From the Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, SUNY Stony Brook, Stony Brook, New York 11794-5215
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ABSTRACT |
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The synaptonemal complex (SC) is a proteinaceous
structure formed between pairs of homologous chromosomes during
prophase I of meiosis. The proper assembly of axial elements (AEs),
lateral components of the SC, during meiosis in the yeast,
Saccharomyces cerevisiae, is essential for wild-type levels
of recombination and for the accurate segregation of chromosomes at the
first meiotic division. Genetic experiments have indicated that the
stoichiometry between two meiosis-specific components of AEs in
S. cerevisiae, HOP1 and RED1, is
critical for proper assembly and function of the SC. A third
meiosis-specific gene, MEK1, which encodes a putative serine/threonine protein kinase, is also important for proper AE
function, suggesting that AE formation is regulated by phosphorylation. In this paper, we demonstrate that Mek1p is a functional kinase in vitro and that catalytic activity is an essential part
of the meiotic function of Mek1 in vivo. Immunoblot
analysis revealed that Red1p is a
MEK1-dependent phosphoprotein.
Co-immunoprecipitation experiments demonstrated that the interaction
between Hop1p and Red1p is enhanced by the presence of
MEK1. Thus, MEK1-dependent phosphorylation of Red1p facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby enabling the formation of functional AEs.
Sexual reproduction requires the formation of haploid gametes so
that fertilization can reconstitute the diploid chromosome number of
the organism. The chromosome number of the cell is divided precisely in
half by meiosis, a highly conserved and specialized type of cell
division. Specifically, the reduction in chromosome number is
accomplished by the segregation of homologous chromosomes to opposite
poles at the first meiotic division. Proper disjunction requires
crossovers (typically manifested cytologically as chiasmata) that
provide the physical connections between homologs necessary for proper
orientation on the Meiosis I spindle (1). Mutants that interfere with
the formation of crossovers produce chromosomally imbalanced gametes
that are either inviable themselves or fuse to create inviable zygotes
(2).
In most organisms, two requirements must be met for wild-type levels of
crossovers to occur during meiosis. First, there must be recombination
machinery to physically exchange the DNA from non-sister chromatids.
Second, a tripartite proteinaceous structure called the synaptonemal
complex (SC)1 must form
between homologous chromosomes (3, 4). SC formation is initiated by the
condensation of sister chromatids along a protein core called an axial
element (AE). Synapsis occurs when AEs are held together by the
introduction of central region components to make mature SCs.
In Saccharomyces cerevisiae, deletion of RED1, a
gene encoding a component of AEs, eliminates AE formation and therefore
red1 mutant phenotypes may indicate functions for AEs (5).
In red1 diploids, spore viability is drastically reduced,
despite only a 4-fold reduction in crossing over, suggesting a role for
AEs in packaging crossovers in such a way that the chiasmata can be used for directing Meiosis I chromosome segregation (5, 6). RED1 also plays a role in a checkpoint that monitors whether
recombination is progressing correctly or not. For example, mutations
in the meiosis-specific recA homolog DMC1 cause arrest prior
to the first meiotic division, presumably as a result of the formation
of aberrant recombination intermediates (7). In the absence of
RED1, this arrest is abolished despite the fact that the
aberrant recombination intermediates are still being generated. It
appears, therefore, that the packaging of recombination intermediates
into some sort of RED1-dependent chromosomal
structure is necessary for the recombination-monitoring checkpoint to
work (8).
Understanding AE assembly is clearly a key to understanding axial
element function. Genetic and cytological experiments have identified
two other meiosis-specific genes, HOP1 and MEK1
(also known as MRE4), that are important for formation of
functional AEs in yeast. Evidence that the stoichiometry of Red1p to
Hop1p is important in axial element formation comes from genetic
experiments in which overexpression of RED1 in the presence
of limiting amounts of Hop1p created a dominant negative phenotype (9).
This dominant negative phenotype can also be produced by overexpressing
RED1 in the presence of wild-type levels of HOP1
but with limiting amounts of functional Mek1p (10). Mek1p therefore
appears to influence the Hop1p:Red1p stoichiometry. This paper presents
biochemical data supporting the model that the interaction of Hop1p and
Red1p with each other is promoted by phosphorylation of Red1p by Mek1p (10).
Yeast Strains and Media--
The genotypes of the strains used
in this study are shown in Table I. All
the strains are isogenic derivatives of the SK1-related diploid NH144
(11). Diploid strains mutated for RED1 or MEK1 were created by first transforming S2683 and RKY1145, the two haploid
parents of NH144, with the appropriate DNA fragments (12) followed by
mating. For the red1::LEU2 mutation, a 4.9-kb
BglII/PstI fragment was purified from pNH119 (13)
and used for transformation. This insertion allele exhibits a null
phenotype with respect to spore viability (other phenotypes have not
been tested). A 3.6-kb XbaI/BamHI fragment from
pTS1 carries the mek1 Plasmids--
Plasmids for this study were made by standard
procedures (16) using the Escherichia coli strain BSJ72 and
are listed in Table II. To construct the
mek1
To fuse the MEK1 gene to GST (glutathione
S-transferase) (18), PCR was employed to generate
a fragment containing the entire MEK1 gene. The resulting
fragment was digested with BglII and ligated into the
BamHI site of pRD56 to create pNH168.
