Institute of Molecular Biology, University of
Oregon, Eugene, Oregon 97403-1229
Received 3 July 2000/Returned for modification 15 August
2000/Accepted 13 October 2000
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INTRODUCTION |
The yeast Saccharomyces
cerevisiae contains three related protein kinases that are members
of the PAK (p21-activated kinase) family, proteins that interact with,
and presumably are regulated by, Cdc42p, a p21 GTPase required to
establish polarity of the actin cytoskeleton (9, 17). The
roles of these PAKs are seemingly quite different. In haploid cells,
Ste20p participates in at least three signal transduction pathways: the
pheromone response pathway (22, 23, 37), the invasive
growth pathway (42), and the HOG pathway
(35). Cla4p plays a role, albeit a poorly understood one,
in the budding process (2, 7, 15); no specific function has yet been established for Cla4p. The third PAK, Skm1p, is expressed only in meiotic cells (29). Surprisingly, given their
apparent distinct functions, loss of both STE20 and
CLA4 is lethal (7), suggesting that these two
protein kinases share an essential function(s). To investigate this
essential function, especially as it relates to STE20, we
carried out a screen for mutants that are lethal in a
cla4
mutant background. This effort has identified 10 complementation groups (NCS, for need CLA4 to
survive). Here we report on one of these groups, which encodes a
regulatory subunit for type 2A protein phosphatases (PP2As).
There are five known members of the yeast PP2A family
(54). PP2As are multimeric enzymes capable of catalyzing
the hydrolysis of phosphate groups from phosphoseryl, phosphothreonyl,
and phosphotyrosyl moieties. The catalytic (C) subunits are encoded by
PPH21, PPH22, PPH3, SIT4,
and PPG1 and share as much as 86% sequence similarity at
the amino acid level (54). Enzymatic activity, substrate specificity, and subcellular localization are modulated through the
interaction of the C subunit with an army of regulatory subunits to
form a trimeric holoenzyme. Tpd3p is thought to be the A subunit, based
on homology to the mammalian counterpart and on the ability to interact
physically with at least three of the yeast PP2A C subunits (8,
34). However, the formation of a Tpd3p-PP2A dimeric core complex
has not been demonstrated. In mammals, the dimeric core complex
interacts with one of several B-type regulatory subunits, B (PR55), B'
(PR61), and B" (PR72) (32). In yeast, only the B (Cdc55p)
(14) and B' (Rts1p) (48, 57) subunits have been identified. Finally, the activity of some PP2As can be
altered by a different type of regulatory subunit, a phosphotyrosyl phosphatase activator (PTPA), which stimulates the phosphotyrosyl phosphatase activity of PP2A C subunits in vitro (5, 56). S. cerevisiae has two putative PTPA subunits, encoded by
YIL153w and YPL152w (39). Given the
single A subunit, two B-type subunits, five C subunits, and two PTPA
subunits, 30 PP2A holoenzymes could in principle be present in the
yeast cell. With such a wide array of possible holoenzymes, it is not
surprising that the molecular and cellular mechanisms of PP2A function
are poorly understood.
Here we describe the identification and characterization of
NCS1, which encodes a protein related to mammalian PTPA
subunits. NCS1 (PTPA1) has previously been shown
to play a role in lowering the mutagenesis rate in cells treated with
known DNA mutagens (38) and is allelic to RRD1
(39). We find that NCS1 also plays a role at
the G2/M transition. Cells lacking NCS1 and
CLA4 arrest with grossly elongated buds, a phenotype that
appears to be an exacerbation of the G2 delay observed in
cells lacking CLA4. The abnormal morphology of
cla4 and ncs1 cla4 strains and the
lethality of ncs1 cla4 strains can be overcome by
changing the tyrosyl residue at position 19 of Cdc28p to phenylalanine,
mimicking the activated state of Cdc28p, or by deleting the Cdc28p
regulatory kinase, SWE1. Yeast has a second PTPA homolog,
encoded by YPL152w (also designated NOH1
[NCS1 homolog], PTPA2, and RRD2)
(38, 39). Deletion of both NCS1 and
NOH1 is lethal and results in the accumulation of unbudded,
uninuclear cells, demonstrating that PTPA function is important for bud
emergence. Both Ncs1p and Noh1p bind to the catalytic domain of Sit4p,
a PP2A-like protein phosphatase that plays a role in the regulation of
genes expressed late in the G1 phase of the cell cycle and
also in bud emergence (11, 55). Thus, Ncs1p and Noh1p may
regulate Sit4p activity and impinge on events that happen in late
G1 and at the G2/M transition of the cell cycle.
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MATERIALS AND METHODS |
Microbiological techniques.
Yeast and bacterial strains were
propagated by standard methods (47). Bacterial
transformations, DNA preparations, and plasmid constructions were
performed by standard methods (45). Yeast transformations
were performed by the Li+ ion method (16).
Samples for fluorescence-activated cell sorting (FACS) were prepared as
described by Ma et al. (28) and assayed using a Becton
Dickinson FACScan apparatus. Yeast extract-peptone-dextrose (YEPD) and
synthetic medium supplemented with dextrose (SD) were prepared as
described by Kaiser et al. (18). DNA-modifying enzymes were purchased from New England Biolabs, Inc. (Beverly, Mass.). Unless
stated otherwise, all other reagents were purchased from Sigma Chemical
Company (St. Louis, Mo.).
Yeast strains.
The yeast strains used in this study are
listed in Table 1. All strains except
Y3389 and SY3390 are congenic to the S288C genetic background, using
YPH499 and YPH500 (51) as the initial parents. The
ADE8 locus of YPH499 (and YPH500) was disrupted using the
two-step approach by inserting pSL2534, which contains a disruption of
the ADE8 open reading frame (ORF) marked by URA3,
and then selecting for white, ade2-101 ade8 colonies on
5'-fluoroorotic acid (5'-FOA). The FUS1-lacZ reporter gene
was inserted at the MFA2 locus using pSL1580
(13) to create SY3537 and SY3358. The his3-200 and lys2-801 loci of SY3537 and
SY3358 were repaired by a single-step gene replacement using the
wild-type HIS3 and LYS2 genes, creating SY3359
and SY3357, respectively. Single-step gene disruptions were performed
using DNA fragments generated either by PCR (1) or by
digestion of the relevant plasmid (43).
Deoxyoligonucleotides (Keystone Laboratories, Camarillo, Calif.) were
used to synthesize DNA fragments containing 40 bp of sequence 5' to the
ORF (as ascertained from the Saccharomyces genome database),
a selectable marker, and 40 bp of sequence 3' to the ORF by PCR. The
pRS series of plasmids served as templates to provide the selectable
marker DNA (51). The products of these reactions were used
to make gene deletions through single-step gene replacements in SY3537,
SY3357, SY3358, or SY3359. Plasmid-based single-step gene knockouts
were performed using pEL46-2 (ste20::TRP1) (22), pSL2680 (cln1::URA3),
pSL2681 (cln2::LEU2), AS3g
(pph21::LEU2) (52), AS6g
(pph22::URA3) (52), and DLB333
(swe1::LEU2) (50). pBB131
(CLA4-MYC) and pBB138 (cla4PAK-MYC)
(2) were inserted at the URA3 locus. Deletions
were confirmed by PCR-based chromosome analysis and phenotypic analysis.
