Investigation of MAPK Yeast Mating Pathway Unveils Potential Key Genes CDC73 and CLB6
Oswaldo Martinez
Department of Biology
Massachusetts Institute of Technology
December 12, 2022
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Abstract
Mitogen-activated protein kinase (MAPK) signal transduction pathways are vital for cell
survival and function in eukaryotic organisms. Dysregulation in MAPK pathways can lead to a
variety of human diseases such Parkinson’s disease, making it an important system to
understand. To study the pathway, we chose Saccharomyces cerevisiae, a eukaryotic organism
that relies on a MAPK pathway to undergo sexual mating, as a model organism to understand the
genetic components of the pathway. We mutagenized yeast and used orthogonal assays to find
haploid mutant strains that were unresponsive to ⍺-factor, the yeast mating pheromone, to
discover genes that are required for a mating response. We identified three key genes, Ste5,
CDC73, and CLB6, that play a role in S. cerevisiae’s mating pathway aiding in our overall
understanding of mammalian MAPK signaling pathways. Ste5 was a previously discovered gene
that transcribes a scaffold protein vital for the function of other parts of the pathway. CDC73 and
CLB6 seem to be indirectly affecting the MAPK pathway by affecting the cellular state yeast
cells are in. This inhibits the yeast’s ability to be in the G1 state, stopping from mating to be able
to occur.
Introduction
Saccharomyces cerevisiae is a eukaryotic organism with a genome of 12.8 megabase
pairs, containing approximately 6,000 genes1. S. cerevisiae is a widely used model organism due
to having many ideal properties for studying the eukaryotic genome such as low cost, short life
cycle, and similarities in basic cellular processes to other eukaryotic organisms. Due to the
similarity in cellular processes to mammalian cells, S. cerevisiae has become an integral
organism to understanding cellular functions and pathways. Particularly, we are interested in
studying S. cerevisiae’s mating pathway.
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S. cerevisiae can reproduce asexually and sexually depending on environmental factors
such as available resources or harsh living conditions. For asexual reproduction, yeast cells
replicate by undergoing mitosis where the mother cell divides to produce a daughter cell. This
division happens through the process of budding where the daughter cell starts as a small
growing bud on the mother cell until it grows and separates. This division then leaves a scar on
the cell membrane of the mother cell which can indicate the number of times a mother cell has
budded. Additional to asexual reproduction, diploid yeast can undergo a special state during
unfavorable environmental conditions where the cell will undergo meiosis and sporulation to
create four haploid spore cells2. In this state, the tetrad consisting of the four haploid spore cells
encased in the ascus, a covering for the cells, will not divide or grow. Once conditions are
favorable, the spore will lose the ascus and continue miotic growth and cell division as it was
doing beforehand.
Aside from asexual reproduction, S. cerevisiae can reproduce sexually in haploid state.
Sexual reproduction occurs when both types of haploid yeast are present, mating type a (MATa)
and mating type ⍺ (MAT⍺). Once both types of yeast are in vicinity of each other, the cells
secrete pheromones to signal mating. Once they receive the signal and make contact, they fuse to
form a diploid cell that can undergo meiosis3. The mating pheromone signaling pathway used by
S. cerevisiae is an important target for understanding mitogen-activated protein kinases as they
play a vital role in the pathway.
Mitogen-activated protein kinases (MAPKs) are kinases that mediate intracellular
signaling and are associated with many cellular processes such as cell survival, death,
proliferation, and differentiation4. Additionally, cells use MAPK signaling pathways to respond
to changing environmental conditions and other types of stimuli. MAPKs are vital for the
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function and survival of cells, and understanding the molecular mechanisms by which MAPKs
operate is important to our understanding of various human diseases. Many diseases such as
Alzheimer’s disease and Parkinson’s diseases are implicated with MAPK signaling pathways
illustrating the importance of understanding the S. cerevisiae mating pathway.