The mek1-K199R mutation was introduced into the
GST-MEK1 fusion gene by oligonucleotide site-directed
mutagenesis (19). The 2.9-kb NotI/SalI fragment
from pNH168 containing GAL1p-GST-MEK1 was cloned into
NotI/SalI-digested pVZ1 to generate pNH205. The presence of the mutation creates an FspI restriction site
that was used for screening plasmids with the desired mutation. The plasmid containing the mek1-K199R allele was designated pNH206.
To place the GST-MEK1 and GST-mek1-K199R alleles
under control of the MEK1 promoter, the MEK1
promoter was first cloned into the 2µ vector, YEplac181. A 924-bp
fragment starting at position
To assay for complementation of the mek1
The mek1-974 mutation was rescued onto a plasmid by gap
repair (20). The MEK1 plasmid, pB131, was cut with
SacI and HpaI and transformed into the
mek1-974 diploid NH104 (10). The resulting plasmid, pLP41,
was used in sequencing reactions with primers located at staggered
intervals along the MEK1 gene. To make the mek1-974 integrating plasmid, pNH235, a 3.2-kb
EcoRI/SalI fragment from pLP41 was ligated into
the URA3 vector, pRS306, digested with EcoRI and
SalI.
The first step in the construction of the RED1-3HA allele
was the introduction of an EcoRI site in place of the
RED1 stop codon by PCR. A 1.3-kb fragment containing the
3'-half of RED1 was amplified, digested with
BglII and EcoRI, and ligated into BglII/EcoRI-digested pNH124 (10) creating pNH208.
A 3.4-kb SalI/EcoRI fragment from pNH208 carrying
the entire RED1 gene with ~1.1 kb of upstream sequences
was cloned into SalI/EcoRI SK-HA3 (21). This
ligation fuses the full-length RED1 gene in-frame at the 3'-end with three copies of the hemagglutinin (HA) epitope in pNH209.
The tagged allele was moved either to a high copy 2µ plasmid (pNH212)
or to a URA3-integrating plasmid (pTS23) by ligating the
3.5-kb SalI/SacI fragment from pNH209 into YEp352
and pRS306, respectively.
Antibodies--
The mouse monoclonal Sporulation--
2-ml Yeast extract-peptone-dextrose cultures
were inoculated with single colonies of the appropriate diploid and
grown to saturation at 30 oC with aeration. Strains
containing plasmids were inoculated with colonies grown on selective
medium. Plasmid stability was monitored immediately prior to transfer
to sporulation medium and >80% of the cells were typically found to
contain plasmid. The yeast extract-peptone-dextrose cultures were
diluted 1:1000-1:3000 into yeast peptone acetate medium and grown for
approximately 16 h with aeration at 30 oC. When
cultures reached an OD660 = 1-2, the cells were pelleted, washed with water, and resuspended in 2% potassium acetate to a final
concentration of 3 × 107 cells/ml. The cells were
shaken at 30 oC for 3 h and then harvested by
centrifugation for 10 min at 5000 rpm in a GSA rotor. Prior to
centrifugation, a 1-ml aliquot was removed to the 30 oC
roller, and the percent sporulation was assessed by light microscopy the following day. Sporulation typically reached levels of >90%.
Kinase Assays--
After 3 h in sporulation medium, 20-ml
aliquots of cells were washed with water, and the cell pellets were
quick frozen at Immunoprecipitation of Hop1p and Red1-3HAp from Meiotic
Extracts--
Sporulating cells were harvested, washed one time with
water, and resuspended in 0.3 ml of lysis buffer (25 mM
Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM EDTA) per
20 ml of cells. To reproducibly detect Red1-3HAp, it was necessary to
prepare extracts the same day as the cells were sporulated,
i.e. the cells could not be frozen first. The reason for
this phenomenon is not understood but is probably not because of
degradation of the Red1-3HA protein. After extracts were prepared, the
proteins could be frozen at
The amount of Hop1p and Red1-3HAp present in the immunoprecipitates
was quantitated as described in the instructions for the ECL kit
(Amersham Pharmacia Biotech). A series of dilutions from the soluble
extracts used in the experiment was fractionated on the same gel and
probed simultaneously with the appropriate antibodies. After the ECL
reaction, the films were scanned using a Bio-Rad imaging densitometer.
A standard curve of protein concentration was generated for Hop1p and
Red1-3HAp using the Molecular Analyst, Version 1.1 software (Bio-Rad).
The amount of Hop1p and Red1-3HAp present in the immunocomplexes was
then determined using the standard curves. To compare the efficiency of
the co-immunoprecipitation in MEK1 and mek1 Phosphatase Experiments--
For each sample, 20 ml of
sporulating YTS3/pNH212 cells were used for
immunoprecipitation of Red1-3HAp. The immunoprecipitates were treated
with Overexpression of HOP1 Suppresses mek1-974 in the SK1 Background
whereas Overexpression of RED1 Exacerbates the mek1-974 Mutant
Phenotype--
Genetic interactions between MEK1,
HOP1, and RED1 in the A364A strain background led
to the proposal that phosphorylation of either Hop1p or Red1p by Mek1p
facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby
ensuring the proper balance between the proteins in the assembly of
axial elements during meiosis (10). To test this model biochemically,
it was desirable to use the SK1 strain background in which sporulation
proceeds rapidly, relatively synchronously, and with high efficiency
(25). To ensure that the genetic dosage effects of HOP1 and
RED1 overexpression in the presence of the leaky
mek1-974 mutant occur in SK1 as well as A364A, it was
necessary to repeat the dosage experiments in SK1. The
mek1-974 mutation was cloned by gap repair using the diploid NH104 (10), and the entire gene was sequenced. A single transition mutation of Gly to Ala at nucleotide 502 was found. This
mutation creates a valine to methionine substitution at amino acid 168, which is located immediately before the GXG motif in the
first conserved kinase domain (26). The mek1-974 allele was
introduced into the SK1 background by two-step gene replacement, thereby creating the homozygous diploid, NH211. This diploid was transformed with the same plasmids used in the previous analysis (10),
the transformants were sporulated, and tetrads were dissected to assess
spore viability.