Plasmid construction.
Plasmids used are listed in Table
2. pRS315ADE8 was constructed
by inserting a 2.0-kb EcoRI/HindIII
ADE8-containing fragment from pSL2535 (constructed by
Charlie Boone) into a similarly digested pRS315 vector. A 4.0-kb
NruI/SacI CLA4 genomic fragment from
pATL3 (gift from Fred Cross) (2) was first made blunt
using the Klenow fragment then inserted into SmaI-cut
pRS315ADE8 to create pRS315ADE8CLA4. pRS316ADE8CLA4 was generated by inserting the 6.0-kb
HindIII/XbaI fragment from
pRS315ADE8CLA4 into a similarly cut pRS316 vector. CLA4 knockout plasmids
pKScla4::HIS3 and
pKScla4::TRP1 were constructed using
homologous recombination (1, 27) by first replacing the
ORF of a CLA4 gene, harbored in plasmid
YEp13CLA4, with HIS3 or TRP1 (data not
shown). The cla4 null allele was then amplified by PCR using
deoxyoligonucleotides containing BamHI sites at their 5'
ends. The PCR product was inserted into the BamHI site of
pKS (Stratagene). Plasmid YEpURA3ADH-SIT4-HA was created
using homologous recombination to replace the LEU2 gene of
YEpLEU2ADH-SIT4-HA (8) with URA3
(1, 27). Likewise, YCpHIS3cla4-75 was generated
from YCpTRP1cla4-75 (7) by using homologous
recombination to replace TRP1 with HIS3.
YEp13NCS1 was constructed by inserting a PCR product that
encompassed 276 bp upstream and 135 bp downstream of the
NCS1 putative start and termination codons. The
deoxyoligonucleotides used to generate the approximately 1.6-kb
fragment contained flanking BglII sites which permitted
insertion into the BamHI site of YEp13. YEp13NCS1-3xMYC was created by the
deoxyoligonucleotide-mediated method of Schneider et al.
(46) and included the entire NCS1 ORF
fused to the MYC triple-repeat sequence. To generate
pRS415NCS1-3xMYC, an approximately 2.0-kb PCR fragment
was amplified using YEp13NCS1-3xMYC as a template and
the original NCS1 deoxyoligonucleotides as primers. The
fragment was inserted into the BamHI site of pRS415. All
plasmids were shown to complement the ncs1cla4
synthetic lethality. All fragments generated by PCR utilized a 5:1
mixture of TAQ (Promega, Madison, Wis.) and Vent (New England Biolabs)
DNA polymerases.
Galactose-inducible triple-hemagglutinin (HA)-tagged yeast expression
constructs pGAL-3xHA-NCS1 and pGAL-3xHA-NOH1 were
created by generating blunt-ended ORFs by PCR using
deoxyoligonucleotides specific for YIL153w and
YPL152w (Research Genetics) and inserting the ORFs into
p705-3 (10) that had been cut with HpaI. Both pGAL-3xHA-NCS1 and pGAL-3xHA-NOH1 could
complement the synthetic lethality of an ncs1noh1
yeast strain.
pmal-c2-SIT4 was created by PCR amplification of the
SIT4 ORF from genomic DNA (SY3357), using
deoxyoligonucleotides specific for the SIT4 ORF and
containing flanking BamHI sites. The approximately 1.0-kb
fragment was then inserted into the BamHI site of
pmal-c2 (New England Biolabs).
pmal-c2-SIT4(N) was constructed by partial digestion of pmal-c2-SIT4 using NcoI, which cuts
once (bp 483) within the SIT4 ORF, filling in the 5'
overhang with the Klenow fragment of DNA polymerase I, and religating
the newly formed blunt ends. pmal-c2-SIT4(C) was
generated by partial digestion of pmal-c2-SIT4 using
BglII followed by complete digestion using EcoRI.
The free plasmid ends were made blunt using Klenow fragment, and the
newly formed ends were religated.
Yeast mutagenesis and NCS screen.
Yeast cells
were mutagenized by either of two methods. First, cells were spread
onto YEP plates containing 8% dextrose (to improve red color
development). Cells were radiated with UV light (75 µJ), which
yielded 50% viability, and incubated at 30°C for 3 to 7 days. Cells
were also mutagenized using the method and libraries of Burns et al.
(4). The affected locus was identified essentially as
described by Burns et al. (4) except that pRSQ306 (6) rather than YIp5 was inserted to retag and identify
the locus.
Whole-cell extracts.
Yeast whole-cell extracts were prepared
from mid-log-phase cultures (optical density at 600 nm
[OD600] of 1.0 to 2.0). All manipulations were carried
out at 4°C. Fifty-milliliter cultures were centrifuged, and the cells
were suspended in lysis buffer (20 mM Tris-Cl [pH 7.5], 1 mM EDTA,
1× protease inhibitor cocktail [Boehringer Mannheim catalog no.
1697498], 1 mM phenylmethylsulfonyl fluoride, phosphatase inhibitors
[25 mM sodium fluoride, 0.25 mM sodium orthovanadate, 15 mM sodium
pyrophosphate, and 15 mM p-nitrophenyl phosphate]). In
cases where detergent was required, the lysis buffer was supplemented
with Triton X-100 to a final concentration of 0.5% (vol/vol). Yeast
cells were broken by vortexing with glass beads. The extracts were
centrifuged at 2,000 × g to remove the glass beads and
unbroken cells.
For antiphosphotyrosine detection, Cdc28p was enriched from yeast
whole-cell lysates using p13SUC1
protein-conjugated agarose. The precipitates were washed three times
with lysis buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel, and subjected to Western
analysis. Cdc28p was detected using an antibody that recognizes the
PSTAIRE amino acid sequence of Cdc28p (Santa Cruz Biotechnology, Inc.,
Santa Cruz, Calif.) at a dilution of 1/1,000. Cdc28p containing phosphotyrosine was detected using 4G18 (Upstate Biotechnology, Inc.,
Lake Placid, N.Y.) at a dilution of 1/1,000. Western blots were washed
with 10 mM Tris-Cl (pH 7.5)-150 mM NaCl-0.2% Tween 20.
Protein concentrations were determined using the Bio-Rad (Hercules,
Calif.) protein assay reagent as instructed by the manufacturer, with
bovine serum albumin as a standard.
To prepare bacterial extracts, bacteria were centrifuged at
5,000 × g and resuspended in phosphate-buffered saline
(50 mM phosphate, 150 mM NaCl [pH 7.0]) supplemented with 1 mM
phenylmethylsulfonyl fluoride. Cells were broken by two passes through
a French press apparatus, and the debris removed by centrifugation at
10,000 × g. Affinity chromatography using amylose
resin (New England Biolabs) was performed in batch at 4°C with
constant agitation for at least 1 h. After the incubation, the
resin was washed three times with 10 bed volumes of phosphate-buffered saline.
