Exploring the effects of YAK1 on cellular growth rates of Saccharomyces cerevisiae and
Saccharomyces bayanus
Project Definition:
The YAK1 gene found in the yeast Saccharomyces cerevisiae has been described as a
gene important to S. cerevisiae for sensing and responding to environmental stimuli and was first
identified more than twenty years ago (Garrett and Broach, 1989). The protein YAK1 encodes,
Yak1p, is a kinase. Kinases are a type of enzyme that alters the activity of other proteins. Yak1p
helps to decrease the growth rate of yeast when resources are scarce, allowing the organism to
focus its efforts on being more robust (Hartley et al., 1994). However, the specific molecular
mechanisms of this interaction remain poorly understood. Previous research has demonstrated
that overexpression of YAK1 in conjunction with other mutations causes cells to stop growing
completely (Garret et al., 1991). The North Central College Yeast Lab has expanded on these
observations to demonstrate that overexpression of YAK1 reduces the growth rates of yeast
strains who carry no additional mutations (Pratt et al., 2007). Loss of YAK1 dramatically
increases cell susceptibility to heat shock due to its role in the Ras signaling pathway (Hartley et
al., 1994).
YAK1 bears a striking resemblance to five proteins found in human cells, called the
DYRK family, which have been implicated in Down Syndrome pathology (Park et al. 2009),
Parkinson’s disease (Kim et al., 2006), cell death and cancer (Friedman, 2007). The DYRK
family is known to regulate other proteins and transcription factors, and DYRK1A in particular
was highly selected for in the evolutionary divergence occurring between humans and
Neanderthals (Green et al., 2010). Increasing our understanding of YAK1 in yeast may expand
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our knowledge of the human DYRK genes, including their role in human disease as well as their
evolutionary significance.
A close relative of S. cerevisiae, Saccharomyces bayanus, has a gene with YAK1
homology that has been rendered defective by the presence of a stop codon in the middle of
YAK1’s kinase domain. A stop codon would stop the cell machinery of S. bayanus in the
middle of producing the YAK1 protein. S. bayanus has been shown to produce the truncated
protein, which is missing most of the kinase domain and the entire C-terminus. Denoted
“Yak1a”, little is known regarding whether or not this protein has a function in S. bayanus. S.
cerevisiae and S. bayanus are thought to have descended from a common ancestor before
evolving into two separate species approximately 20 million years ago. S. bayanus fills a
different ecological niche than S. cerevisiae despite their close relation. S. cerevisiae grows
optimally at 30°C while feeding on dextrose, a fermentable carbon source, while S. bayanus
experiences optimal growth at 25°C while feeding on glycerol, a non-fermentable carbon source
(Lee et al., 2008). The North Central Yeast Lab has hypothesized that the removal of functional
YAK1 from S. bayanus contributed to the evolutionary phenomena that transformed one
ancestor yeast into the two daughter species S. cerevisiae and S. bayanus.
In order to explore YAK1’s function in S. cerevisiae and S. bayanus, we have genetically
modified YAK1 in both species with a variety of different modifications. We have altered
endogenous YAK1 in some strains and added YAK1 from other species. These YAK1 mutants
will be subjected to growth curve trials in order to determine their growth rates. Measuring the
growth rates of the mutants is an efficient way to determine YAK1’s effect on the organism’s
optimal niche. My research would attempt to answer several questions about YAK1’s role in
controlling growth rates:
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
Does giving S. bayanus a functional copy of S. cerevisiae YAK1 affect its growth rate?

How does disabling the kinase domain of S. cerevisiae YAK1 affect the growth of S.
cerevisiae with and without the presence of its normal YAK gene?

How does disabling the kinase domain of S. cerevisiae YAK1 and cloning it into S.
bayanus affect the growth of S. bayanus?

Does giving S. bayanus YAK1 to S. cerevisiae affect growth rates with and without its
own functional YAK1 gene?

How does giving both S. cerevisiae and S. bayanus only the C-terminus of S. cerevisiae
YAK1 affect growth rates?