As in the A364A background, the mek1-974 mutant is
partially functional in SK1, producing 33.2% viable spores compared
with ~1% for the deletion (Ref. 27 and Table
III). The spore viability defect is
complemented by MEK1 on a CEN plasmid (Table III,
NH211). The presence of excess HOP1 greatly improved the
mutant phenotype, resulting in 74% viable spores. In contrast,
overexpression of RED1 decreased the number of viable spores
from 33.2 to 16.8% (Table III, NH211). Both the HOP1
suppression and the RED1 exacerbation of the
mek1-974 spore inviability phenotype are statistically significant by Detection of Red1p in Meiotic Cells Using the Hemagglutinin Epitope
Tag--
Because attempts to use polyclonal antibodies directed
against Red1p (28) to detect the Red1p protein by immunoblot analysis were unsuccessful,3 an
epitope-tagged version of the protein was created. The RED1 gene was fused in frame at the 3'-end to three copies of the
hemagglutinin epitope. The fusion gene was then integrated at
URA3 in two different isogenic SK1
red1::LEU2 haploids. This insertion allele of
RED1 exhibits a null phenotype with regard to spore
viability (13). The haploids were mated to create a
red1::LEU2 diploid that is homozygous for the
RED1-3HA allele (YTS3::pTS23). Alternatively, the
RED1-3HA gene on a high copy number 2µ vector was
transformed into YTS3 (YTS3/pNH212). After 3 h in sporulation
medium, a time point when Red1-3HAp and Hop1p concentrations are
maximal,3 protein extracts were made from YTS3/YEp352,
YTS3::pTS23, and YTS3/pNH212. No protein of the appropriate
molecular weight for Red1-3HAp was detected with the
Tetrad dissection was performed to determine the ability of the
RED1-3HA allele to complement the spore viability defect of red1::LEU2 in YTS3. YTS3 carrying vector alone
produced 3.8% viable spores. When RED1-3HA is homozygous
in YTS3::pTS23, no complementation was observed (Table III,
YTS3/YEp352). Over-expression of the RED1-3HA allele
restored a significant amount of RED1 although the
complementation was not as good as overexpression of the untagged
allele of RED1 (Table III, YTS3/YEp352). Because the
overexpressed RED1-3HA allele was detectable and provided a
substantial level of RED1 function (50% spore viability
versus 3.8%), this construct was used in all subsequent experiments.
Red1-3HAp Is a Phosphoprotein--
To determine whether the
slower migrating species of Red1-3HAp are because of phosphorylation,
the Red1-3HA protein was immunoprecipitated and tested for sensitivity
to Mek1p Has Kinase Activity in Vitro and the Kinase Activity Is
Essential for the Meiotic Function of MEK1--
A good candidate for
the kinase which phosphorylates Red1p is Mek1p. To determine whether
the Mek1p kinase homology is meaningful, Mek1p kinase activity was
tested in vitro. The MEK1 gene was fused to the
3'-end of the affinity tag GST and the GST-MEK1 fusion allele was placed under the control of the MEK1 promoter. As
a negative control, an allele of MEK1
(mek1-K199R) was created in which the codon for an invariant
lysine residue present in domain II of the kinase (amino acid 199) was
mutated to arginine. This lysine to arginine mutation has been shown to
reduce or abolish kinase activity in a number of other kinases (30).
The mek1
Complementation tests were performed to assay the function of the two
MEK1 alleles. The GST-MEK1 gene in YTS4
complemented the mek1 The Bulk of Red1-3HAp Phosphorylation Is Dependent on
MEK1--
To test whether Red1-3HAp is a potential substrate for
Mek1p, the protein was examined in MEK1 (YTS3/pNH212)and
mek1
When the soluble fraction is examined, a low level of phosphorylated
Red1-3HAp is sometimes observed. Because the spore viability of the
mek1 Hop1 and Red1-3HAp Co-immunoprecipitate from Meiotic Cell
Extracts--
Co-immunoprecipitation experiments were performed to
determine whether Hop1p and Red1-3HAp are physically associated in
meiotic cells. YTS3/pNH212 was sporulated for 3 h, protein
extracts were made, and equal amounts of protein were incubated with
either
The Hop1p and Red1p immunoprecipitated samples were divided in half,
fractionated by SDS-PAGE, transferred to nitrocellulose and probed with
either The Absence of MEK1 Reduces the Efficiency of the Hop1p/Red1-3HAp
Co-immunoprecipitation--
To test whether the
MEK1-dependent phosphorylation of Red1-3HAp has any
effect on the ability of Red1-3HAp to interact with Hop1p, the
co-immunoprecipitation experiment was performed using extracts from the
mek1 Genetic studies in the A364A background led to a model which says
that Mek1p phosphorylation facilitates the assembly of Red1p and Hop1
in a particular stoichiometry necessary to generate functional AEs
during SC formation (10). To test this model biochemically, it was
desirable to use the SK1 strain background, because the cells sporulate
efficiently and synchronously. However because mutants in
MEK1 and HOP1 exhibit more severe phenotypes in
the SK1 background compared with A364A, it was possible that the model based on the findings from the A364A strain background would not apply
to the SK1 background. For mek1 The mek1-974 allele used for the dosage experiments in
A364A was cloned by gap repair and introduced into SK1. The
mek1-974 allele is partially functional in SK1, the same as
in A364A. Overexpression of HOP1 in the SK1
mek1-974 diploid resulted in partial suppression of the
spore viability defect. In contrast, overexpression of RED1
decreased spore viability. Co-overexpression of HOP1 and RED1 was previously shown to result in a phenotype similar
to neither gene being present, consistent with the idea that the two
proteins can titrate out each other. In SK1, the HOP1/RED1 co-overexpression phenotype more resembled that of HOP1
alone, suggesting that this strain background may be more sensitive to the levels of HOP1, a result consistent with the more severe
recombination phenotype exhibited by hop1 mutants in this
strain. The reproducibility of the dosage results in SK1 indicated that
it would be an appropriate strain in which to test the stoichiometry
model biochemically.