In vitro binding assay.
Lysates were first cleared of
particulate matter by centrifugation at 100,000 × g.
Approximately 100 µg of total protein in a volume of 300 µl of
lysis buffer was mixed with 50 µl (50% slurry) of maltose binding
protein (MBP)-Sit4p-decorated amylose resin and incubated for 1 h
at 4°C. After the incubation, the resin was washed four times with
lysis buffer supplemented with 0.5% Triton X-100, resuspended in 50 µl of sample buffer, and subjected to SDS-PAGE (10% gel)
(21); the proteins were detected by Western analysis.
Coimmunoprecipitation assays and Western blotting.
Yeast
whole-cell extracts were normalized to a total protein concentration of
1 mg/ml. A 0.5-ml aliquot of the lysate was mixed with 20 µl of
protein A-conjugated agarose (50% slurry) to remove material that
binds nonspecifically to protein A-agarose. After removal of the
agarose, the lysate was incubated for 1 h at 4°C with 20 µl of
an anti-HA monoclonal antibody (12CA5) that had been cross-linked to
protein A-agarose. The agarose beads were washed three times with lysis
buffer and resuspended in 50 µl of sample buffer. One-third to
one-half of the sample was analyzed by SDS-PAGE (10% gel). Proteins
were transferred to nitrocellulose and detected using either anti-HA or
anti-MYC monoclonal antibody. Horseradish peroxidase-conjugated goat
anti-mouse secondary antibodies were used as instructed by the
manufacturer (Bio-Rad). Signals were visualized by exposure to Kodak
film (X-Omat AR) (typical exposure times were between 1 and 5 min).
Microscopy.
Yeast cells were grown in either YEPD or SD
medium to a density of approximately 107 cells/ml. After
centrifugation, the cell pellets were resuspended in either water or TE
(10 mM Tris-Cl [pH 7.5], 1 mM EDTA) and visualized using a Zeiss
Axioplan II photomicroscope with a 100× oil immersion objective.
Antibodies were used at a dilution of 1/10 (
-Cdc3p; a generous gift
from John Pringle) (19) or 1/20 (-tubulinYOR1/34; a generous gift from John Chant)
(36).
Generation of ncs1-2.
NCS1 mutants were
generated essentially as described by Muhlrad et al. (33).
Using deoxyoligonucleotides specific for the pRS415 polylinker, the
NCS1 gene was amplified using pRS415NCS1-3xMYC as
a template. The PCR mixtures contained 1× buffer (Promega), 2 mM
MgCl2, 0.1 to 0.25 mM MnCl2, 1 µM each
deoxyoligonucleotide, three deoxynucleoside triphosphates at 0.25 mM,
and 1 deoxynucleoside triphosphate at 0.05 mM. The products of the
reactions were subcloned into pRS414 using homologous recombination
(27) in strain SY3414. The transformants were grown on
medium supplemented with 5'-FOA to select for ncs1 mutants
that could still complement the loss of NCS1 at 25°C.
Temperature-sensitive alleles of NCS1 were identified from
the 5'-FOAr subset of ncs1 mutants by their
inability to complement the loss of NCS1 at 37°C.
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RESULTS |
Screen for mutations that are lethal in the absence of
CLA4.
To identify potential activators or targets of the
presumed STE20 essential function, we sought new mutations
that are synthetically lethal with cla4 (Fig.
1). We used a variation of the red/white colony sectoring assay to identify synthetic lethal mutations (20). Two strains, SY3360 (MATa
leu2-1 ura3-52 his3-200 trp1-63 ade2-101 ade8
cla4::TRP1) and SY3361 (MAT
leu2-1 ura3-52 lys2-801 trp1-63 ade2-101 ade8
cla4::TRP1), were transformed with
pRS316ADE8CLA4, a low-copy-number plasmid, to
create SY3362 (from SY3360) and SY3363 (from SY3361). ade2
ade8 colonies are white, whereas ade2 ADE8 colonies are
red. Therefore, as a result of occasional plasmid loss under
nonselective conditions, colonies of SY3362 and SY3363 are composed of
red (plasmid-containing) and white (plasmid-free) pie-shaped sectors
(sector+ phenotype). Occasional plasmid loss also allows
SY3362 and SY3363 to sport mitotic segregants able to grow on medium
supplemented with 5'-FOA, a toxin that kills cells expressing the
URA3 gene. Therefore, mutations that result in the
requirement for CLA4 (or ADE8) will yield red
(sector
), 5'-FOAs colonies.
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FIG. 1.
Schematic representation of the basis for the genetic
screen used to isolate mutants that are synthetically lethal in the
absence of CLA4. Xi (activator of
STE20), Xii (component of a
non-STE20-related pathway), and Yi
(target of STE20) represent classes of mutants that are
hypothesized to be recovered by the genetic screen.
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Two independent techniques were used to generate mutations in
NCS genes. First, strains SY3362 and SY3363 were mutated by UV irradiation, yielding 77 sector, 5'-FOAs
colonies for SY3362 and 28 sector, 5'-FOAs
colonies for SY3363 out of approximately 200,000 cells from each parent
strain. Of the 77 mutants generated from SY3362, 61 were recessive, 14 were dominant, and 2 were apparently unable to mate with SY3363 (these
mutants were later shown to carry a mutation at LYS2, an
auxotrophic marker used in SY3362 for the selection of diploids when
mated to SY3363). Mutation of SY3363 yielded 13 recessive and 10 dominant mutant strains. Mutations were organized into complementation
groups either by mating the SY3362 mutants to the SY3363 mutants or by
first backcrossing the SY3362 mutants to SY3363 to generate an mating-type strain and then mating the newly constructed strain with
the SY3362 mutants.
The second mutagenesis technique relied on transformation into SY3362
of linear DNA fragments isolated from a LEU2 transposon mutagenesis library (4). The procedure yielded only one
mutant (out of approximately 30,000 transformants) in which (i) the
LEU2 marker segregated 2:2 when backcrossed to the parental
strain, demonstrating that the phenotype is due to an insertion at only one locus, and (ii) the LEU2 marker cosegregated with the
mutant phenotype when crossed to SY3363. This gene is designated
NCS1, and transposon-generated allele is designated
ncs1::LEU2. Since no alleles of
NCS1 were found with the UV mutagenesis procedure, and only
one allele by the insertion mutagenesis procedure, we assume that the
screen has yet to be saturated.
As a first step to learn about NCS1 function, we examined
the growth and morphology of ncs1 mutants.
ncs1 single mutants grew at wild-type rates at all
temperatures tested and had wild-type morphology. In addition,
ncs1 mutants were not defective for any known
STE20 function tested: they responded to pheromone, mated with wild-type efficiency, and underwent filamentous growth as assessed
by agar invasion.