Answering these questions would provide an expanded insight into the function of Yak1p
and possibly increase our understanding of the speciation event that led S. cerevisiae and S.
bayanus to occupy two different ecological niches. The process of constructing growth curves
for so many strain comparisons is very slow at North Central’s Yeast Lab: with the equipment
available here, I am only able to run one growth curve per week. I hope to accelerate the pace of
my project by pursuing it at the University of Minnesota, where Dr. Judy Berman has offered me
the opportunity to use an instrument that will increase my productivity by nearly 100-fold.
Methodology:
My proposal is to pursue these research questions at an expedited pace by using the
extensive resources available at the University of Minnesota. In order to better understand
YAK1’s role in controlling the growth rates of S. cerevisiae and S. bayanus, I will construct
growth curves comparing various S. cerevisiae and S. bayanus strains with various YAK1
mutations. A growth curve is constructed by using a spectrophotometer to measure the optical
density of an aqueous cell culture. Spectrophotometers pass light through a sample of the culture
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and measure the amount of light transmitted through the solution. From that measurement, one
can determine the approximate density of cells in the culture. Taking the measurements over
several time intervals and recording the optical densities of the culture at those times allows one
to construct a curve from which a growth rate can be determined. Yeast growth is logarithmic,
beginning with a phase of cell doubling until resources are depleted and the culture enters
stationary phase, where growth levels off. The slope of this line is the growth rate.
Figure 1: Example of an idealized growth curve set made by Mark McClellan with the Tecan
plate shaker/incubator/spectrophotometer, the instrument I will be using to construct my curves.
Constructing growth curves is a tedious exercise. Working on my project over the
summer, I was able to construct two growth curves, each taking a week respectively to complete.
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Dr. Berman has offered to allow me to use an instrument in her lab at the University of
Minnesota capable of running 96 growth curves at a time. This would greatly increase the
efficiency of my data collection, allowing me to run enormous sample sizes in comparatively
short time.
In order to answer multiple research questions simultaneously, I will be constructing
growth curves for a multitude of YAK mutants, half in S. cerevisiae and half in S. bayanus. The
mutants will be examined alongside wildtype yeast that have normal YAK1 as a control. In
order to allow for the inclusion of error bars in the growth curves, I will be running the assays in
triplicate to allow for statistical analyses of the results. The cultures will be grown at 22°C
(room temperature) and in dextrose, the preferred media of S. cerevisiae.
In order to attempt to address the multitude of questions I hope to answer, I will be using
a variety of yeast strains in my growth curve trials.
S. cerevisiae
364
wildtype
normal YAK1 control
1231
wildtype, ura+
Normal YAK1, allows growth
without uracil
1312
yak1::TRP
YAK1 deletion mutant
1232
pADH-ScYAK1
YAK1 high overexpression
mutant
1310
YEp-ScYAK1
YAK1 overexpression mutant
1311
YEp-Scyak1-k398r
YAK1 kinase-dead mutant
1306
YEp-SbYAK1
S. bayanus YAK1 along with
normal S. cerevisiae YAK1
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1326
YEp-ScYAK1-C
C-terminus of YAK1 added to
normal YAK1
1327
YEp-Scyak1- k398r -C
Kinase-dead YAK1 with YAK1
C-terminus and normal YAK1
1330
yak1::TRP YEp-ScYAK1-C
YAK1 deletion with YAK1 Cterminus
1331
yak1::TRP YEp-Scyak1k398r -C
YAK1 deletion mutant with
Kinase-dead YAK1 and YAK1
C-terminus
ySb8
wildtype
normal YAK1 control
ySb10
wildtype, ura+
normal YAK1, allows growth
without uracil
ySb12
yak1::HIS
YAK1 deletion mutant
ySb11
pADH-ScYAK1
High-expression cerevisiae
YAK1 along with normal
bayanus YAK1
ySb24
YEp-ScYAK1
cerevisiae YAK1 along with
normal bayanus YAK1
ySB25
YEp-Scyak1- k398r
cerevisiae YAK1 kinase-dead
mutant
ySb26
YEp-SbYAK1
YAK1 overexpression
ySb40
YCp-ScYAK1
Single copy of the S. cerevisiae
YAK1 gene
ySb42
YEp-ScYAK1-C
cerevisiae YAK1 C-terminus
ySb43
YEp-Scyak1- k398r -C
cerevisiae YAK1 C-terminus,
kinase-dead
S. bayanus
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Time permitting, I would also hope to run additional experiments using glycerol instead
of dextrose as the yeast’s carbon source in order to explore whether or not growth media plays a
role in YAK function. It might also be helpful to conduct further trials at 25 and 30°C, the
preferred temperatures of S. bayanus and S. cerevisiae, respectively. I plan to follow the
following research schedule during my two weeks at University of Minnesota:
December
Week 1
Week 2
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
5
6
7
8
9
10
11
Arrive in
Meet lab
Place
Berman lab
Berman
Berman lab
Minneapolis
staff, start
cultures in
work
lab work
work
cultures
Tecon
13
14
15
16
17
Berman
Berman
Berman lab
Collect
Leave
lab work
lab work
work
final
Minneapolis
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results
I discussed the schedule with Dr. Johnston, who believes the above timetable to be
reasonable for the trials I plan to run. The growth curves require some initial setup, but will run
automatically most of the time. In the mean time, I will be given work to do by one of Dr.
