From the
In a search for mutants exhibiting altered activity of the yeast
transcription factor, Mcm1, we have identified the SLN1 gene,
whose product is highly related to bacterial two-component
sensor-regulator proteins. sln1 alleles identified in our
screen increased Mcm1p-mediated transcriptional activation, while
deletion of the SLN1 locus severely reduced Mcm1p activity.
Our data establish that Mcm1p is a downstream target of the Sln1
signaling pathway. Yeast Sln1p was recently shown to be involved in
osmoregulation and to depend on the Hog1 MAP kinase (Maeda, T.,
Wurgler-Murphy, S., and Saito, H. (1994) Nature 369,
242-245). We show that SLN1-mediated regulation of Mcm1p
activity is independent of the Hog1 MAP kinase, and suggest that the
role of SLN1 is not restricted to osmoregulation.
Prokaryotic organisms use a simple modular strategy for response
and adaptation to their physical and chemical environments. In its
simplest form, two components, a sensor and a response regulator, are
combined to sense and transmit information using a high energy
phosphohistidine intermediate
(1) . The two-component regulatory
system was recently demonstrated in eukaryotes by the isolation of
genes in Arabidopsis thaliana (2) and
Saccharomyces cerevisiae (3, 4, 5) encoding proteins containing many of the expected consensus
features. We show here that one of the downstream targets of the yeast
Sln1p two-component pathway is the transcription factor, Mcm1.
Mcm1
is a constitutively expressed DNA-binding protein that is essential for
yeast viability
(6) . The optimum site for Mcm1p binding (P
site) consists of a 16-base pair perfect palindrome
(7, 8, 9) . At low affinity (partially
degenerate) binding sites, Mcm1p activity is both amplified and
regulated by interactions with accessory proteins that are thought to
act by altering the conformation of Mcm1p
(10) . In contrast, at
high affinity (palindromic) P sites, Mcm1p binds in an active
conformation in the apparent absence of additional proteins
(10, 11) .
The N-terminal 97 amino acids of Mcm1
carry the DNA binding, dimerization, and protein interaction functions
of the protein
(12, 13, 14) . This so-called
``MADS box'' domain is 70% identical to an equivalent domain
of human serum response factor (SRF).
Various SLN1 subclones were generated
from the SLN1 library plasmid (pGY111). pGY112 was generated
by subcloning the 2.8-kb BamHI fragment from pGY111 into the
BamHI-linearized LEU2 2-µm shuttle vector, pRS425
(Stratagene). pGY113 was constructed by religation of pGY111 in the
absence of the 2.8-kb BamHI fragment. pGY121 consists of the
3.5-kb pGY111 PstI fragment in PstI-linearized
pRS425. pGY140 was constructed by subcloning the 4.3-kb pGY111
ClaI fragment into pGEM5Zf(+) (Promega) to generate
pGY129 and then reisolating that fragment with EagI ends for
subcloning into the EagI site of pRS425. To construct pGY151,
the 6.7-kb pGY111 BbuI fragment was first subcloned into
BbuI-digested pGEM5Zf(+) to generate pGY138. Next, the
5-kb pGY138 EcoRV fragment was subcloned into
EcoRV-cut pGEM5Zf(+) generating pGY144, and finally, a
5-kb pGY144 EagI fragment was subcloned into
EagI-linearized pRS424 (Stratagene), a TRP1 2-µm
shuttle vector. The insert sequences in pGY151 extend from the
BbuI site to the EcoRV site in SLN1 (Fig. 1). The SLN1 integrating vector, pGY141 was
constructed by inserting a 2.2-kb XhoI- SalI fragment
containing the LEU2 gene into the polylinker SalI
site of pGY138.
The PTP2 plasmid, pGY153, was constructed by subcloning a 5-kb
BamHI fragment from pRSYP2
(22) into the 2-µm
TRP1 pRS424 vector (Stratagene). JF1361 and JF1360 were
generated by transformation of JF819 and JF1359 with a 5.1-kb
BamHI fragment isolated from pGEM-YP2:: LEU2 (22) . Putative PTP2 disruptions were confirmed by
Southern hybridization analysis.