To investigate the phenotype of ncs1::LEU2
cla4 cells (Fig. 2), we
constructed a strain of that genotype carrying a plasmid-borne temperature-sensitive CLA4 allele, cla4-75. At
the restrictive temperature, these cells arrested growth as cells
containing a hyperpolarized bud, reminiscent of the cla4
and ste20 cla4 morphology. In the case of
ste20 cla4 <YCpHIS3cla4-75> cells, the
hyperpolarized morphology is accompanied by a second defect in bud
growth. In wild-type cells, a pair of septin rings flank the mother/bud
junction. New growth, for example, fusion of secretory vesicles with
the plasma membrane, occurs on the daughter side of the septin rings.
In ste20 cla4 <YCpHIS3cla4-75>
double-mutant and cln1 cln2 cla4
<YCpHIS3cla4-75> triple-mutant cells, however, growth
occurs on the mother side, and as a result the septin proteins are
located along the length of or at the tip of the bud (Table 3; also see reference 7).
ncs1::LEU2 cla4
<YCpHIS3cla4-75> mutants did not exhibit this defect.
Septin mislocalization was observed in only 3% of cells, comparable to
what is seen in cla4 cells (Table 3). The absence of a
septin localization defect is peculiar to ncs1 cla4,
as other ncs cla4 <YCpHIS3cla4-75> mutants
tested exhibit the localization defect (data not shown). A septin
localization defect also was not seen for cla4 ncs1-2 strains, which contain a temperature-sensitive NCS1 allele
(4% septin mislocalization). Together, these data suggest either that NCS1 is involved in orchestrating only a subset of
STE20 functions that are essential in a cla4
background or that loss of NCS1 reveals a separate,
non-STE20-related function of CLA4.
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FIG. 2.
Phenotypic analysis of
ncs1::LEU2 cla4 strains. (A) Loss of
NCS1 is lethal in the absence of CLA4. Strains
SY3357 (wild type), SY3364 (ncs1::LEU2
cla4::TRP1 <pRS316ADE8CLA4>),
SY3365 (ncs1::LEU2
cla4::TRP1 <YCpHIS3cla4-75>), and
SY3360 carrying the cla4-75 allele
(cla4::TRP1 <YCpHIS3cla4-75>)
were grown in synthetic medium at 25°C to an OD600 of 1.0 to 2.0. A serial dilution (1/10) was performed starting with 10,000 cells. Cells were spotted onto prewarmed YEPD plates at 25 and 37°C
and allowed to grow for 3 days. (B) Morphological examination of
ncs1::LEU2 cla4::TRP1
strains. Strains SY3364, SY3365, and SY3360 (carrying
YCpHIS3cla4-75) were grown in synthetic medium at 25°C and
shifted to 37°C for 4 h.
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Cla4p has at least two biochemical activities: the ability to bind
Cdc42p via the PAK domain, and protein kinase activity. To determine
whether either activity is required for viability in an
ncs1::LEU2 cla4 mutant, we constructed
two sets of ncs1::LEU2 cla4 strains.
The first set contained a plasmid-borne cla4-75 allele and
also harbored another allele of CLA4 that encodes a version
of CLA4 lacking the PAK domain (SY3380). This strain was temperature sensitive for growth (Fig.
3A), implying that the PAK domain mutant
could not provide the CLA4 function that is essential in the
ncs1 cell. The second set contained the
URA3-marked plasmid, pRS316ADE8CLA4 (SY3378), and
either an empty vector (pRS315ADE8), a plasmid encoding a
kinase-dead version of CLA4
(pRS315ADE8cla4K549R), or wild-type CLA4
(pRS315ADE8CLA4). The ability to lose the URA3 linked CLA4 was assayed by growth on medium containing
5'-FOA. The cla4K549R allele was unable to replace wild-type
CLA4 (Fig. 3B). Taken together, the inability of
cla4(PAK) and cla4K549R to
substitute for wild-type CLA4 implies that both Cla4p
activities are required for viability in a strain lacking
NCS1.
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FIG. 3.
CLA4 functions are required for
viability in ncs1 cells. (A) Cla4p lacking the PAK
domain cannot support viability in the absence of Ncs1p. Strains
SY3364, SY3365, SY3380 (cla4::TRP1
ura3::cla4PAK-MYC::URA3
<YCpHIS3cla4-75>), and SY3379
(ncs1::LEU2 cla4::TRP1
ura3::cla4PAK-MYC::URA3
<YCpHIS3cla4-75>) (sectors 1 to 4, respectively) were grown at 25°C on YEPD medium; single colonies were
assayed for growth by streaking onto prewarmed YEPD medium and
incubating for 3 days at 37°C. (B) Cells lacking Ncs1p require an
active Cla4p kinase. Yeast strain SY3378
(ncs1::HIS3 cla4::TRP1
<pRS316ADE8CLA4>) was transformed with pRS315 (sector a),
pRS315ADE8cla4K549R (which encodes a kinase-dead Cla4p),
(sector b), or pRS315ADE8CLA4 (sector c), and the
ability of pRS315ADE8cla4K549R to substitute for
pRS316ADE8CLA4 was assayed by growth on synthetic
complete medium lacking leucine (left) or supplemented with 5'-FOA
(right) for 3 days at 30°C.
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ncs1::LEU2 cla4 cells arrest
prior to the G2/M transition.
The hyperpolarized bud
phenotype of cla4 mutants can be interpreted as a delay
in the switch from apical bud growth to isotropic growth that occurs
late in G2 (17, 24, 25, 41) and is reminiscent
of defects conferred by alterations in the expression of
SWE1 and CLB2, known regulators of Cdc28p kinase
activity. This G2 delay is thought to be the result of
activation of a cell cycle checkpoint that monitors the presence and
growth of the bud. Phosphorylation of Tyr19 in Cdc28p by Swe1p inhibits
Clbp-Cdc28p kinase activity and prevents progression to anaphase. We
therefore examined other cell cycle events in cla4,
ncs1::LEU2, and cla4 ncs1::LEU2 mutants. Using FACS analysis, we
analyzed the DNA content of a cla4 mutant to determine
whether there was indeed a delay in the G2 phase of
the cell cycle. Asynchronous cultures of four strains, wild
type, cla4, cla4 CDC28Y19F, and
cla4 swe1
were grown to mid-log phase, and their
DNA was stained with propidium iodide (Fig.
4A). The wild-type culture contained a high percentage of cells with a 1C DNA content whereas the
cla4 culture contained a high percentage of cells with a
2C DNA content, implying an accumulation of cells that have replicated
their DNA and have yet to undergo mitosis. Mutation of CDC28
to CDC28Y19F or deletion of SWE1 restored the
1C/2C ratio of cla4 mutants to that seen for the
wild-type strain. We examined the bud morphology of these strains and
found that CDC28Y19F and swe1 suppressed the
hyperpolarized bud phenotype (data not shown).
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FIG. 4.