Berman’s lab technicians, Mark McClellan. I will collect the results of my experiment on my
final lab day and analyze the data in light of our previous research when I return to North Central.
Results:
The data I collect will be organized into a professional poster and interpreted in the light
of the North Central College Yeast Lab’s previous research into the relationship between YAK1
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and the speciation of S. cerevisiae and S. bayanus. The poster will be presented at the Rall
Symposium and NCUR.
Relevant Experience:
I have taken BIO 102 and I am currently enrolled in BIO 260. These two courses carry a
heavy emphasis on genetics and molecular biology, the two major themes I need to familiarize
myself with in order to complete this research project. In addition, I participated in the Summer
Research Program 2010 in Dr. Johnston’s Yeast Lab for six weeks. During this time, I was
introduced to my project when I began measuring the optical density of yeast cultures grown at a
variety of temperatures and with different carbon food sources. I made the plasmids used to
make Sc strain 1331 and Sb strain 43 and constructed initial growth curves that showed some
promise. I also attended the Midwest Yeast Meeting 2010 with Dr. Johnston in order to watch
other yeast geneticists present their work, where I was introduced to Dr. Berman and offered the
opportunity to work in her lab over D-term. The Midwest Yeast Meeting was a valuable
experience because I was able to watch a plethora of professional presentations of research in the
field of study my project falls into.
Personal Application:
Based on my experience with summer research, I am considering research as my primary
career interest and I’m planning on attending graduate school. Completing this project at Dr.
Berman’s lab will allow me to experience graduate school before I actually get there. I want to
make sure that working in a lab is in fact the career I want to pursue, something I can only know
by learning firsthand what a big research lab is like. In order to give me the full graduate school
experience, Dr. Berman will be handing me tasks related to her research in addition to my own
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project, allowing me to try out other techniques and familiarize myself with instruments not
available at the North Central lab.
References:
Friedman, E. 2007. Mirk/Dyrk1B in cancer. J. Cell. Biochem. 102:274-279.
The author, Eileen Friedman, received her PhD at Johns Hopkins University and is a
professor of pathology at Upstate Medical University in New York. The article discusses
the Mirk/Dyrk1B gene’s function in the cellular repair of cancerous tumors. This article
was useful in finding relevant medical concerns for the study of YAK1, which shares
homology with the DYRK family.
Garret, S. and J. Broach. 1989. Loss of Ras activity in Saccharomyces cerevisiae is suppressed
by disruptions of a new kinase gene, YAK1, whose product may act downstream of the
cAMP-dependent protein kinase. Genes Dev 3: 1336-48.
The bulk of the article revolves around the discovery of YAK1’s role in the RAS/cAMP
signal transduction pathway and the construction of a tentative model to characterize how
it functions. The preliminary model presented here lead to the author’s second YAK1centered paper, which further characterizes YAK1’s role in the regulation of growth rates.
Garret, S., M.M. Menold and J.R. Broach. 1991. The Saccharomyces cerevisiae YAK1 gene
encodes a protein kinase that is induced by arrest early in the cell cycle. Mol Cell Biol 11:
4045-52.
The follow-up article to Garrett’s previous work on YAK1 firmly establishes YAK1 as a
protein kinase working in opposition to A kinase to regulate yeast growth. This article is
important for its characterization of YAK1’s function in S. cerevisiae as a negative
growth regulator.