Through the use of a large-scale forward genetic screen, we will identify the key genes
that play a role in S. cerevisiae’s mating pathway which will illuminate our view on mammalian
MAPK signaling pathways. We will use transposon mutagenesis to create a variety of yeast
mutants and filter them through phenotypic assays, such as the halo and sporulation assay, to
determine which mutations are directly related to our target. We will then take our samples and
sequence them to directly find which genes are involved in the MAPK signaling pathway. This
will provide potential areas of interest to target diseases that are connect to this pathway.
Methods
mTn3 Trasnposon Digestion
We performed the mTn3 transposon digestion using the DNA library and plasmid template the
from Burns paper1. We used NotI as our restriction enzyme for the procedure. Our digestion mix
consisted of 1 µg of mTn3 transposon-mutagenized genomic library plasmid DNA, 1X CutSmart
Buffer from NEB, and 60 units of NotI-HF from NEB. We centrifuged the mix at 6000 rpm for
15 seconds, and placed them in a thermocycler at 16°C for 16 hours.
Gel Electrophoresis for Confirmation of Digestion
We ran a gel electrophoresis using 1x TAE buffer and a 0.8% agarose gel. We loaded a 1-kb
DNA ladder, an undigested control, and three digested samples into the gel.
Transformation of mTn3 Library into Yeast
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We transformed the yeast using a standard lithium acetate method5. The yeast strain used was
PPY295 (genotype is in Table 4). The transformation mixes consisted of 200 µg of salmon
sperm DNA and 0.8 ug of the corresponding NotI digested mTn3 DNA. We had two controls
with 200 µg of salmon sperm DNA and either 20 µL of H2O or 400 ng of YCplac111 plasmid.
The selection plates used were SC-Leu plates.
α-Factor Screening
To screen for α-factor resistant mutants, we used replica plating to transfer the mutant colonies
onto new plates. The plates used for screening were SC-Leu and SC-Leu plates with α-factor.
Additionally, we used colonies from these plates to streak onto new SC-Leu with α-factor plates
after sufficient growth time
Halo Assay
To continue screening our mutants for false positives, we conducted a halo assay. For each
potential α-factor resistant mutant, we divided a plate into 4 equal sections with a single filter
disk in each section, the cells were dispersed evenly on the plate using melted top agar. The filter
disks had 10 µL of varying α-factor concentrations applied to them, 0 M, 10-3 M, 10-4 M, and 105
M. Additionally, we had two control plates of non-mutated PPY295 and ste11Δ (genotype in
Table 4). All the plates were incubated in at 30°C for two days. To analyze the results, we used a
ruler to measure the diameters of the halos that appeared on the plates, with two measurements
being take 90° apart from each other. This measurement was averaged to find the final halo
diameter size.
Mating Assay
As another test for false positives, we conducted a mating assay. For yeast strains PPY295,
PPY144, and ste2Δ, we chose 10 colonies from each strain and resuspended them in 20 µL of
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YPD media. For the mating combinations, we did 3 types: PPY295 + PPY144, ste2Δ + PPY144,
and PPY144 + corresponding mutant (genotypes for PPY295, PPY144, and ste2Δ are in Table
4). For the three types of combinations, we added 5 µL of the corresponding sample into 20 µL
of YPD media and plated 5 µL of the mix. The plate used was a SC-His plate that was divided
into 11 equal parts, 8 combinations and the three yeast strains. The plate was incubated at 34°C
for two days. For analysis, we observed which combinations resulted in growth indicating
mating occurred.
Sporulation Assay
For the sporulation assay, we used a sporulation plate (1% Potassium acetate + 30 µg/mL
adenine, 20 µg/mL histidine, 60 µg/mL leucine, 30 µg/mL lysine, 40 µg/mL tryptophan, 20
µg/mL uracil). We divided the plate into 8 equal sections, two for PPY295 and PPY917, and 6
for our mutants (PPY295 and PPY917 genotypes are in Table 4). We collected two colonies
from each sample (plated on Sc-Leu + α-factor plates) and plated them in their corresponding
section. The plate was incubated at room temperature for a week. For analysis, we smeared a
sample of each section onto a glass slide with 5 µL of water and used a phase contrast
microscope at 40x magnification to count the number of tetrads and normal cells in view. We
then calculated a sporulation rate by dividing the number of tetras by the total number of cells
counted for each sample.