The cla4 ncs1::LEU2
defect results in a G2 delay that is suppressed by deletion
of SWE1. (A) Strains SY3357, SY3360, SY3392, and SY3391 were
grown to mid-log phase in YEPD medium at 30°C. The cells were fixed,
stained with propidium iodide, and subjected to FACS analysis. (B)
Strains expressing wild-type SWE1 (SY3362, SY3378, SY3368,
and SY3504) or lacking SWE1 (SY3392 carrying
pRS316ADE8CLA4, SY3403, SY3404, and SY3505) were constructed
and grown on YEPD medium at 25°C. After 3 days, dilutions of cells
were spotted onto rich medium (YEPD) or onto synthetic complete medium
supplemented with 5'-FOA and incubated for 3 days at 25°C. (C)
Deletion of CLA4 increases the phosphotyrosine content of
Cdc28p. Whole-cell extracts were prepared from exponentially growing
strains SY3357, SY3390, SY3393, SY3360, and SY3398. Cdc28p was enriched
from the extracts using p13SUC1-conjugated
agarose beads. The beads were washed, and the eluted proteins were
analyzed by Western analysis using antibodies that recognize
phosphotyrosine (4G18) and the PSTAIRE amino acid motif of Cdc28p. The
band that appears in the Cdc28Y19F sample of the upper panel
(antiphosphotyrosine blot) differs in mobility from Cdc28p. We
therefore conclude that it is not Cdc28p but another protein that is
phosphorylated on a tyrosine residue(s), but only in Cdc28Y19F cells.
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Does the ability to bypass the elongated bud phenotype of
cla4 cells restore viability to ste20
cla4, cln1 cln2 cla4, or ncs1
cla4 cells? To test this possibility, cla4
swe1 cells were crossed to either ste20 cla4
<pRS316ADE8CLA4>, cln1 cln2 cla4
<pRS316ADE8CLA4>, or ncs1 cla4
<pRS316ADE8CLA4>. The diploids were then sporulated, and
the ability of spores to survive without wild-type CLA4
assayed by growth on medium supplemented with 5'-FOA. As expected,
cla4 cells were able to grow on medium supplemented with
5'-FOA whether or not SWE1 was deleted (Fig. 4B).
Strikingly, deletion of SWE1 suppressed the lethality of the
ncs1 cla4 double mutation. This result contrasts with
that obtained for ste20 cla4 and cln1 cln2
cla4 mutants: swe1 did not restore viability to
these mutants. Analogous results were obtained using the
CDC28Y19F allele instead of swe1. Although
CDC28Y19F did not suppress the lethality of ste20
cla4, we examined whether it suppressed the hyperpolarized bud
morphology by constructing a ste20 cla4 CDC28Y19F strain carrying plasmid borne cla4-75. At the restrictive
temperature, the buds formed by this strain had nearly wild-type
morphology and the septin ring was located at the mother/daughter
junction of the bud neck (data not shown), indicating that
CDC28Y19F suppresses both the morphology defect and the
defect in septin ring mislocalization.
To confirm that the G2 delay seen in cla4
strains was at the level of Cdc28p activation, we determined the
extent of Cdc28p phosphorylation at position 19 by Western analysis
using an antibody directed against phosphotyrosine (see Materials and
Methods). As anticipated, a modest amount of phosphotyrosine that
comigrated with Cdc28p was detected in the wild-type strain (Fig. 4C,
lane 1), while no phosphotyrosine-associated Cdc28p was detected in the strain expressing CDC28Y19F (lane 2).
Cells lacking MIH1, which encodes the phosphatase that
dephosphorylates residue Tyr19 (Y19) (44), had
relatively high (approximately 11-fold higher than wild-type)
levels of phosphotyrosine-associated Cdc28p (lane 3).
cla4 cells also had relatively high (approximately 34-fold higher than wild-type) levels of phosphotyrosine-associated Cdc28p (lane 4). Interestingly, the increase in the amount of phosphotyrosine Cdc28p was higher in cla4 mih1 mutants
than in either single mutant (approximately 50-fold above the wild-type level) (lane 5). On the other hand, the ratio of phosphorylated Cdc28p
to total Cdc28p was no greater in extracts from
ncs1::LEU2 mih1 cells than in extracts
from mih1 cells (data not shown). These data suggest that
the regulation of inhibitory phosphorylation of Cdc28p in a
cla4 strain can occur independently of Mih1p. Moreover, the lack of difference in Cdc28p phosphorylation
between ncs1::LEU2 mih1 and
mih1 cells implies that the effect of Ncs1p on Cdc28p is
dependent on Mih1p and that NCS1 may regulate
MIH1.
As another measure of G2 progression, we examined mitotic
spindle length. The formation of short spindles occurs during S phase
and is dependent on the activity of Clb3p and Clb4p forms of Cdc28p
kinase but not on DNA replication (12, 40). The mitotic
cyclin forms of Cdc28p kinase, Clb1p-Cdc28p and Clb2p-Cdc28p, promote
the elongation of the spindles toward the distal poles of both mother
and daughter cells (12). Elongation of the spindles and
the switch from apical to isotropic bud growth appear to be concurrent
and dependent on mitotic cyclin/Cdc28p kinase activity. The relative
position and length of the mitotic spindle, therefore, may act as an
effective cytological marker for the switch from apical to isotropic
growth. Because cla4 cells exhibit a G2
delay, we asked whether they also have a defect in spindle elongation or whether such a defect might be apparent in
ncs1::LEU2 cla4 double mutants.
A set of strains was synchronized in G1 by the presence of
-factor for 1.5 h and then shifted to 37°C for 4 h to
deactivate the temperature-sensitive cla4-75 allele. After
the initial incubation at 37°C, the cells were washed and incubated
at 37°C for 1.25 h in medium lacking -factor. At that time,
more than 73% of the cells of each culture were budded. The
cells were fixed and stained with -tubulin. In the wild-type
strain (SY3357), 74% (181 of 244) of the cells had elongated spindles
projecting into the daughter bud. Strains lacking either
NCS1 (SY3364) or CLA4 (SY3360 containing cla4-75) yielded elongated spindles in 67% (140 of 208) or
81% (171 of 210) of the cells, respectively. The
ncs1::LEU2 cla4 cla4-75 strain
(SY3365), however, produced elongated spindles in only 20% (70 of 346)
of the cells.
In the foregoing experiments, we observed numerous cells with elongated
buds as well as elongated spindles in the cla4 strain. This observation seems to be at odds with the reported coupling of
spindle elongation and the switch from apical to isotropic bud growth
(24, 31). One possible explanation is that the overlap in
the functions of Ste20p and Cla4p is enough to allow Ste20p to
partially act on behalf of Cla4p. This redundancy may provide enough
kinase activity to allow spindle elongation but not enough to promote
the switch from apical to isotropic bud growth. Alternatively,
CLA4 may function to couple spindle elongation to the switch
from apical to isotropic bud growth. The possibility that the
cla4-75 allele is leaky and provides sufficient activity for
one function and not the other is unlikely because a strain lacking
CLA4 produced similar results (data not shown).
