higher levels of
FLO11 expression.
KRH1 and KRH2 negatively regulate processes
controlled by the cAMP/PKA pathway
Gpa2p activity also has effects in strains that are not capable of
undergoing pseudohyphal or invasive growth. For example, cells of a
non-filamentous strain that contain a constitutively active allele of
GPA2 display a decrease in sporulation efficiency and an increase in
heat shock sensitivity (Xue et al.,
1998). Increased sensitivity to heat shock is a phenotype
associated with cells that have increased activity of growth control pathways,
including both the cAMP/PKA pathway and other redundant pathways
(Cameron et al., 1988
;
Toda et al., 1987a
). To
determine whether the KRH1 and KRH2 genes play a role in
heat shock sensitivity, survival following a heat shock was measured for
wild-type, krh1
, krh2
and krh1
krh2
cells in a W303 background. Whereas 48% of wild-type
cells in stationary phase survived after a 50°C heat shock, only 1.0% of
krh1
krh2
cells survived after this treatment
(Fig. 2A). Single
krh1
and krh2
mutants displayed intermediate
survival levels of 2.8% and 22.4%, respectively. This finding confirms that
the KRH1 and KRH2 gene products are negative regulators of
the signaling pathway because an increase in heat shock sensitivity occurs
either when constitutively active GPA2 is present
(Xue et al., 1998
) or when the
KRH1 and KRH2 genes are deleted.
|
The effect of krh1 krh2
mutations on the
ability of cells to sporulate was also examined. Cells containing intact
KRH1 and KRH2 genes displayed 51% sporulation efficiency
(Fig. 2B).
krh1
, krh2
and krh1
krh2
cells all showed a decrease in sporulation efficiency.
The largest effect was seen in krh1
krh2
cells, which had a sporulation efficiency of 6%. Single krh1
and krh2
mutants displayed intermediate levels of sporulation
of 26% and 35%, respectively. Therefore, deletion of the KRH1 and
KRH2 genes results in multiple physiological changes that are
associated with increased activation of the cAMP/PKA pathway.
The interaction between Krh1p and Gpa2p revealed by the two-hybrid assay
raised the possibility that Krh1p and Krh2p are proteins that interact with
G subunits in general. Therefore, it was possible that these
proteins also interact with Gpa1p, the G
subunit that
mediates the pheromone response pathway. krh1
,
krh2
and krh1
krh2
cells were
tested for their ability to undergo cell cycle arrest in response to pheromone
by measuring the density of cell growth in an area surrounding a filter disk
containing
-factor (halo assay). No differences were detected in this
assay among wild-type, krh1
, krh2
or
krh1
krh2
cells (data not shown). Therefore,
the function of KRH1 and KRH2 appears to be specific to the
Gpa2p pathway.
KRH1 and KRH2 act downstream of GPA2
If Krh1p and Krh2p couple Gpa2p to downstream components, then deletion of
the KRH1 and KRH2 genes would be expected to eliminate the
effects of a constitutively active form of Gpa2p. To test this idea, the
effect of Gpa2p activation on FLO11 RNA abundance was determined by
isolating RNA from strains containing either vector or the constitutively
active GPA2R273A allele. In wild-type cells,
FLO11 RNA levels were significantly higher in cells containing the
GPA2R273A allele than in cells containing vector alone
(Fig. 3A, lanes 1,2). These
findings are in agreement with previous results that indicate that
GPA2 is involved in the transition to filamentous growth
(Kübler et al., 1997;
Lorenz and Heitman, 1997
). The
presence of the GPA2R273A allele had no effect on
FLO11 RNA abundance in the krh1
krh2
strain (lanes 3 and 4). One interpretation of this result is that transmission
of the signal generated by activated Gpa2p is largely prevented by deletion of
KRH1 and KRH2.
|
To determine whether the KRH1 and KRH2 genes are required
for the effect of Gpa2p activation on heat shock sensitivity, survival
following a heat shock was measured in wild-type and krh1
krh2
strains. Heat shock treatment of wild-type cells
containing the constitutively active GPA2R273A allele
caused a decrease in survival of approximately 70-fold compared with
vector-containing cells (Fig.