The hog1-
Plasmid YIp5- mcm1-
Probes for Northern hybridization analysis included: a 1.6-kb
EcoRI fragment containing MF
To identify new genes that modulate the transcriptional
activity of Mcm1p, cells carrying a plasmid-borne, CYC1-lacZ fusion gene directed by a high affinity P site
(UAS(P)- lacZ) (pGY48) were subject to EMS mutagenesis. A wild
type strain carrying the UAS(P)- lacZ reporter has a pale blue
phenotype on media containing the chromogenic
Since
the increased UAS(P)- lacZ activity of the nrp2 mutants was a recessive phenotype ( nrp2-1/NRP2,
), the gene was cloned from a centromere based genomic
library (P. Hieter) by complementation of the defect leading to the
X-gal phenotype. Among 10,000 transformants, a single plasmid, pGY111,
was identified that rescued the blue colony phenotype of the
nrp2-2 mutation. pGY111 also complemented the X-gal
phenotype of the nrp2-1 and nrp2-3 mutants. The complementing gene was localized to a 5-kb
BbuI- EcoRV fragment within the 10-kb insert of pGY111
by testing the complementing activity of subclones and deletions (Fig.
1). The DNA sequence in the vicinity of the BamHI site in the
BbuI- EcoRV complementing clone was determined and
found to be identical to a region of the previously isolated SLN1 gene
(5) .
Evidence that the X-gal phenotype of nrp2 mutants was due to mutation of the SLN1 gene was provided
by the mutant phenotype of an nrp2-1/sln1-
The Sln1 protein
exhibits a high degree of similarity to both the sensor and response
regulator modules of bacterial two component regulators
(5) . In
bacterial two-component regulation, signal transmission depends on
autophosphorylation of a conserved histidine in the sensor and
phosphotransfer to a conserved aspartate in the response regulator, the
function of which is to relay the signal. Activity of the pathway
depends on the half-life of the phosphoaspartate, which is determined
by the relative rates of phosphotransfer and phosphohydrolysis carried
out by the two modules
(1, 33) .
To evaluate the
phenotype of a SLN1 null mutation, a marked SLN1 gene
disruption was constructed. Consistent with the phenotype previously
described
(5) , the sln1-
To confirm the
effect of the sln1-
The increased P
site activity in the nrp2 mutants was not reflected in
increased MF
Although the physiological
role of Sln1p is currently unclear, recent studies demonstrate that
Sln1p function is mediated by the Hog1 MAP kinase in the osmosensing
pathway and depends on the activity of protein tyrosine phosphatase
( PTP) genes
(34) . To understand the basis of the
effect of sln1 mutations on Mcm1p activation, we investigated
the relationship of PTP2 and HOG1 to the activity of
the UAS(P)- lacZ reporter. The growth phenotype of the
sln1-
The recent isolation of mutations in the
HOG1 MAP kinase gene as suppressors of the growth defects of a
sln1-
The identification of the well
characterized Mcm1 transcription factor as a target of a two-component
regulator reveals for the first time a specific transcriptional outcome
of this newly recognized signaling pathway in yeast. We find that
Sln1p-mediated regulation of P activity is independent of the
osmosensing MAP kinase pathway, implying that Sln1p has multiple
signaling functions. We anticipate that the analysis of additional
nrp mutants will help to define the signal and refine our
understanding of the post-translational events involved in signal
transmission.
Assay
methods and calculations are described in the notes to Table II.
Activities are the average of three or more
trials.
Assay methods and calculations are described in the notes
to Table II. All values were normalized to the activity of the wild
type strain measured in the absence of
salt.
We thank G. Gussin, D. Weeks and Lois Weisman for
their critical reading of this manuscript; M. Gustin, I. Ota, A.
Varshavsky, K.-L. Guan, C. Christ, and B. K. Tye for plasmids and
strains; S. Eliason for experimental assistance; and members of the
Fassler and Deschenes laboratories for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
Like
Mcm1, SRF activity is regulated and modified by its interactions with
accessory proteins
(15) . In addition, SRF-mediated activation
is responsive to growth factor signals via kinase-based signal
transduction cascades. To investigate the possibility that Mcm1p
activity is modulated in response to extracellular signals, mutations
leading to altered activity were sought. We previously found that
mutations in the SPT13 ( GAL11) gene increased
Mcm1-mediated activation from a high affinity (palindromic) P site
(16) . Additional negative regulators of palindromic P site
activity were sought, and one gene, NRP2, identified in this
screen, is the subject of this report.