A basic assumption of the stoichiometry model is that Mek1p is a
serine/threonine protein kinase. This assumption was proven to be
correct by demonstrating that Mek1p has autophosphorylation activity
in vitro. This activity is greatly reduced by substitution of a conserved lysine in the catalytic domain for arginine To determine whether either Hop1 or Red1 is a
MEK1-dependent phosphoprotein, the two proteins
were analyzed by immunoblot analysis for mobility shifts that may be
indicative of phosphorylation. Extensive attempts to detect a
phosphorylated form of Hop1p were unsuccessful. In contrast, a modified
form of Red1-3HAp that is sensitive to phosphatase treatment was
observed. The reduced amounts of this modified form in
mek1 Genetic evidence exists for Hop1p and Red1p homo-oligomers as well as
for Hop1p/Red1p hetero-oligomers (10, 13, 31). The Hop1p homo-oligomer
has recently been demonstrated biochemically using purified protein
(22). To test whether Hop1p and Red1p interact in meiotic cells,
co-immunoprecipitation experiments were performed. The Hop1p antibodies
co-precipitated both the phosphorylated and unphosphorylated forms of
Red1-3HAp. The presence of unphosphorylated Red1-3HAp in the Hop1p
immune complex is not surprising given that Mek1p phosphorylation is
not essential for the Hop1p/Red1p interaction because stretches of SC
are observed even in mek1 It is important to note that these experiments were all performed with
an allele of RED1 that is being overexpressed. Because Smith
and Roeder (28) have shown that overexpression of RED1 can
result in a more continuous staining pattern of Red1p along the
chromosome, it may be that the stoichiometries observed here do not
reflect the exact stoichiometries present in strains containing endogenous levels of Red1p.
Although Mek1p phosphorylation of Red1-3HAp is not essential for the
Hop1p/Red1p interaction, we have proposed that it facilitates the
formation of the hetero-oligomer in the proper stoichiometry (10). This
idea was tested by performing the Hop1p/Red1-3HAp co-immunoprecipitation experiments in a mek1 The genetic data presented in (9) are consistent with the
interpretation of the co-IP experiment that MEK1 facilitates Hop1p/Red1p hetero-oligomer formation. However, the possibility cannot
be ruled out that the diminished co-immunoprecipitation of Red1-3HA
with Hop1p and vice versa in the mek1 The mechanism for assembling AEs by phosphorylation-facilitated
protein-protein interactions may be conserved in evolution. Homologs
for Mek1p and Hop1 exist in S. pombe and C. albicans (Table IV). Within the
amino terminus of Mek1p, but outside of the kinase homology, is a
conserved sequence of 56 amino acids called the forkhead-associated
domain (FHA) (33). This domain is found almost exclusively in nuclear
proteins, including Dun1p and Spk1p, two protein kinases of S. cerevisiae involved in the cellular response to DNA damage (33).
Interestingly, in Drosophila there is an ovarian-specific
protein kinase called loki that also contains an FHA
domain.4 It is intriguing to
speculate that loki is the functional homolog of Mek1p in flies,
especially given that SC formation only occurs in Drosophila
females. The Asy1 gene of Arabidopsis thaliana
encodes a protein with significant homology to Hop1p (Table IV), and
asy1 mutant plants exhibit phenotypes (greatly reduced
synapsis and chiasma
formation)5 similar to
hop1 mutants in yeast (32). Cor1/SPC3 may be the structural
analog to Red1p in mammalian cells. Both proteins are meiosis-specific
and are predicted to contain extensive coiled coil domains in their C
termini (34, 35). Furthermore, the C termini of both proteins mediate
homodimerization in the two-hybrid system (10, 36). SPC3 has been shown
to be phosphorylated and, furthermore, that the pattern of
phosphorylation is dynamic throughout meiotic prophase (37). It is
possible that the early phosphorylation events observed for SPC3
fulfill the same role in establishing the correct protein stoichiometry
in mammalian AEs that it plays in the Hop1p/Red1p interaction.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
::LEU2 allele, and a 2.9-kb BamHI fragment from pTS21 was used to introduce
mek1
::URA3. These deletions remove
75 base pairs upstream of the ATG as well as approximately 75% of the
MEK1 coding sequence. The presence of the
red1::LEU2,
mek1
::LEU2, and
mek1
::URA3 alleles was confirmed both genetically and by Southern blot analysis. The ade2-Bgl
mutation was introduced by two-step gene replacement (12) using pR943 (generously supplied by B. Rockmill, Yale University) cut with SpeI. Haploids containing the ade2-Bgl mutation
and appropriate other markers were obtained by crosses. These haploids
were then mated to give homozygous diploids. The mek1-974
mutation was introduced into two different
mek1
::LEU2 haploids by two-step gene
replacement using pNH235 digested with BssHI to target
integration to the mek1
::LEU2 locus.