3B), confirming previous results
(Xue et al., 1998
). However,
krh1
krh2
cells containing either vector or
the GPA2R273A plasmid displayed the same survival levels
(Fig. 3B). Elimination of the
effects of the GPA2R273A allele by the krh1
krh2
mutations is consistent with a model in which Krh1p and
Krh2p act downstream of the Gpa2p
-subunit.
Signal generated by lack of KRH1 and KRH2 does not
require SCH9
Previous results from our laboratory suggested that the Sch9p kinase acts
downstream of Gpa2p (Xue et al.,
1998). It was therefore of interest to test whether deletion of
the SCH9 gene blocks the signal resulting from deletion of the
KRH1 and KRH2 genes. If Krh1p and Krh2p act immediately
downstream of Gpa2p, then components that act further downstream in the same
pathway would be expected to eliminate the signal generated by the absence of
the negative regulators. To determine the effect of an sch9
mutation on the phenotype conferred by the krh1
and
krh2
mutations, the heat shock sensitivity of strains
containing different mutations was measured. Cells containing an
sch9
mutation did not display a heat shock sensitive phenotype
(Fig. 4A), in agreement with
previous results (Xue et al.,
1998
). Heat shock treatment of the triple mutant
krh1
krh2
sch9
strain caused a
large decrease in survival, similar to the effect seen in a
krh1
krh2
strain
(Fig. 4A). Therefore, the
sch9
mutation does not eliminate the phenotype caused by
deletion of the KRH1 and KRH2 genes. This finding suggests
that Sch9p does not act downstream in a pathway that is negatively regulated
by Krh1p and Krh2p.
|
The data presented above are consistent with the idea that Krh1p and Krh2p
act downstream of Gpa2p to negatively regulate a signaling pathway that does
not require Sch9p. If this were the case, then krh1
krh2
mutations would be expected to suppress phenotypes
present in gpa2
sch9
double mutants by
activating the downstream pathway. To test this possibility, strains were
constructed that contained either deletions of KRH1 and
KRH2, deletions of GPA2 and SCH9, or all four
mutations together. krh1
krh2
mutant strains
did not display an apparent growth defect
(Fig. 4B). gpa2
sch9
mutant strains displayed a severe growth defect and did
not produce visible colonies after 2 days of growth, as described previously
(Kraakman et al., 1999
;
Lorenz et al., 2000
). Strains
containing all four mutations showed substantial suppression of the growth
defect seen in the gpa2
sch9
strain. These
results indicate that loss of KRH1 and KRH2 results in
activation of a pathway that partially compensates for the lack of
SCH9.
Signal generated by lack of KRH1 and KRH2 or
activation of Gpa2p requires TPK2
Activation of the RAS/cAMP pathway suppresses the growth defect
conferred by an sch9 mutation
(Hartley et al., 1994
;
Toda et al., 1988
). Although
the Ras proteins function independently of Gpa2p
(Colombo et al., 1998
;
Xue et al., 1998
), suppression
of the sch9
growth defect by krh1
krh2
mutations could be explained by activation of downstream
components of the RAS/cAMP pathway under these conditions. One
candidate for a downstream component that is activated in krh1
krh2
cells is the cAMP-dependent protein kinase Tpk2p. In
yeast, cAMP-dependent protein kinase is encoded by three genes, TPK1,
TPK2 and TPK3 (Toda et al.,
1987b
). However, only the TPK2 gene product positively
regulates FLO11 transcription
(Pan and Heitman, 1999
;
Robertson and Fink, 1998
). To
determine whether TPK2 is required for the signal generated by
krh1
krh2
mutations, the basal level of
FLO11 RNA was measured in cells containing different combinations of
mutations. FLO11 RNA was present at a basal level in wild-type cells,
but was undetectable in cells containing a tpk2
mutation
(Fig. 5A, lanes 1,2), as
described previously (Pan and Heitman,
1999
; Robertson and Fink,
1998
). FLO11 RNA was also undetectable in
krh1
krh2
tpk2
triple mutant
cells (lane 4), demonstrating that TPK2 is required for the signal
generated in krh1
krh2
cells. These results
imply that the signal transmitted through Krh1p and Krh2p inhibits the
activity of the Tpk2p kinase, either directly or indirectly.