Cla4p physically interacts with Cdc28p.
Given the genetic
interactions between CLA4 and CLN1/CLN2 and
between CLA4 and CDC28, we examined whether Cla4p
and Cdc28p exist in a complex. To test this idea, we performed a Cdc28p
pull-down experiment. Extracts were prepared from a strain expressing
an epitope-tagged version of Cla4p, Cla4-MYCp (2), and
Cdc28p was precipitated by using agarose-conjugated
p13SUC1, a protein known to associate with
cyclin-dependent kinases. The MYC antibody recognized a single protein
of approximately 93 kDa (Fig. 5, lane 2).
Cla4-MYCp was detected in the p13SUC1
precipitates (lane 2), suggesting a direct physical association between
Cla4p and Cdc28p or protein that interacts with Cdc28p. Cla4-MYCp was
not precipitated when p13SUC1 was not present on
the agarose beads (lane 1). To ensure that the interaction between
Cla4-MYCp and Cdc28p is specific for Cdc28p and not another protein
that could be recognized by the
p13SUC1-conjugated agarose beads, we performed a
similar experiment using cells expressing Cdc28-HAp (53).
Precipitation with the antibody that recognizes the HA epitope led to
coprecipitation of Cla4p (lane 6). We estimate the amount of Cla4-MYCp
that associates with Cdc28-HAp to be approximately 1 to 5% of the
starting material, whereas the amount of Cla4-MYCp that was pulled down
with the p13SUC1-agarose to be 20 to 30% of the
starting material. The difference between the experiments may in part
be due to the different methods used to isolate Cdc28p:
p13SUC1 can recognize all Cdc28p molecules in
the extracts, whereas the HA antibody recognizes Cdc28-HAp but not
wild-type Cdc28p.
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FIG. 5.
Cla4p binds to Cdc28p. Cdc28p was enriched from
whole-cell extracts of strains SY3409 and SY3357 (lanes 2 and 3, respectively) using p13SUC1-agarose beads or
from extracts of SY3357 carrying pSF19 (CDC28-HA) (lane 4),
SY3409 (lane 5), and SY3409 carrying pSF19 (lane 6), using HA
antibody-conjugated agarose. Neither Cdc28p nor Cla4-MYC could be
enriched from extracts using undecorated agarose beads (lane 1;
SY3409). Top, Western (immunoprecipitation [IP]) analysis of
Cla4-MYCp associated with Cdc28p-coated beads; middle, amount of
Cla4-MYCp present in the whole-cell extract (WCE); bottom, amount of
Cdc28p associated with the agarose beads.
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NCS1 encodes a protein with sequence similarity to the
PTPA family of enzymes.
The LEU2-marked transposon
based insertion was mapped to ORF YIL153w on chromosome IX
by rescuing the marked locus and sequencing the flanking regions of DNA
(2). The marker insertion was at codon 86 of the ORF (Fig.
6A). The presumed protein sequence of YIL153w shows high sequence identity to the PTPA family of
protein phosphatase regulators (5). The family includes
members from Homo sapiens (38% identity to Ncs1p),
Drosophila melanogaster (29% identity to Ncs1p), and
Schizosaccharomyces pombe (54% identity to Ncs1p). The PTPA
family of regulators, including the yeast homologs, have been shown to
increase the phosphotyrosyl phosphatase activity of PP2As in vitro
(56); however, the in vivo function of these proteins is
not known.
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FIG. 6.
Schematic representation of NCS1 null alleles
and phenotypic analysis of ncs1::LEU2
noh1::HIS3 double-mutant strains. (A) Schematic
representation of the NCS1 alleles,
ncs1::LEU2 and
ncs1::HIS3, used throughout this study.
The putative translation start site is shown with an arrow. The
termination codon is denoted by an asterisk. (B) Analysis of
ncs1 noh1 double mutants. Tetrads dissected from the
meiotic progeny of diploid MATa/MAT
ncs1::LEU2/NCS1
NOH1/noh1::HIS3 were placed on YEPD medium and
incubated at 30°C for 3 days. (C) Phenotypic analysis of cells
lacking PTPA function. The budding index (percent budded cells/total
number of cells) of asynchronously growing cultures was measured for
strains SY3357, SY3383, and SY3382 after growth of cultures in YEPD
medium at 25°C followed by a shift to 37°C for 5 h. WT, wild
type.
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A second gene, YPL152w, also encodes a protein with sequence
similarity to the PTPA family. NCS1 and YPL152w
(which we designate NOH1; also called RRD2
[39]) show a great deal of sequence similarity (179 of
393 amino acid residues) but are only 25% identical. As an initial
examination of PTPA function, we sought to determine the effect of loss
of PTPA function on cell growth. Although loss of either
NCS1 or NOH1 had no effect on growth or
morphology, deletion of both genes led to inviability. In particular,
the spores of the double mutants germinated but could undergo fewer than five rounds of division (Fig. 6B). Examination of the
double-mutant microcolonies revealed cells that were rounded and
unbudded. To investigate this phenotype further, a
temperature-sensitive NCS1 allele (ncs1-2) was
created by the error-prone PCR technique (Materials and Methods). The
plasmid-borne recessive ncs1-2 allele
(pRS414ncs1-2) was transformed into the MATa
ncs1::LEU2 strain, and the resulting
strain was mated to a MAT noh1::HIS3
strain. The resulting diploid was sporulated, and
ncs1::LEU2 noh1::HIS3
segregants containing plasmid borne ncs1-2 were identified.
These segregants were unable to grow at 37°C, and approximately 82%
of the cells arrested with a round and unbudded morphology (Fig. 6C).
Based on the synthetic lethality exhibited by three sets of mutants,
ste20 cla4, ncs1::LEU2
cla4, and ncs1::LEU2 noh1, we
hypothesized that STE20 and NOH1 might show
genetic interactions. However, we were unable to detect new phenotypes
for ste20 noh1 double mutants, nor did we detect new
phenotypes when ste20 ncs1::LEU2 and
noh1 cla4 double mutants were constructed (Table
4), providing further evidence that the
overlap in NCS1 and CLA4 function is physiologically different from the overlap in STE20 and
CLA4 function.
Strains lacking the gene for the PP2A-like phosphatase,
SIT4, require CLA4.
The homology between Ncs1p
and the PTPA family of proteins suggests that Ncs1p may regulate a
PP2A. Indeed, van Hoof et al. (56) have shown that the
proteins encoded by both of the S. cerevisiae PTPA homologs
can increase the specific activity of a PP2A, PP2AD, for a
phosphotyrosyl-containing substrate. However, PP2AD is a
mammalian enzyme, and we sought to identify a yeast target of Ncs1p. We
speculated that the relevant target would show genetic interactions
with CLA4. In S. cerevisiae there are four PP2As
and one PP2A-like phosphatase, Sit4p. We first tested SIT4
by mating SY3361 (MAT cla4) to strain SY3389
(MATa sit4::HIS3). After
sporulation and tetrad (n = 20) dissection, no
sit4 cla4 double mutants were recovered (Table 4). In
contrast, strains lacking both SIT4 and NCS1
appeared phenotypically similar in terms of growth and morphology to a
strain lacking only SIT4 (Table 4). SIT4 exhibits
synthetic lethality with two other genes, SSD1 and
CLN3; we therefore asked whether NCS1 would show
interactions with these genes. We were able to construct
ncs1::LEU2 ssd1 and
ncs1::LEU2 cln3 double-mutant strains.
