Analysis of cAMP signaling in Saccharomyces cerevisiae using
sugar transitions
Abstract
The cAMP pathway in Saccharomyces cerevisiae is activated upon addition of a sugar and
has many downstream effects on the cell. Activation of this pathway has already been
studied on cells growing in ethanol, but not in cells growing on a sugar. Here we analyze
yeast cells using flow cytometry that had been growing on trehalose and transition them to a
different sugar in order to figure out if this still activates the cAMP signalling. This was done
by analysing WT cells with yEPAC, a FRET-based biosensor. Cells were transitioned to
fructose, galactose, glucose, sucrose, and xylose. Results show that cells give the highest
cAMP response to sucrose, and therefore, sucrose is the most preferred carbon source of
Saccharomyces cerevisiae to grow on.
Introduction
Saccharomyces cerevisiae, better known as budding yeast, are very versatile organisms
especially because their adaptation mechanisms to environmental changes are very well
developed [1]. Environmental changes such as variation in nutrients are sensed by yeast by
numerous specialized cellular receptors that sense nutrient concentration changes [2]. One
of such pathways is the cAMP-PKA pathway, where cAMP gets activated upon addition of a
carbon source that can be used in glycolysis, resulting in activation of PKA. PKA activation
leads to many downstream effects such as inhibition of stress response [3]. Recent paper by
Botman et al. [4] has investigated yeast cells in fermentation, where they grow on ethanol,
transitioning to many different environments. Although literature lacks information about
yeast in different types of sugar. Therefore this experiment investigates the adaptation
mechanism of yeast cells to several sugars such as fructose, galactose, glucose, sucrose,
and xylose. The aim is to figure out if the peak seen in past analysis comes from the
transition from fermentation to glycolysis or from the transition from a sugar the cells grow
slower on to an easier for them to grow on sugar. To achieve this, the wild type strain WT
(303-1A) + yEPAC was used which contains several auxotrophs such as leucine,
tryptophan, uracil, adenine and histidine. The yEPAC part is a FRET-based biosensor for
cAMP measurements in yeast and therefore will give a peak in flow cytometer analysis after
adding alternative sugars. A CYR1 negative control was used which is almost in the last step
of the cAMP-PKA pathway and has no step parallel to it and can therefore inhibit the whole
stress response. In the end, the cAMP levels were measured using a flow cytometer to see
the response to the changing environment.
Materials and Methods
Controls
A positive and negative control were used for the experiment. The positive control was a WT
(W303-1A) + yEPAC strain with active cAMP signalling. This strain has several auxotrophs
such as leucine, tryptophan, uracil, adenine and histidine. The yEPAC plasmid was acquired
based on Lithium Acetate yeast transformation in Tutucci [5], and contained a uracil marker.
As a negative control, a CYR1 mutant was used, as this makes it so there is no cAMP
signalling and therefore no peak in flow cytometer analysis after adding alternative sugars.
The WT + mTQ2 strain was used as a negative control for the overlap between red and blue
fluorescence.
Sugars
Several sugars which were going to be used for sugar transitions in the flow cytometer were
dissolved in advance: fructose, galactose, glucose, sucrose, and xylose. All the sugars were
dissolved in a yeast nitrogen base (YNB), in which all auxotrophs are covered except for
uracil, with a stock 1.11x concentration, 9.009 mL was used to get a final concentration of
1x.
Every time 1000 mM YNB was taken to dissolve 1.8016 g of the sugars glucose, fructose
and galactose, and 1.5013 g xylose and 3.4230 g galactose. These amounts are based on
the molecular weights, to get a final concentration of 1M in each solution.
1
sugar transitions
Abstract
The cAMP pathway in Saccharomyces cerevisiae is activated upon addition of a sugar and
has many downstream effects on the cell. Activation of this pathway has already been
studied on cells growing in ethanol, but not in cells growing on a sugar. Here we analyze
yeast cells using flow cytometry that had been growing on trehalose and transition them to a
different sugar in order to figure out if this still activates the cAMP signalling. This was done
by analysing WT cells with yEPAC, a FRET-based biosensor. Cells were transitioned to
fructose, galactose, glucose, sucrose, and xylose. Results show that cells give the highest
cAMP response to sucrose, and therefore, sucrose is the most preferred carbon source of
Saccharomyces cerevisiae to grow on.
Introduction
Saccharomyces cerevisiae, better known as budding yeast, are very versatile organisms
especially because their adaptation mechanisms to environmental changes are very well
developed [1]. Environmental changes such as variation in nutrients are sensed by yeast by
numerous specialized cellular receptors that sense nutrient concentration changes [2]. One
of such pathways is the cAMP-PKA pathway, where cAMP gets activated upon addition of a
carbon source that can be used in glycolysis, resulting in activation of PKA. PKA activation
leads to many downstream effects such as inhibition of stress response [3]. Recent paper by
Botman et al. [4] has investigated yeast cells in fermentation, where they grow on ethanol,
transitioning to many different environments. Although literature lacks information about
yeast in different types of sugar. Therefore this experiment investigates the adaptation
mechanism of yeast cells to several sugars such as fructose, galactose, glucose, sucrose,
and xylose. The aim is to figure out if the peak seen in past analysis comes from the
transition from fermentation to glycolysis or from the transition from a sugar the cells grow
slower on to an easier for them to grow on sugar. To achieve this, the wild type strain WT
(303-1A) + yEPAC was used which contains several auxotrophs such as leucine,
tryptophan, uracil, adenine and histidine. The yEPAC part is a FRET-based biosensor for
cAMP measurements in yeast and therefore will give a peak in flow cytometer analysis after
adding alternative sugars. A CYR1 negative control was used which is almost in the last step
of the cAMP-PKA pathway and has no step parallel to it and can therefore inhibit the whole
stress response. In the end, the cAMP levels were measured using a flow cytometer to see
the response to the changing environment.
Materials and Methods
Controls
A positive and negative control were used for the experiment. The positive control was a WT
(W303-1A) + yEPAC strain with active cAMP signalling. This strain has several auxotrophs
such as leucine, tryptophan, uracil, adenine and histidine. The yEPAC plasmid was acquired
based on Lithium Acetate yeast transformation in Tutucci [5], and contained a uracil marker.
As a negative control, a CYR1 mutant was used, as this makes it so there is no cAMP
signalling and therefore no peak in flow cytometer analysis after adding alternative sugars.
The WT + mTQ2 strain was used as a negative control for the overlap between red and blue
fluorescence.
Sugars
Several sugars which were going to be used for sugar transitions in the flow cytometer were
dissolved in advance: fructose, galactose, glucose, sucrose, and xylose. All the sugars were
dissolved in a yeast nitrogen base (YNB), in which all auxotrophs are covered except for
uracil, with a stock 1.11x concentration, 9.009 mL was used to get a final concentration of
1x.
Every time 1000 mM YNB was taken to dissolve 1.8016 g of the sugars glucose, fructose
and galactose, and 1.5013 g xylose and 3.4230 g galactose. These amounts are based on
the molecular weights, to get a final concentration of 1M in each solution.
1