Leu
5FoaR colonies were then mated to create
the mek1-974 diploid NH211. The red1::LEU2
mek1
::LEU2 homozygous diploid NH177 was
constructed by crossing a MATa
mek1
::LEU2 haploid with MAT
red1::LEU2 haploid and dissecting tetrads from the
resulting heterozygous diploid. Leu+ spore colonies that
exhibited a non-parental di-type pattern for LEU2 were then
mated to produce the double homozygote. To make the
RED1-3HA homozygous diploid YTS3::pTS23,
S2683red1 and RKY1145red1 were transformed with pTS23 digested with
StuI to target integration of the plasmid to
URA3. The resulting transformants were mated to create the
diploid. For YTS1:pLP36 and YTS1::pLP37, the plasmids pLP36
and pLP37 were digested with StuI to target integration to
URA3 and transformed into YTS1. Standard yeast genetic
methods were used (14). Solid media have been described (15). YPA
contains 1% yeast extract, 2% bactopeptone, and 2% potassium
acetate. Sporulation was performed using 2% potassium acetate.
Saccharomyces cerevisiae SK1 strains
::URA3 and
mek1
::LEU2 deletion alleles, the
polymerase chain reaction (PCR) was used to generate fragments of
URA3 or LEU2 with SacI and
PvuII ends. The PCR fragments were digested with
SacI and PvuII and ligated to pB131 (17) cut with
SacI and HpaI to generate pTS21
(mek1
::URA3) and pTS1 (mek1
::LEU2), respectively.
Plasmid list
896 relative to the MEK1 ATG
and ending at +28 was amplified by PCR. The oligonucleotide was
engineered to put an NdeI site at the ATG of MEK1
with a SalI site downstream. The fragment was digested with
BamHI and SalI and ligated into
BamHI/SalI-digested YEplac181 to make pDW14. The
2.9-kb NdeI/SalI fragments containing either
GST-MEK1 (pNH168) or GST-mek1-K199R (pNH206) were
then cloned into pDW14 cut with NdeI and SalI to
make pDW15 and pDW16, respectively. The MEK1 and
mek1-K199R alleles were completely sequenced to confirm that
no unknown mutations were introduced by either the PCR or site-directed
mutagenesis. Sequencing was performed using the ABI Amplitaq sequencing
kit (Perkin-Elmer) followed by electrophoresis on an ABI model 373A
automated sequencer.
by
mek1-K199R, both the MEK1 and
mek1-K199R alleles were put under control of the MEK1 promoter without the GST fusion. To accomplish this, a
5.5-kb EcoRI fragment from pR4C4 (10) was first cloned into
EcoRI-digested pVZ1 to generate pLP31. After digestion of
pLP31 with SpeI and SalI, the vector backbone was
ligated with the 1.2-kb SpeI/SalI fragment from
either pNH206 or pNH168, resulting in the substitution of approximately
80% of the MEK1 coding sequence. The resulting plasmids,
pLP34 (mek1-K199R) and pLP35 (MEK1) were then
digested with NotI and SalI, and the 2.6-kb
fragments were ligated into pRS306 to make pLP36 and pLP37, respectively.
-HA antibody is
contained in the 12CA5 ascites fluid that was purchased from Babco.
Affinity-purified
-Gstp antibodies were generously provided by D. Kellogg (University of California Santa Cruz). Antibodies directed
against the full-length native Hop1 protein were generated as follows.
The Hop1 protein was overexpressed in vegetative yeast cells and
purified to >95% homogeneity (22). 0.5 mg of purified Hop1 protein
was injected into two rabbits followed by a boost of 0.25 mg three
weeks later (performed by Babco). The resulting sera were found to
cross-react with a band of ~67 kDa, the predicted molecular mass of
Hop1p, when cells carrying the galactose-inducible
GAL10p-HOP1 gene (22) were grown in galactose. This band was
absent when the cells were grown on glucose. The 67-kDa band was also
not observed using the preimmune sera from these rabbits. Further
evidence that these antibodies are specific for Hop1p is that the
67-kDa protein is only observed in sporulating cultures when
HOP1 is
present.2
70 oC. Kinase assays were performed as
described (23).
70 oC and thawed with no
loss in the ability to detect Red1-3HAp by immunoblot analysis.
Protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin) and phosphatase inhibitors (10 mM NaF, 10 mM
Na4P2O7, 1 mM
Na3VO4) were present throughout the lysis and
wash steps. One gram of glass beads was added, and the tubes were
vortexed three times for 30 s with 2-min intervals on ice. The
lysate was removed to a clean tube, and detergents were added to final
concentrations of 1% Triton X-100, 0.5% sodium deoxycholate, and
0.1% SDS. The lysate was rocked for 15 min at 4 oC and
cleared by centrifugation at 14,000 × g for 10 min.