Yeast Strains and Media
All yeast strains used
in this work (Table I) are from our strain collection or were
constructed for this study except RC634
(17) . The media were
prepared as described by Sherman et al. (18) and
included synthetic complete medium (SC) lacking one or more specific
amino acids ( e.g. SC-uracil) and rich medium (YPD). Plates for
the detection of -galactosidase contained 50 µg of
5-bromo-4-chloro-3-indolyl
-
D-galactopyranoside
(X-gal)/ml and were prepared as described by Larson et al. (19) . All yeast strains were grown at 30 °C.
EMS Mutagenesis
JF819 carrying the pGY48 reporter
(below) was grown to saturation in SC-uracil. Cells were washed and
resuspended in 0.1
M sodium phosphate, pH 7.0, prior to
incubation with EMS (0.29% v/v) for 20 min at 30 °C. Following
inactivation of the EMS, cells were diluted with 0.1
M phosphate buffer and plated on SC-uracil media at 30 °C.
Colonies arose at roughly 40% the expected numbers. Mutants were
selected on the basis of their blue phenotype following replica plating
to SC-uracil media containing 50 µg/ml X-gal.
Plasmids
The reporter plasmid, pGY48, a derivative
of plasmid pLG670Z
(20) , carries a CYC1-lacZ fusion
gene lacking the CYC1 UAS. A 16-base pair palindromic Mcm1
binding (P) site oligonucleotide was cloned into the UAS position of
the CYC1 promoter to create pGY48
(16) . pLG669Z
carries a CYC1- lacZ fusion gene that contains the
intact CYC1 UAS
(20) . The 20B plasmid carries a
GAL1-lacZ fusion gene in which the upstream regulatory
sequences -473 to -632 of GAL1 have been deleted
(21) . As a result, reporter gene expression is not
glucose-repressible.
Figure 1:
Restriction map and complementation
analysis of the nrp2 complementing clone (pGY111) containing
the SLN1 gene. Yeast sequences are shown as solid and
vector sequences as dotted lines.
A marked deletion of the SLN1 gene was
constructed by removal of an internal 2.1-kb HpaI fragment
from plasmid pGY138, followed by insertion of a 2.2-kb
XhoI- SalI LEU2 fragment or a 0.85-kb
EcoRI- BglII TRPI fragment at the point of
the deletion to generate plasmids pGY143 and pGY148. The disrupted
SLN1 gene was isolated on a 7.5-kb (pGY143) or a 6.2-kb
(pGY148) BbuI restriction fragment and used to transform a
wild type diploid generated by mating strains JF819 and JF1220. GY61
and GY67 are haploid progeny of the disrupted sln1::LEU2 diploid. JF1350-1355 are haploid progeny of the
sln1
::TRP1 disrupted diploid.
::TRP1 disruption vector, pGY150, was constructed from pJB30
(23) by deletion of an internal 0.4-kb EcoRI fragment,
and replacement with a 0.85-kb BglII- EcoRI TRP1 fragment. A 3.0-kb BamHI- ClaI fragment was
isolated and introduced into JF1331 to generate JF1362 and into the
nrp2-1 mutant, JF1359, to generate GY91.
hog1-
transformants were distinguished by their failure
to grow on solid media containing 0.9
M NaCl
(23) .
DEQ
(12) was used in the
two-step transformation of the wild type strain JF820 to generate the
mcm1 truncation mutant (JF1335) used as an Mcm1-deficient
control in the halo and Northern (RNA) hybridization experiments.
1 isolated from
plasmid p69A
(24) , a 3.8-kb EcoRI fragment containing
the ACT1 gene isolated from plasmid pACT1 (F. Winston), and a
2-kb NdeI fragment that includes the MCM1 coding
region isolated from plasmid pGA1761
(25) .
Halo Assay
The halo assay was as described
previously
(26) except that synthetic media was used to permit
growth of the sln1- strains. The tester lawn was RC634
( MATa sst1-3)
(17) .