Shmoo Assay
We used yeast cells growing in log phase for the assay to obtain results quickly. We designed the
experiment to have a control for each mutation as well as PPY295 and ste11Δ (PPY295 and
ste11Δ genotypes are in Table 4). For α-factor treated samples, we added 20 µL of α-factor 10-3
M solution to 2 mL of liquid culture and incubated the tubes for two hours at 30°C. For analysis
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of the assay, we plated 5 µL of our sample onto glass slides and counted the amount of shmooing
cells and non-shmooing cells using a phase contrast microscope on 40x magnification. We then
calculated a shmoo formation rate by dividing the number of shmooing cells in each sample by
the total number of cells counted for that sample.
Yeast Transformation of the Recovery Plasmid
We transformed the yeast using a standard lithium acetate method5. For the recovery plasmid, we
used the pRSQ2 plasmid described in the Ross-Macdonald paper6. We induced our yeast cells to
grow into log phase for the procedure. For the strains used, we used our chosen mutants with a
“no DNA” control and “pCUG” control. The pCUG plasmid is 5.5kb long with URA3+, AmpR,
CEN, and ARS, we used 962.5 ng of the plasmid.
Genomic DNA Isolation
The method for yeast genomic isolation is the same as that described in Philippsen’s paper7.
DNA Purification
For purification, we used EcoRI-HF and CutSmart Buffer from NEB. For the digestion reactions,
we had a composition of 10 µg of our mutant gDNA, 1X CutSmart Buffer, and 20 Units of
EcoRI-HF. The tubes were centrifuged for 15 seconds at 6000 rpm and incubated in a
thermocycler at 37°C for 16 hours. We ran a 0.8% agarose gel to confirm that our genomic DNA
was successfully digested.
gDNA Ligation
For our ligation mix, we used 6.25 µg digested gDNA, 400 units of T4 DNA ligase, 0.25 mmol
of ATP, and 1X T4 DNA Ligase Buffer. The tubes were centrifuged for 15 seconds at 6000 rpm
and incubated in a thermocycler at 16°C for 16 hours.
Ligation Reaction Purification
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We used the protocol from the E.Z.N.A Cycle Pure manual (Omega 2012).
Inverse PCR Reaction
The primers used were primer lacZ: 5’ - gtaaccgtgcatctgcaag – 3’, and primer ori: 5’ –
ccatgattacgccaagctc - 3’. For our PCR mutant solutions, we used 1 µM of our primers, 1X
Phusion Master Mix, and 6.25 µg of purified ligated gDNA. We also had a no DNA control and
pRSQ2 control, using 99 ng of the plasmid. The thermocycler program was 95°C for 2 minutes,
repeated for a total of 30 times (95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90
seconds), 72°C for 10 minutes, and left to rest at 4°C indefinitely. We then ran a 0.8% agarose
gel to confirm our results. Additionally, we purified our PCR reaction using the E.Z.N.A Cycle
Pure Kit (Omega) protocol.
PCR Product Sequencing
We used the M13(-40) primer for sanger sequencing and sent our samples at a concentration of
20 ng/µL, measured using a spectrophotometer, and were sent to Quintara Biosciences. We
analyzed the sequencing using NCBI BLAST website.
Results
Sporulation Assay
After mutagenizing our yeast and plating various colonies. We selected 6 colonies to streak onto
plates and use to determine which of our 6 samples were ⍺-factor resistant and haploid. One of
the first steps we took was to conduct a sporulation assay to determine if our samples were
haploid as cells that were diploid would exhibit behavior familiar to ⍺-factor resistant yeast cells
while not being resistant. This would lead to false results for future steps. For the sporulation
assay results, we saw tetrads form in mutations 5 and 6 with sporulation rates of 0.24 and 0.059
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respectively (Table 1). While for mutations 1-4, we did not see any tetrads form. This indicates
that mutations 1-4 are haploid while mutations 5 and 6 contain diploid cells.