|
Krh1p and Krh2p appear to act downstream of Gpa2p and upstream of the
cAMP-dependent protein kinase Tpk2p. To investigate further the ordering of
the signaling pathway, the requirement for TPK2 in signaling through
activated Gpa2p was determined. Wild-type cells carrying the
GPA2R273A allele contained significantly more
FLO11 RNA than cells carrying vector alone
(Fig. 5B, lanes 1,2), as
described above. However, no detectable FLO11 RNA was present in
tpk2 mutant cells carrying either vector or the
GPA2R273A allele (lanes 3,4), indicating that
TPK2 acts downstream of GPA2.
The effect of a tpk2 mutation on the altered cell
morphology conferred by the krh1
krh2
mutations was also examined. Whereas krh1
krh2
mutants grown to saturation in rich medium formed chains of elongated cells,
krh1
krh2
tpk2
mutants formed
clusters of more rounded cells (Fig.
5C). Therefore, the tpk2
mutation largely
suppresses the cell morphology phenotype of krh1
krh2
cells. In addition, krh1
krh2
tpk2
cells displayed significantly less
haploid invasive growth than krh1
krh2
cells
when the plate washing was done after 3 days of growth (data not shown).
The effect of TPK2 overexpression was determined in both wild-type
and krh1 krh2
strains. In wild-type cells,
TPK2 overexpression resulted in a large increase in the abundance of
FLO11 RNA (Fig. 5D,
lanes 1,2). However, TPK2 overexpression had no effect on the high
level of FLO11 RNA present in cells containing deletions of the
KRH1 and KRH2 genes (lanes 3,4).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The association between GPA2 mutant phenotypes and cAMP signaling
has led to the suggestion that yeast adenylyl cyclase is directly activated by
the G subunit Gpa2p. This situation would be analogous to
the direct activation of mammalian adenylyl cyclase by the G
subunit G
s. In contrast to this idea, we have shown that the
novel proteins Krh1p and Krh2p appear to act immediately downstream of Gpa2p.
Moreover, Krh1p and Krh2p require the cAMP-dependent kinase Tpk2p for their
signaling function, in agreement with previous observations that show an
association between GPA2 and cAMP signaling. Therefore, it seems
likely that the effect of Gpa2p on cAMP signaling is mediated by Krh1p and
Krh2p. However, overexpression of TPK2 had no effect in cells
containing krh1
krh2
mutations. Therefore,
further experiments will be needed to determine whether this result is due to
maximal activation of the pathway in krh1
krh2
cells or to the existence of a complex relationship between Krh1p, Krh2p and
Tpk2p.
Krh1p and Krh2p do not display any obvious sequence identity to other known
proteins, but they do contain six repeats that display some similarity to
kelch repeats (Fig. 6). Kelch
repeats are segments of about 50 amino acids that contain a characteristic
double glycine motif (Adams et al.,
2000). The double glycine is situated C-terminal to four
hydrophobic residues and N-terminal to conserved tyrosine and tryptophan
residues that are separated from it by spacer regions. The repeats in Krh1p
and Krh2p also contain a double glycine motif situated C-terminal to four
hydrophobic residues. However, only one of these repeats
(Fig. 6, repeat 3) contains the
conserved tyrosine and tryptophan residues, and the spacer region between
these residues and the double glycine repeat is smaller than that seen in the
consensus for kelch repeats. The other repeats are missing either the tyrosine
or tryptophan at the appropriate position. Therefore, they can be thought of
as variant kelch repeats. The crystal structure of a protein containing seven
kelch repeats has revealed that each repeat forms a four-stranded
ß-sheet, resulting in a protein that consists of a seven-bladed
ß-propeller (Ito et al.,
1994
). This 3D structure is similar to that of G protein
ß-subunits, which contain seven WD domains that form a seven-bladed
ß-propeller (Sondek et al.,
1996
; Wall et al.,
1995
). These observations raise the interesting possibility that
Krh1p and Krh2p bind to Gpa2p in a manner similar to that by which
Gß subunits bind to G
subunits. Given that
Gpa2p is a positive regulator of the signaling pathway and that Krh1p and
Krh2p are negative regulators, it would be predicted that binding of Gpa2p to
Krh1p and Krh2p blocks their ability to inhibit downstream steps in the
pathway.
|
The KRH1 gene was identified previously by a mutation that causes
cells to continue to grow when incubated in alkaline sporulation medium
(Ohkuni and Yamashita, 2000).