RNA Isolation and Northern Hybridization
Analysis
Cells were grown to a concentration of 1-2
10
/ml in SC-uracil media. RNA was prepared by the
method of Carlson and Botstein
(27) . Electrophoresis, blotting
and hybridization were performed as described previously
(28) .
P-Labeled probes were prepared using random primers
(29, 30) .
-Galactosidase Assays
-Galactosidase
assays were performed using cleared lysates
(31) as described
previously
(32) . Strains were cultured in SC-uracil media
except in the salt induction experiments, overnight cultures grown in
SC-uracil were diluted into rich media (YPD) and grown for two to three
doublings to a final density of 1
10
/ml. Sodium
chloride was added as a solid to a final concentration of 0.9
M. At the indicated times, 10-ml aliquots were removed and
-galactosidase assays performed.
Western Analysis
Strains were harvested in
exponential growth at the times indicated following the addition of
solid NaCl to 0.9
M. 50 µg of each extract, prepared as
described previously
(23) , was subjected to 10%
SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose, and
probed with anti-phosphotyrosine antibody (1:2000 dilution, 4G10;
Upstate Biotechnology Inc.). Immune complexes were visualized by ECL
chemiluminescence (Amersham Corp.).
-galactosidase
substrate, X-gal. Following mutagenesis, colonies were identified that
exhibited increased blue color on X-gal media and increased activity in
liquid
-galactosidase assays. Mutants with increased reporter gene
activity were designated NRP, which stands for Negative
Regulators of P site activity. One complementation group,
NRP2, consisting of three alleles, is the subject of this
report. Each of the three nrp2 mutations increased activity of
the UAS(P)- lacZ reporter approximately 5-fold (Table II). The
nrp2 mutations increased the activity of the closely related
(UAS( CYC1)- lacZ) reporter only 1.1-1.7 fold;
hence, the large effect of the nrp2 mutations appears to
depend on the UAS(P) element (). The activity of the
unrelated (UAS( GAL1)- lacZ) reporter was decreased
slightly () in two of the nrp2 strains.
diploid
(). Linkage between SLN1 and nrp2 was
also tested. The SLN1 integrating plasmid, pGY141, was
linearized at the unique BamHI site within the SLN1 gene and Leu
integrants of an nrp2-3 diploid were isolated. Diploid integrants exhibited a white
phenotype on X-gal plates, and following sporulation, the mutant (blue
on X-gal plates) phenotype segregated 2:2 in dissected tetrads. A white
Leu
spore colony was then mated with JF819
( SLN1
). The absence of the mutant phenotype
among the progeny of this diploid (0/44) was consistent with linkage
between the SLN1 and nrp2 loci.
mutant failed to grow or
formed only microcolonies on YPD media, and grew slowly on synthetic
complete media. The activity of the UAS(P)- lacZ reporter
activity in sln1-
strains measured under permissive
growth conditions (SC-uracil, 30°C) was 10-fold less than in
SLN1
strains (). Although
deletion of SLN1 decreased P site activity, the original
recessive nrp2 activating alleles increased P site activity.
One possible explanation for these phenotypes is that Sln1, like
bacterial two-component systems
(1) , harbors both positive and
negative activities, and that the negative Sln1 function can be
provided in trans. The reduced UAS(P)- lacZ reporter
activity observed in sln1-
mutants suggests that SLN1 is required for full P-mediated activation.
mutation on P-mediated activation,
expression of the Mcm1-regulated MF
1 gene encoding
mating pheromone was analyzed. Deletion of SLN1 caused a
reduction in MF
1 levels as seen by halo assay (Fig.
2 A) and by Northern (RNA) hybridization analysis
(Fig. 2 B). In these experiments the mcm1
DEQ (12) mutant was included as a negative control. The
activity of the UAS(P)- lacZ reporter in this Mcm1 truncation
mutant is 18% of that in a wild type strain
(12) . Densitometric
analysis indicated that MF
1 levels were reduced in the
sln1-
mutants to 25% of wild type levels. The reduction
in MF
1 expression was a specific result of the sln1 defect and was not attributable to the slow growth of the sln1 mutant strain since expression of an Mcm1-independent gene,
ACT1, was unaffected in the mutant (92.5% of wild type)
(Fig. 2 B).