Filtering False Positives for ⍺-factor Resistance
To continue determine which mutation samples to use, we continued using orthogonal assays
with the shmoo assay being the next. This assay was used to determine if our mutation samples
were either ⍺-factor resistant or false positives. From our results, we see that shmoos formed in
our mutation 2 sample, with a shmoo formation rate of 0.3 (Table 2). This result leads us to
believe that mutation 2 may be a false positive for ⍺-factor resistance as shmoos formed in the
presence of ⍺-factor indicating there was a response from our yeast cells to it. Continuing our
process of eliminating false positive samples, we conducted a mating assay and halo assay to
continue testing our samples for ⍺-factor resistance. For the mating assay, we were looking to
see for growth when our mutants were in the presence of PPY144, which is able to make with
MATa yeast cells. From imaging the plate, we see growth in mutants 2, 5, and 6 which indicates
that these samples are false positive and do respond to ⍺-factor (Figure 1). We did not see any
growth in mutants 1, 3, and 4. For the halo assay, we again were looking for response to ⍺-factor
which would be visible by growth stoppage leading to the presence of halos. From our plates we
only saw halos in mutation 2, which were very light but still accounted for, which indicates that
the sample was somewhat responsive to ⍺-factor (Figure 2). Overall, looking at all of the
orthogonal assays, we determined that mutations 1, 3, and 4 were the best to use as they never
indicated in our assays that the samples were false-positives for ⍺-factor resistance or diploid
(Table 3).
Analyzing Inverse PCR Products from Plasmid Recovery
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For the next steps after determining which mutation strains to use, we conducted plasmid
recovery to obtain the mutated gene DNA. The schematic for how the plasmid recovery was
done as well as the ligation steps afterward is illustrated in Figure 4. After using plasmid
recovery, we conducted inverse PCR to amplify our target sequence and analyzed our results
with a gel electrophoresis. From our gel, we determined that the inverse PCR was partially
successful. From our PCR lanes for mutants 1, 3 and 4, we see that only mutation 3 and 4 has
some very light presence of DNA in the lanes while mutant 1 does not (Figure 3). This indicates
that the obtained DNA amount is very small and is not in abundance. However, this did not pose
a problem for sequencing and we were able to identify three genes: Ste5, CDC78, and CLB6.
Discussion
From our experimental process, we were able to identify 3 genes: Ste5, CDC73, and
CLB6. These genes were isolated from our mutants that passed our screening process from the
results of the orthogonal assays. We chose mutants 1, 3, and 4 to recover the gene from due to
the lack of response to ⍺-factor.
To start with Ste5, it is one of the “sterile” genes which are named after the sterile
phenotype mutations to these genes cause, and it is a scaffold protein in the MAPK pathway8.
This finding indicates that Ste5 is an integral part of the MAPK pathway as the phenotype we
saw was a lack of response to ⍺-factor in our mutant sample. Since Ste5 is a scaffold protein, the
interactions between other proteins in the pathway and Ste5 are vital for a response to occur once
the signal is received.
The next gene we isolated was CDC73, which is a component of the Paf1p complex and
modulates the activity of RNA polymerases 1 and 29. It is a vital protein for cell division, and
due to the importance of cell division response in mating, it is most likely affecting G1 cell cycle
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arrest. This effect would lead to the inability for the yeast cells to mate as they would not be able
to enter this phase to continue the cellular process of mating once they receive the ⍺-factor
pheromone. However, more work is needed to characterize the effects of CDC73 on the
MAPK signaling pathway.
The final gene we isolated was CLB6 which is a B-type cyclin that is involved in DNA
replication when the cell is in S phase10. CLB6 seems to be present in late G1 phase which is
when mating occurs in yeast cells. This means that mutations to this gene may disrupt the
function of the cell to respond properly to the mating pheromone. However, more work should
be done to understand the impact of the mutation on the overall MAPK pathway in the cell.