The supernatant was divided into two tubes to which either 1-2 µl of
-Hop1p or
-HA antibodies was added, followed by rocking for 2 h at 4 oC. To precipitate the immune complexes, 40 µl of protein A-Sepharose (Amersham Pharmacia Biotech) slurry
(equilibrated 1:1 with lysis buffer) were added. The tubes were then
rocked again for an additional hour at 4 oC. The Sepharose
beads were washed four times with 0.5 ml of lysis buffer and
resuspended in 30 µl of 2× SDS protein sample buffer. The beads were
heated at 95 oC for 5 min and loaded in duplicate onto a
6% SDS-polyacrylamide gel. After fractionation, the proteins were
blotted to nitrocellulose and probed for Hop1p and Red1-3HAp using
-Hop1p and
-HA antibodies, respectively. The presence of the
antibodies was determined using the ECL kit from Amersham Pharmacia Biotech.
extracts, the following formula was used.
(Eq. 1)
protein phosphatase (New England Biolabs) as described
(24).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2 analysis (p < 0.001).
The one difference observed between SK1 and A364A was the result
obtained when both HOP1 and RED1 were co-overexpressed on the same plasmid. In A364A,
HOP1/RED1 co-overexpression restored the spore
viability observed for vector alone. In SK1, a suppression phenotype
similar to that for HOP1 alone was found (Table III,
NH211).
Spore viability assayed by tetrad dissection
-HA antibodies
in YTS3 containing the vector alone (Fig.
1A). These cells were
proceeding through meiosis, as evidenced by there being a wild-type
amount of Hop1p in the YTS3/YEp352 extract (Fig. 1B). A
RED1-3HA-dependent
-HA cross-reacting band
was observed in YTS3::pTS23. This protein represents
Red1-3HAp as this band is absent from extracts of NH144, an isogenic
diploid homozygous for the untagged RED1 gene, as well as
from vegetative cultures of YTS3::pTS23.3
Interestingly, in YTS3/pNH212, where the RED1-3HA allele is
present in high copy number, several Red1-3HAp bands are apparent
(Fig. 1A).
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Fig. 1.
Detection of Red1-3HAp and Hop1p in meiotic
extracts. The red1::LEU2 diploid, YTS3, was
transformed with vector alone (YEp352), pTS23 (two integrated copies of
RED1-3HA), or pNH212 (2µ RED1-3HA). Cells
were sporulated for 3 h, and total protein extracts were made. 20 µg of total extract were fractionated by SDS-PAGE, blotted to
nitrocellulose, and probed with either -HA antibodies (panel
A) or
-Hop1p antibodies (panel B).
protein phosphatase (New England Biolabs). After washing, the
immunoprecipitates were resuspended in phosphatase buffer, treated with
or without phosphatase, fractionated by SDS-PAGE, and blotted, and the
Red1-3HAp was detected using the
-HA antibody. In the sample where
no phosphatase was added, three discrete bands were observed (Fig.
2). When
protein phosphatase was
added, the slower mobility bands were no longer visible. The addition
of phosphatase inhibitors prior to the phosphatase prevents the loss of
the shifted bands (Fig. 2), indicating that it is the phosphatase
activity which accounts for the disappearance of the modified bands and
not a contaminating protease. One concern was that the phosphorylation
is occurring on the HA epitope rather than Red1p. That this is not the
case is demonstrated by the findings of Bailis and Roeder (29) who have
recently shown that Red1p is a phosphoprotein using antibodies against
the endogenous Red1p.
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Fig. 2.
Detection of a
MEK1-dependent phosphorylated form of
Red1-3HAp in meiotic extracts. Red1-3HAp was immunoprecipitated
from 3 mg of soluble protein extracted from YTS3/pNH212 after 3 h
in sporulation medium. Immunocomplexes were treated with and without
protein phosphatase and phosphatase inhibitors. Red1-3HAp was
detected by immunoblot analysis using
-HA antibodies.
::URA3 diploid YTS4 was
transformed with YEplac181 (2µ), pDW15 (2µ GST-MEK1), or
pDW16 (2µ GST-mek1-K199R). Cells were sporulated for
3 h, protein extracts were made, and the Gst-Mek1p and
Gst-mek1-K199R fusion proteins were purified using
glutathione-Sepharose beads. Kinase activity was assayed by
autophosphorylation. The relative amount of Gst-Mek1p and
GST-mek1-K199R present during the assay was determined by probing the
same blot used for autoradiography with antibodies directed against
Gstp. A radioactive band migrating at the predicted molecular mass for
Gst-Mek1p was observed; this band is absent in YTS4/YEplac181 (Fig.
3A). Very little
autophosphorylation activity was detectable for the Gst-mek1-K199R
protein despite the fact that more protein was present than for
Gst-Mek1 (Fig. 3B). The reduction in phosphorylation of the
mek1-K199R protein demonstrates that most of the observed kinase
activity is because of Mek1p and not to a co-precipitating kinase or to
the Gst moiety.
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Fig. 3.
Autophosphorylation of Gst-Mek1p from meiotic
extracts. Cells were sporulated for 3 h and extracts from
YTS4 (mek1 /mek1
) containing vector alone
(YEplac181), GST-MEK1 (pDW15), or GST-mek1-K199R
(pDW16) were treated with glutathione-Sepharose to affinity purify the
GST fusion proteins. The purified proteins were treated with
[
-32P]ATP:ATP to assay for autophosphorylation. The
proteins were examined by phosphoimager analysis to detect incorporated
radioactivity (panel A). The same blot was subsequently
probed with
-Gstp antibodies (panel B).
spore viability defect while the
GST-mek1-K199R allele did not (Table III, YTS4).