The ncs1::LEU2 ssd1 double mutant was
wild type for growth at 25 and 37°C (Table 4) and had no
morphological abnormalities (data not shown). The
ncs1::LEU2 cln3 double mutant, on the
other hand, grew at wild-type rates at 25°C but was temperature
sensitive for growth at 37°C (Table 4), accumulating unbudded cells
at the restrictive temperature.
Of the PP2A family, Pph21p and Pph22p contribute the majority of the
PP2A activity. As for sit4 strains, strains lacking PPH21 and PPH22 are severely impaired for growth
or dead, depending on the genetic background (54). In our
genetic background (S288C), pph21 pph22 double mutants
are inviable (Table 4), dividing only two to three times after
germination. As done for SIT4, we constructed diploids
heterozygous at the CLA4 locus and the PPH21 and
PPH22 loci. After sporulation, analysis of the tetrads
revealed no observable growth phenotype in the pph21
cla4 or the pph22 cla4 double mutant compared
to the respective congenic pph21, pph22,
and cla4 strains (Table 4). Likewise, no genetic
interactions between CLA4 and the other two PP2A genes,
PPH3 and PPG1, were observed (Table 4). Although
these data do not rule out an interaction between Pph21p, Pph22p,
Pph3p, or Ppg1p and Ncs1p, they do suggest that the essential function
shared by CLA4 and NCS1 does not involve PPH21, PPH22, PPH3, or
PPG1.
Ncs1p and Noh1p physically interact with Sit4p.
Given the
genetic interactions between CLA4 and both NCS1
and SIT4, we asked whether the PTPA proteins and Sit4p
interact biochemically. A binding assay was conducted using fragments
of Sit4p that had been expressed in bacteria as malE-encoded
MBP fusion proteins and then attached to amylose resin. These fragments included the full-length Sit4 protein, a C-terminal truncation encoding
only the first 164 amino acids, and an N-terminal deletion of the first
161 amino acids leaving only the Sit4p catalytic domain. As a source of
Ncs1p and Noh1p, we prepared whole-cell extracts from a yeast strain in
which Ncs1p and Noh1p were overexpressed from the GAL1
promoter as HA fusion proteins. The expression of either
pGAL-3xHA-NCS1 or pGAL-3xHA-NOH1 was able to
restore viability to ncs1::LEU2 noh1
double mutants (data not shown). The HA-Ncs1p or HA-Noh1p-containing
extracts were incubated with amylose resin decorated with Sit4p, and
the bound PTPA protein was detected by Western analysis. As seen in
Fig. 7B, both HA-Ncs1p and HA-Noh1p associate with full-length Sit4p as well as the C-terminal catalytic domain. HA-Ncs1p, but not HA-Noh1p, exhibited weak interaction with the
N-terminal regulatory domain of Sit4p. These data show that the
S. cerevisiae PTPA proteins are capable of interacting with
PP2As, possibly regulating the phosphatase activity through interaction
with the phosphatase catalytic domain.
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FIG. 7.
In vitro association of PTPA proteins with Sit4p. (A)
Schematic representation of bacterially expressed MBP-Sit4p fusion
proteins used in the assay. 1, MBP; 2, MBP-full-length Sit4p (F); 3, MBP-N-terminal regulatory domain of Sit4p (amino acids 1 to 164) (N);
4, MBP-C-terminal catalytic domain of Sit4p (amino acids 161 to 302)
(C). (B) Yeast PTPA proteins associate with the MBP-Sit4p fusion
proteins, as shown by Western analysis using the HA antibody 12CA5 as a
probe (top) and the -MBP antibody (New England Biolabs, Inc.) as a
loading control) (bottom). WCE, whole-cell extract.
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We extended our analysis of the interactions between Ncs1p and PP2As to
include an examination of potential physical interactions in vivo.
Specifically, we compared the association of Ncs1p with Pph21p and
Sit4p. Strains carrying plasmids YEpURA3ADH-SIT4-HA and
YEp13NCS1-3xMYC or plasmids YEp24PPH21-HA and
YEp13NCS1-3xMYC were grown to mid-log phase, and extracts
were prepared. The protein phosphatases were precipitated with
antibodies to the HA epitope, and the potential association with Ncs1p
was assessed by Western analysis. Ncs1p was present in the Sit4p
precipitates but not the Pph21p precipitates (Fig.
8, lanes 5 and 6). Of course, this result
does not exclude a physical interaction between Ncs1p and Pph21p; the
interaction may simply be below our limits of detection. These results
suggest that Ncs1p and Sit4p are part of a complex in vivo and are
consistent with the notion that Sit4p is a target of Ncs1p.
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FIG. 8.
Ncs1p coimmunoprecipitates with Sit4p from whole-cell
extracts (WCE). Lane 1, SY3357 (wild type; WCE); lane 2, SY3357 (wild
type; immunoprecipitate [IP]); lane 3, SY3394 (ncs1)
carrying YEp13NCS1-3xMYC (IP); lane 4, SY3394
(ncs1) carrying YEp13NCS1-3xMYC (WCE);
lane 5, SY3394 (ncs1) carrying YEp13NCS1-3xMYC
plus YEp24PPH21-HA (IP); lane 6, SY3394 (ncs1)
carrying YEp13NCS1-3xMYC plus YEpURA3ADH-SIT4-HA
(IP).
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DISCUSSION |
Yeast contains two related PAKs, Ste20p and Cla4p, that have
individual functions but also appear to share an additional essential function. This latter conclusion is based on the synthetic lethality of
ste20 and cla4 mutations (7,
15). We sought new mutations that are lethal in a
cla4 background in an effort to identify putative targets
or regulators of STE20 function and more generally to learn
about the essential function(s) shared by CLA4 and
STE20. This effort identified 10 complementation groups, one
of which, NCS1, is the focus of this report. Ncs1p is a
member of the conserved PTPA family of proteins, which are capable of
increasing the phosphotyrosyl phosphatase activity of PP2As in vitro
(5). We show that Ncs1p binds to, and thereby presumably
regulates, the PP2A-like phosphatase, Sit4p. Because loss of
SIT4 is lethal in a cla4 background, we infer
that loss of the Ncs1p-Sit4p interaction is responsible for the
lethality of ncs1 cla4 strains. NCS1
function has been explored in previous studies, which showed that cells
lacking NCS1 have a higher mutagenesis rate, are more
resistant to rapamycin and caffeine, and are more sensitive to vanadate
than are wild-type cells (39). We show that together with
CLA4, NCS1 has a role in the cell cycle that is
manifest at the G2/M transition, perhaps by regulating
Cdc28p kinase activity. In addition, together with NOH1, a
gene that encodes a closely related PTPA that also binds to Sit4p,
NCS1 has a role in the G1 phase of the cell cycle.