Figure 2:sln1- decreases
Mcm1-activated expression of endogenous genes. A, halo assay.
MAT
strains were grown permissively on synthetic medium
and then transferred to a lawn of MATa cells that are
supersensitive to
mating pheromone. The size of the zone of
non-growth reflects MF
gene transcript levels.
B, Northern (RNA) hybridization. Equal amounts (15 µg) of
total RNA from the indicated MAT
strains were separated
by formaldehyde-agarose gel electrophoresis. A single filter was
hybridized to a
P-labeled restriction fragments
containing sequences from the MF
1 gene (from plasmid
pHK2; Ref. 24), the ACT1 gene from plasmid pACT1 (F. Winston),
and the MCM1 gene from plasmid pGA1761 (25). Hybridization to
0.7-kb ( MF
1), 1.3-kb ( ACT1), and several 1 to
1.6-kb ( MCM1) transcripts was detected as expected. Ethidium
bromide staining of the same gel is shown to the right.
MAT
strains: wild type, JF820; sln1-
::TRP1,
JF1350; sln1-
::LEU2, GY61 and GY67;
mcm1-
DEQ, JF1335 (12); nrp2-1,
GY31.
The effect of sln1- on
P-mediated activation was not due to reduced MCM1 message or
protein levels. MCM1 transcript (Fig. 2 B) and
protein (data not shown) levels were comparable in sln1-
and SLN1
strains. These results indicate
that Sln1p is essential for full Mcm1p activity, and not for its
expression. One way that P-mediated activation could be modulated
involves changes in the affinity of the Mcm1 protein for its binding
site; however, gel mobility shift experiments in which sln1-
and SLN1
extracts were incubated with a
palindromic P site probe revealed no SLN1-dependent changes in
the level of Mcm1p complex formed (data not shown).
gene expression. In fact, MF
1 expression was somewhat decreased in nrp2-1 strains. This may indicate that, while the imperfect P sites (PQ)
(7) present in the promoters of
-specific genes like
MF
1 are dependent on Sln1p for activity, PQ-mediated
activation is insensitive to the special activating form of Sln1p
encoded by the nrp2 mutants. The possibility that an
nrp2-activated Mcm1p or Mcm1p complex has reduced affinity for
PQ sites merits further investigation.
mutation is suppressed by increased dosage of the
PTP2 gene
(34) . However, introduction of a high copy
plasmid carrying the PTP2 gene only very weakly suppressed the
nrp2-1 phenotype and the effect of ptp2-
was equally modest (Table III). Hence, Ptp2 is unlikely to be a
normal component of this pathway. Alternatively, the small effect of
PTP2 dosage on UAS(P)- lacZ activity may be a
reflection of the involvement of additional related protein phosphatase
activities in the pathway.
mutant
(3) led to the suggestion that
phospho-Sln1p negatively regulates the yeast osmosensing pathway. The
role of Hog1p in Sln1p-mediated activation of the UAS(P)- lacZ reporter was measured in wild type cells exposed to 0.9
M NaCl. Although the expected increase in Hog1p tyrosine
phosphorylation was observed, no change in P reporter activity was
found under these conditions (Fig. 3, )
(3, 23) . P site activity was also not significantly
changed by deletion of the HOG1 gene in either
SLN1
() or nrp2-1 strains (data not shown). The fact that P-mediated activation is
neither positively nor negatively regulated by HOG1 suggests
that Sln1p may be separately involved in control of osmoregulation and
in control of Mcm1p activity.
Table:
Yeast strains used in this
work
Table:
Effect of NRP2 mutations
on P-mediated activation of lacZ reporter gene
-Galactosidase
activity was measured in unclarified or clarified glass bead
supernatants. Numbers are the average of at least four trials on at
least two different transformants. Normalized activity = (Miller
units (mutant)/Miller units (wild type)
100. Standard
deviations were less than 30% of the
average.
Table:
Effect of PTP2 on P site activation
Table:
Effect of salt on UAS(P)-lacZ
expression
-
D-galactopyranoside; EMS,
ethyl methanesulfonate; kb, kilobase pair(s); UAS, unactivated
sequence; P, Mcm1 binding site.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.