GST-MEK1 also complemented when integrated into the
chromosome, showing that overexpression is not a requirement for
activity.2 In addition, MEK1 and
mek1-K199R (without GST) were integrated at URA3
in the mek1
::LEU2 diploid YTS1. Full
complementation of the spore viability defect was seen in the
YTS1::pLP37 diploid carrying MEK1 compared with
YTS1 alone (94.5% versus 5.0% viable spores; Table III,
YTS4). In contrast, the mek1-K199R allele fails to
complement (Table III, YTS4). Therefore, mutation of the invariant lysine to arginine at position 199 creates an allele of MEK1
that exhibits a null phenotype with regard to spore viability. Because this mutation also greatly reduces Mek1p kinase activity in
vitro, we conclude that the kinase activity of Mek1p is essential
for its meiotic function.
(NH177/pNH212) diploids. Extracts were prepared from
cells after 3 h in sporulation medium. Because the total amount of
Red1-3HAp present in mek1
diploids is
reduced,3 nearly three times as much total protein from the
mek1
was loaded onto the gel compared with
MEK1. Although there is a similar amount of the
unphosphorylated form of Red1-3HAp in both strains, the vast majority
of phosphorylated Red1-3HAp is absent in the mek1
diploid (Fig. 4). Red1-3HAp
phosphorylation is therefore dependent on MEK1. This
experiment does not, however, address whether this dependence is
because of direct phosphorylation of Red1-3HAp by Mek1p or whether an
intermediate kinase is involved.
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Fig. 4.
Detection of Red1-3HAp in MEK1
and mek1 diploids. Total cell extracts from
YTS3/pNH212 (MEK1/MEK1) and NH177/pNH212
(mek1
/mek1
) were examined for Red1-3HAp
using immunoblot analysis and
-HA antibodies. For YTS3/pNH212, 35 µg were loaded on the gel; for NH177/pNH212, 90 µg were used.
diploid overexpressing RED-3HA is
very low (1.3%, 77 asci), any phosphorylated Red1-3HAp that is
present is apparently non-functional. Whether this "promiscuous"
phosphorylation of Red1-3HAp is because of the overexpression of the
protein or whether it normally occurs is not known.
-HA antibodies or
-Hop1p antibodies to immunoprecipitate
Red1-3HAp and Hop1p, respectively. The specificity for Hop1p
immunoprecipitation was shown by the fact that no Hop1 is detected in
immunoprecipitates from sporulating cells deleted for HOP1
(Fig. 5, compare lanes 5 and
6) nor is Hop1 precipitated if the
-Hop1p antibody is
omitted (Fig. 5, lane 4). Similarly, no Red1-3HAp is
observed when the
-HA antibody is used for immunoprecipitation
in diploids carrying the untagged RED1 gene (Fig. 5, compare
lanes 2 and 3) or if the
-HA antibody is left
out when precipitating from diploids containing the
RED1-3HA gene (Fig. 5, lane 4).
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Fig. 5.
Specificity of Hop1 and Red1-3HAp
immunoprecipitation. Cells from various diploids were sporulated
for 3 h and soluble extracts prepared. Hop1p and Red1-3HAp were
immunoprecipitated from these extracts using either -Hop1p or
-HA
antibodies. Lanes 1, 2, 4, and
5, YTS3/pNH212 (HOP1/HOP1
red1::LEU2/red1::LEU2/2 µ RED1-3HA); lane 3, YTS3/p1b-1 (HOP1/HOP1
red1::LEU2/ red1::LEU2/2µ
RED1); lane 6, DW10::pTS23
(hop1::LEU2/hop1 ::LEU2
RED1-3HA/RED1-3HA; and lane 7,
YTS3::pTS23 (HOP1/HOP1
red1::LEU2/red1::LEU2 RED1-3HA/RED1-3HA.
Lanes 1-3 were probed with
-HA antibodies to detect
Red1-3HAp; and lanes 4-7 were probed with
-Hop1p
antibodies.
-HA antibodies or
-Hop1p antibodies to look for
co-immunoprecipitation of Red1-3HAp with Hop1p and vice versa. In YTS3/pNH212, Hop1p co-immunoprecipitated with Red1-3HAp (Fig. 6A, lane 3).
The converse is also true; when Hop1p was first precipitated with
-Hop1p antibodies, Red1-3HAp was detected in the complex (Fig.
6B, lane 1).
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Fig. 6.
Hop1p and Red1-3HAp co-immunoprecipitation
from meiotic extracts. Cells from YTS3/pNH212
(MEK1/MEK1) and NH177/pNH212
(mek1 /mek1
) were sporulated for 3 h.
Hop1p and Red1-3HAp were immunoprecipitated from soluble extracts
prepared from these cells (2.8 mg for YTS3/pNH212 and 6.6 mg for
NH177/pNH212) using
-HA antibodies (panel A) or
-Hop1p
antibodies (panel B). Immunoprecipitates were then probed
with either
-HA (to detect Red1-3HA) or
-Hop1p antibodies as
noted beneath the pictures. The cross-reacting band at the
bottom of panel B, lanes 3 and
4 is because of detection of the antibody used in the
immunoprecipitation.
strain, NH177/pNH212. Because there is less soluble
Hop1p and Red1-3HAp in the absence of MEK1, twice as much
protein was used for the immunoprecipitation compared with the
MEK1 diploid. When the
-Hop1p antibodies were incubated with the protein extracts, nearly equivalent amounts of Hop1p were
precipitated from both strains (Fig. 6B, lanes 3 and 4). However, when these precipitates were probed with
the
-HA antibodies, a 4.3-fold reduction in the amount of Red1-3HAp
co-precipitating was observed with the mek1
extract (Fig.