Interaction of Cla4p with Cdc28p.
CLA4 has been reported
to have important roles in maintaining actin cytoskeletal polarity
during both G1 and G2 phases of the cell cycle
(15); its enzymatic activity is maximal in G2 (2). A second role during G1 is to promote
normal septin organization (26). We have shown that
cla4 mutants exhibit a G2-delay that appears
to reflect activation of a morphogenesis checkpoint because the delay
can be overcome by mutations in CDC28 (CDC28Y19F)
or SWE1. Moreover, these mutations largely restore normal
morphology to cla4 strains. In principle, activation of
the checkpoint could be the consequence of perturbation of actin
cytoskeleton or septin organization. In either event, we suggest that
CLA4 is intimately tied to the checkpoint mechanism, because
we can detect a Cdc28p-Cla4p complex. Thus, these reports that a
protein known to interact with septins also interacts with Cdc28p
parallel the finding that Swe1p, known to interact with and regulate
Cdc28p, also interacts with the septin ring (30).
The morphogenesis checkpoint can be activated by increasing the
activity of Swe1p, which phosphorylates Cdc28p residue Y19 and thereby
inactivates it, or by decreasing the activity of Mih1p, the phosphatase
that dephosphorylates Y19 (3, 44, 50). We have observed an
increase in Cdc28Y19p phosphorylation in cla4 mutants,
consistent with the view presented above that a checkpoint is activated
in these mutants. Does Cla4p influence Mih1p or Swe1p activity? Cells
lacking both CLA4 and MIH1 have higher levels of
phosphorylated Cdc28Y19p than do cells lacking either protein singly.
Moreover, cla4 mih1 cells have a growth defect
compared to cla4 or mih1 cells. Together,
these results imply that Cla4p and Mih1p regulate Cdc28p by different
mechanisms. Because Swe1p is the only enzyme, other than Mih1p, shown
to catalyze Cdc28Y19p phosphorylation/dephosphorylation events, by
default we suggest that Cla4p regulates Swe1p. This is an attractive
possibility given that both proteins interact with Cdc28p and with the
septin ring (7, 26, 30, 41, 49).
NCS1 plays a role in G1 and at the
G2/M transition.
One perspective of CLA4
function has come from the analysis of cla4 ste20
mutants (7, 15). These studies revealed that the septin
ring that separates the mother cell from the growing bud did not
function properly in the double mutant. In particular, new surface
growth occurred on the mother side of the ring rather than on the
daughter side. In contrast, we find that the septin ring is maintained
at the bud neck in cla4 ncs1 mutants, implying that at
least with respect to this property the septin ring is normal. The
different phenotypes of ncs1 cla4 and ste20
cla4 mutants suggest two possible relationships between
NCS1 and STE20. First, if NCS1 is in
some way related to STE20, NCS1 must orchestrate only a subset of STE20 functions. Alternatively,
NCS1 and STE20 functions may be unrelated. In
this case, the lethality of ncs1 cla4 and
ste20 cla4 strains must have different explanations; that is, loss of NCS1 reveals a different cla4
defect than does the loss of STE20. We favor the latter
possibility because CDC28Y19F suppresses the lethality of
ncs1 cla4 mutants but not ste20 cla4
mutants, even though CDC28Y19F restores bud morphology and the septin ring to its normal bud neck location in ste20
cla4 cells.
The foregoing discussion suggests that NCS1 has a function
that is evident at the G2/M transition in the cell cycle.
We believe that NCS1 also has a function in the
G1 phase. This conclusion follows from the observation that
cells lacking both identified PTPA subunit genes, NCS1 and
NOH1, arrest as unbudded cells.
Sit4p as a target of PTPA function.
What are the targets of
NCS1 (and NOH1) action? Having isolated the yeast
PTPA gene (NCS1) in our genetic screen, we sought to
identify the phosphatase target(s) of Ncs1p activity. We reasoned that
one kind of target should (i) be a PP2A, (ii) interact physically with
Ncs1p (and potentially Noh1p), and (iii) have mutant phenotypes in
common with ncs1, for example, synthetic lethality with
cla4. Only one of the five known yeast PP2As, Sit4p,
satisfies these three criteria. Rempola et al. have argued that Pph22p
might be a target of Ncs1p (Rrd1p) because overexpression of
PPH22 can partially suppress the lethality associated with
loss of PTPA function (39). However, the degree of
suppression and the absence of data showing a physical interaction
between the PTPA subunits and Pph22p detract from their argument. In
addition, we were unable to detect an in vivo interaction between Ncs1p
and Pph21p, the nearest homolog of Pph22p, or between PPH22
and CLA4.
The lack of genetic interactions between CLA4 and other
phosphatase structural genes, however, does not rule out the
possibility that Ncs1p interacts with other phosphatase catalytic
subunits. Indeed, we believe Ncs1p has other targets, based on two
observations. First, both Ncs1p and Noh1p interact with the catalytic
domain of Sit4p. However, the phenotype associated with the loss of
both NCS1 and NOH1 (growth arrest) is more severe
than the phenotype associated with the loss of SIT4 in our
strain background. If Sit4p were the only target of PTPA function, then
one would expect that loss of both PTPA genes and the loss of
SIT4 would confer the same phenotype(s) or, if the
phenotypes were different, that the loss of SIT4 would be
more severe given that the Sit4p phosphoserine/threonine as well as
phosphotyrosine phosphatase activity is absent. This is not the case.
Second, the phenotype of a sit4 noh1 strain is no more
severe than that of either single mutant. If Sit4p were the only target
of Ncs1p, then one would expect an noh1 sit4 strain to
be phenotypically similar to an noh1 ncs1 strain. Again, this is not the case. Therefore, we conclude that Sit4p is one,
but not the only, target of PTPA function.
We thank Charlie Boone, Kim Arndt, Danny Lew, Kim Nasmyth, Fred
Cross, Michael Stark, Megan Keniry, Kunliang Guan, and Xiaoli Zhan for
providing plasmids, Mike Snyder for providing the yeast transposon
library, and John Pringle, John Chant, Mike Marusich, and the
University of Oregon Monoclonal Antibody Facility for providing
antibodies. We are forever grateful to Daciana Margineantu, April
Goehring, Diamond Bob Deschenes, Laurie Graham, Liz Conibear, and Tom
Stevens for technical assistance, advice, and reagents. We also thank
members of the Sprague lab for helpful comments and discussions. FACS
analysis was performed by the University of Iowa Flow Cytometry
Facility. DNA sequencing was performed by Yanling Wang.
This work was supported by National Research Service Award GM-18002-03
(to D.A.M.) and by grant GM30027 from the National Institutes of Health
(to G.F.S.).
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Abstract
Introduction