From the class data, we see that other groups have also identified a variety of “sterile”
genes such as STE2 and STE4 which all play a role in the MAPK pathway. This identification of
these genes provides an insight into the molecular machinery that yeast cells use to properly
respond to the mating pheromone. Moreover, this provides a starting point of genes to start
looking at when analyzing human samples that have MAPK disorders. However, with all of our
findings, there is uncertainty of whether the gene identified was the gene responsible for the
phenotype as there could have been multiple transposons that caused loss of function in different
genes.
From our overall findings, we have found potential targets for therapeutics that involve
mutations in the MAPK pathway. The specific characteristics of the proteins involved in the
pathway may illuminate broader understanding of signaling pathways, opening up possibilities
for targeted drug or gene therapy in afflicted patients with diseases such as Alzheimer’s or
Parkinson’s mentioned earlier. For further steps, finding methods to recover the wildtype
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phenotype in our mutant samples will provide a path to start potential treatments for these major
diseases as well as give more information on the specific mechanisms used in MAPK pathways.
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Colonies were plated on media to induce sporulation and were inspected under a phase contrast
microscope. Tetrads and non-tetrads were counted to calculate the sporulation rate. We observed
tetrad formation in mutations 5 and 6 indicating that the yeast colonies were diploid.
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Yeast cells were incubated for 2 hours with either ⍺-factor added at a concentration of 10-3 M or
without any ⍺-factor. After the two-hour incubation, samples were inspected under a phase
contrast microscope, and the amount of shmoos and normal cells were counted to determine a
shmoo formation rate for our samples. We observed shmoo formation in mutation 2 leading us to
determine that it is a false positive for ⍺-factor resistance.
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A summary of the data of all our orthogonal assays to determine which mutation samples to use
for future steps in determining which genes are connected to the target signaling pathway. A (+)
in the table indicates that the sample gave a response that indicates a false-positive. From the
results of our assays, we determined Mut 1, 3, and 4 to be the best candidates as they did not
exhibit any behavior for us to believe that they are false-positive for ⍺-factor resistance.
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Table 4: Yeast Genotypes
Yeast Type
Genotype
PPY295
MATa cry1 his4 leu2 lys2 trp1 tyr1 ura3 cyh2 SUP4-3 bar1-1
Ste11Δ
MATa cry1 ade2 ade3 his4 leu2 lys2 trp1 ura3 SUP4-3 ste11::hisG
PPY144
MATα ade1 arg4 aro2 his7 lys5 met4 ura2
Ste2Δ
MATa cry1 ade2 his4 leu2 lys2 trp1 tyr1 ura3 SUP4-3 ste2Δ::LEU2
PPY917
MATa/MATα cry1/cry1 ade2/ade2 ade3/ade3 his4/his4 leu2/leu2 lys2/lys2
trp1/trp1 ura3/ura3 SUP4-3/SUP4-3
A list of the different genotypes used in the orthogonal assays.
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Figure 1 Depiction of Mating Assay Results. A) The plate image we took of the SC-His plate
that was used for the mating assay. The X on the image depicts that no cells were plated in that
section. B) is a table showing the various strains that were plated in the different sections of our
plate. The mutants were plated with PPY144 to determine if they would respond to ⍺-factor.
Mutants 2, 5, and 6 responded to ɑ-factor showing that they were false-positives.
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Figure 2. Halo Assay Plates. The plate map depicts the general setup of the experiment where
varying amounts of ɑ-factor were plated in each sector through the use of 10 mm disks. The
image below are plate images of all our plates, depicting the 6 mutants and the controls. The only
mutant we saw with halos was Mut 2 which had light halos when treated with ⍺-factor.
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Figure 3. Gel Electrophoresis of the Inverse PCR Products. An image of the 0.8% agarose gel we
ran to determine if our inverse PCR products were produced. From the gel, we determined that
Mut 1 and Mut 4 had very low or no PCR product yield as they do not show up in the gel.
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Figure 4. Scheme for Plasmid Recovery.
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