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Green, R.E., J. Krause, A.W. Briggs, T. Maricic, U. Stenzel, M. Kircher, N. Patterson, H. Li, W.
Zhai, M.H.-Y. Fritz, N.F. Hansen, E.Y. Durand, A.-S. Malaspinas, J.D. Jensen, T.
Marques-Bonet, C. Alkan, K. Prüfer, M. Meyer, H.A. Burbano, J.M. Good, R. Schultz, A.
Aximu-Petri, A. Butthof, B. Höber, B. Höffner, M. Siegemund, A. Weihmann, C.
Nusbaum, E.S. Lander, C. Russ, N. Novod, J. Affourtit, M. Egholm, C. Verna, P. Rudan,
D. Brajkovic, Z. Kucan, I. Gusic, V.B. Doronichev, L.V. Golovanova, C. Lalueza-Fox, M.
de la Rasilla, J. Fortea, A Rosas, R.W. Schmitz, P.L.F. Johnson, E.E. Eichler, D. Falush,
E. Birney, J.C. Mullikin, M. Slatkin, R. Nielson, J. Kelso, M. Lachmann, D. Reich, and S.
Pääbo. 2010. A draft sequence of the Neandertal genome. Science 328: 710-722.
This publication presents the results of a genomic sequencing of the Neandertal genome
from three individuals, allowing the researchers to identify genes important in the
evolution of Neandertals into humans. DYRK1A is mentioned among these genes, a
YAK1 homolog in humans implicated in Downs syndrome, another point of medical
relevance for the project.
Hartley, A.D., M.P. Ward, and S. Garrett. 1994. The Yak1 protein kinase of Saccharomyces
cerevisiae moderates thermotolerance and inhibits growth by an Sch9 protein kinaseindependent mechanism. Genetics 136, 465-74.
The article demonstrates that YAK1 has a key role in the development of heat resistance
and has a broader role than simply suppressing the A kinase growth defect in S.
cerevisiae. It is important for my project because it elaborates on YAK1’s role in
responding to environmental stimuli and further develops its function in S. cerevisiae.
Kim, E.J., J.Y. Sung, H.J. Lee, H. Rhim, M. Hasegawa, T. Iwatsubo, D.S. Min, J. Kim, S.R. Paik,
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K.C. Chung. 2006. Dyrk1a phosphorylates α-synuclein and enhances intracellular
inclusion formation. J. Biol. Chem. 281: 33250-33257.
This article suggests that Dyrk1A regulates α-synuclein through phosphorylation in
immortalized hippocampal neuronal cells, sometimes leading to the formation of
neurotoxic aggregate bodies and cell death. The findings implicate DYRK1A in
Parkinson’s disease, and YAK1 shares strong homology with the DYRK family.
Lee, H.-Y., J.-Y. Chou, L. Cheong, N.-H. Chang, S.-Y. Yang and J.-Y. Leu. 2008.
Incompatibility of nuclear and mitochondrial genomes causes hybrid sterility between
two yeast species. Cell. 135:1065-1073.
Lee et al. work to describe the evolutionary divergence of S. cerevisiae and S. bayanus in
their article, citing differences in mitochondrial DNA as the reason the two species
cannot interbreed and produce viable offspring. This article was essential to my project
because it is of paramount importance to understand the established evolutionary
background of two species before trying to expand on it.
Park, J., Y. Oh and K.C. Chung. 2009. Two key genes closely implicated with the
neuropathological characteristics of Down syndrome: DYRK1A and RCAN1. BMB Rep.
42:6-15.
Park et al. explore the roles two genes, DYRK1A and RCAN1, play in Down syndrome
pathology in hopes of better understanding the molecular mechanisms of the disease.
YAK1’s homology to DYRK1A adds medical relevance to my project background.
Pratt, Z.L., B.J. Drehman, M.E. Miller and S.D. Johnston. 2007. Mutual interdependence of
MSI1 (CAC3) and YAK1 in Saccharomyces cerevisiae. J. Mol. Biol. 368:30-43.
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This article identifies several new interactions between the S. cerevisiae genes MSI1 and
YAK1, expanding our knowledge of YAK1’s function in S. cerevisiae. It was published
out of the North Central College Yeast Lab and provides valuable insight into the
developmental history of my project.
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