6B, lane 2). In three experiments where the
amount of Red1-3HAp co-immunoprecipitating with Hop1 was quantitated,
a 4.3-, 4.1-, and 3.6-fold reduction was observed in the
mek1
diploid compared with MEK1. The reduction in the Red1-3HAp co-IP was not because of a lack of Red1-3HAp in the
mek1
diploid as evidenced by the amount of Red1-3HAp
present in the
-HA immunocomplexes from the mek1
diploid compared with MEK1 (Fig. 6A, lanes
1 and 2). The amount of Hop1p co-immunoprecipitating with Red1-3HAp varied from a reduction of 1.8- (the value for the
experiment shown in Fig. 6, lanes 3 and 4), 1.5-, to 0-fold change in the mek1
diploid compared with
MEK1. To rule out a loading artifact, blots probed with
-Hop1p antibodies were stripped and probed with
-HA antibodies
and vice versa, and the same results were
observed.3 The presence of MEK1 therefore
facilitates the Hop1p/Red1-3HAp interaction in vivo.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
diploids, spore viability is reduced to
1% in SK1 (27) compared with 15% in A364A (10). For hop1 mutants, spore viability is <1% in both strain
backgrounds, but no residual recombination is observed in SK1 (6, 31, 32). To interpret the biochemical results, it was therefore important
to determine whether the same dosage relationships between mek1-974 and overexpression of HOP1 and
RED1 observed in A364A exist in SK1.
a mutation that abolishes function in vivo. Therefore, the kinase
activity of Mek1p is an essential component of its meiotic function.
diploids suggest that Red1-3HAp may be the
substrate for Mek1p although the presence of an intermediate kinase
cannot be ruled out. In MEK1 strains, the Red1-3HA
phosphoprotein was observed only when the RED1-3HA allele
was overexpressed, suggesting that the presence of the HA tag may
partially inhibit the action of the kinase. The Red1-3HA
phosphoprotein is likely to represent the functional form of the
protein, as it is only when this species is present that
RED1 activity is restored in vivo.
diploids (17). In addition,
although the stoichiometry between Hop1p and Red1p is as yet
undetermined, there is usually more Red1-3HAp co-immunoprecipitating
with Hop1p than Hop1p that co-precipitates with Red1-3HA
(e.g. there is an 8-fold difference in Fig. 6). This is the
result expected if the ratio of Red1p:Hop1p is greater than 1:1. If
this is the case then when a molecule of Hop1p is precipitated, it may
bring down not only the phosphorylated Red1-3HAp with which it was
associated but also unphosphorylated Red1-3HAp which was interacting
with the Red1-3HA phosphoprotein.
diploid.
While it is possible to immunoprecipitate equivalent amounts of Hop1p
from meiotic extracts made from MEK1 and mek1
diploids, the level of Red1-3HAp co-precipitating was reduced
approximately 4-fold in the absence of MEK1. When Red1-3HAp
was immunoprecipitated from mek1
and MEK1
extracts, approximately 2-fold less Hop1p was observed in the complex.
The presence of MEK1 clearly promotes the Hop1p/Red1p
interaction. Whether hetero-oligomer formation is enhanced because the
Red1p-3HA phosphoprotein has decreased affinity for Red1-3HAp or an
increased affinity for Hop1p remains to be determined.
Co-immunoprecipitation of Hop1p and Red1-3HAp demonstrates that the
two proteins physically interact in meiotic cells, but these
experiments do not address whether the interactions are occurring on
chromosomes or not. Given that Hop1p and Red1p co-localize on AEs (28),
this idea seems likely.
is an
artifact resulting from the decreased levels of soluble Red1-3HAp
observed when MEK1 is absent. In the latter case, it may be
that the MEK1-dependent phosphorylation of Red1p
serves some other, as yet undefined, function during meiosis. One way
to distinguish between these two possibilities is to identify and
mutate the phosphorylation sites in Red1p and test to see if such
mutants have decreased affinity for Hop1p. Such studies are currently underway.
Comparison of S. cerevisiae Hop1p and Mek1p with different
potential homologs
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ACKNOWLEDGEMENTS |
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We thank Breck Byers, Neta Dean, JoAnne Engebrecht, and Aaron Neiman for helpful discussions and useful comments on the manuscript. We are also grateful to Ann Sutton and Simon Rudge for advice. Aaron Neiman provided the protein alignments given in Table IV. Neta Dean, Doug Kellogg, and Beth Rockmill provided plasmids and/or antibodies. We thank Lisa Ponte and Dana Woltering for excellent technical support.
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FOOTNOTES |
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* This work was supported by a grant from the Pew Charitable Trusts, as well as National Institutes of Health Grant GM50717.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 516-632-8581;
Fax: 516-632-8575; E-mail: nhollin{at}notes.cc.sunysb.edu.
The abbreviations used are: SC, synaptonemal complex; AE, axial element; HA, hemagglutinin; GST, glutathione S-transferase; PCR, polymerase chain reaction; kb, kilobase(s); PMSF, phenylmethylsulfonyl fluoride.
2 N. Hollingsworth, unpublished observations.
3 T. de los Santos, unpublished observations.
4 S. Larochelle and B. Suter, personal communication.
5 A. P. Caryl, F. C. H. Franklin, and G. H. Jones, personal communication.
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REFERENCES |
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