This medium contains acetate as a carbon source and is limiting for nitrogen,
a nutritional condition that induces sporulation in wild-type cells. Previous
results have shown that mutational activation of Gpa2p inhibits sporulation to
a significant degree (Xue et al.,
1998
). The finding that a loss-of-function mutation in the
KRH1 gene causes cells to continue to divide under conditions that
induce sporulation is consistent with our observation that the sporulation
efficiency of krh1
krh2
mutants is very
low.
The KRH2 gene was isolated previously as one of 29 genes that
encode proteins that interact with the nuclear export factor Crm1p in a
two-hybrid assay (Jensen et al.,
2000). Although KRH2 was not characterized further in
that study, interaction with Crm1p could indicate that Krh2p is capable of
nucleocytoplasmic shuttling. It is therefore of interest to note that other
proteins involved in cAMP signaling, such as the catalytic and regulatory
subunits of cAMP-dependent kinase, localize differentially to the nucleus or
cytoplasm in response to different physiological conditions
(Griffioen et al., 2000
).
Differential localization of Krh2p could limit inhibition of the signaling
pathway to a particular cellular compartment, providing an additional level of
regulation to the pathway.
Although it is likely that Krh1p and Krh2p function by transmitting a
signal from Gpa2p to downstream components, the pathway that relays this
signal to its ultimate physiological targets is not clearly understood. For
example, work presented here demonstrates that the Sch9p kinase is not
required for the increase in FLO11 RNA that is observed in cells
lacking KRH1 and KRH2. However, we have shown previously
that SCH9 is required for the increase in heat shock sensitivity
conferred by a constitutively active allele of GPA2
(Xue et al., 1998). Other
experiments exploring the relationship between SCH9 and GPA2
showed that SCH9 is required for the increase in trehalase activity
observed when a nitrogen source is added to cells starved for nitrogen, but
that GPA2 is not required for this process
(Crauwels et al., 1997
;
Kraakman et al., 1999
).
Similarly, GPA2 is required for the increase in cAMP observed when
glucose is added to cells starved for glucose, but SCH9 is not
required for this process (Colombo et al.,
1998
; Crauwels et al.,
1997
). Moreover, double mutant sch9
gpa2
cells have a much more severe growth defect than either
of the single mutants, suggesting that GPA2 and SCH9
function, at least in part, in different pathways
(Kraakman et al., 1999
;
Lorenz et al., 2000
). One
interpretation of the relationship between SCH9 and GPA2 is
that the Sch9p kinase is required only for a subset of the phenotypes
associated with activation of Gpa2p. In that case, it would suggest that there
are branchpoints in the signaling pathway downstream of Gpa2p. Alternatively,
it is possible that Sch9p is required only for signaling through the Gpa2p
pathway under particular growth conditions. Such a result would suggest that
Gpa2p couples to different downstream components depending on growth
conditions.
The uncovering of a novel class of signaling molecules that act downstream
of a G subunit could have implications for G protein
signaling in a wide variety of eukaryotic organisms. A homologue of
KRH2 is present in the genome of the yeast Kluyveromyces
lactis (Ozier-Kalogeropoulos et al.,
1998
), and potential homologues also exist in multicellular
organisms. It is likely that these KRH1 and KRH2 homologues
couple G proteins to downstream signaling components in other organisms.
Therefore, further studies of the function of Krh1p and Krh2p in signaling
through the Gpa2p pathway have the potential to contribute to the
understanding of general mechanisms of G protein signaling.
![]() |
Acknowledgments |
---|
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