examining the agonistic and antagonistic effects of ... · change that releases the associated...
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Hodgson 1
Examining the agonistic and antagonistic effects of various sugars on the surface receptor
protein Gpr1p in Saccharomyces cerevisiae.
an Honors Thesis submitted by:
William D. Hodgson
134 Davis Hollow Rd.
Elizabethton, TN 37643
(423) 291-1799
in partial fulfillment for the degree
Bachelor of Science with Honors
April 27, 2011
Project Advisor: Dr. Stephen Wright
© 2011 William D. Hodgson
Hodgson 2
Table of Contents Page
Abstract 3
Introduction 4
Materials and Methods 10
Results 14
Discussion 27
Acknowledgements 34
References 35
Hodgson 3
Abstract
The purpose of this project is to develop a reliable assay that quantifies cAMP production
in the yeast Saccharomyces cerevisiae in hopes of examining the effects of various sugars as
either agonist or antagonist ligands to Gpr1p. Additionally, the data produced by a cell size
analysis in conjunction with stimulation by sugars gives further evidence for the activity of
Gpr1p. Over the course of this project, a cAMP extraction protocol was developed and used in
conjunction with an ELISA assay to measure the cAMP produced. Activation of the glucose
sensing pathway in Saccharomyces cerevisiae is known to produce a transient increase in cAMP
production so the assay for this nucleotide must be quantifiable. Although it is clear that cAMP
was extracted by our methods, the protocol in this project yields results that are not consistent
with other studies. Since cell size is regulated by Gpr1p, a cell size analysis was also employed
in this project as an indirect measure for Gpr1p activity in wild type and GPR1 delete cells when
exposed to various sugars.
Hodgson 4
Introduction
Saccharomyces cerevisiae, otherwise known as brewer’s yeast, is a single-celled
eukaryotic organism useful for scientific study. This species possesses a class of cell surface
receptors also found in other eukaryotic species known as G-protein coupled receptors (GPCR).
GPCRs are surface proteins that regulate a wide variety of intracellular responses through the
process of signal transduction. Known to be conserved within a variety of species, these proteins
can be used to detect extra-cellular signaling agents such as light, hormones, and drugs (Filmore,
Dohlman).
Saccharomyces cerevisiae possesses two types of GPCRs, the Ste proteins and the
surface receptor Gpr1p. Ste2p and Ste3p are utilized in the detection of the pheromones a-factor
or α-factor. Mating type a (or MATa) cells possess the α-factor receptor Ste-2, and MAT α cells
possess the α-factor receptor Ste-3 (Lemaire et al). Gpr1p, the other surface G-protein found in
Saccharomyces cerevisiae, functions to detect the presence of nutrients in the cells’ environment
(Nakayama et al.). Nutrient sensing is likely to be a vital component in the mating process of this
species. As an energy demanding process, fusion between these single-celled organisms requires
the yeast to have the proper cellular machinery that recognizes the abundance of nutrients their
media as well as the ability to import these nutrients and utilize them.
Hodgson 5
Figure 1. Overview of glucose and pheromone sensing in S. cerevisiae (Versele et al., 2001).
Each surface receptor protein has an associated G-protein that functions inside the cell.
The mating pathways in Saccharomyces cerevesiae are initiated by the Gpa1 protein, the G-
protein associated with the Ste proteins (Nakayama et al, Versele et al.) The pathway
responsible for increasing metabolism upon the detection of sugars is dependent on the
intracellular G-protein Gpa2, associated with receptor Gpr1p (Versele et al.) G-proteins function
as guanine nucleotide exchange factors on the interior cell surface. Upon agonist ligand binding,
the receptor that they associate with undergoes a conformational change that is conveyed to the
G protein. This, in turn, results in a GDP exchange for GTP on the Gα subunit, subsequently
activating it (Johnston, Siderovski). Activation of the Gα subunit also induces a conformational
change that releases the associated Gβγ dimer that, in turn, activates various second messengers
Hodgson 6
within the cell (Figure 2, Johnston, Siderovski). Hydrolysis of GTP to GDP by the Gα subunit
inactivates the signaling cascade (Johnston, Siderovski). Gpa2, unlike Gpa1, has no known
subunits.
Figure 2. G-protein activation (Johnston, Siderovski)
In Saccharomyces cerevesiae, cAMP is used as an intracellular messenger to activate
protein kinase A, which is involved in cell metabolism, stress resistance, and reproduction
(Versele et al). Ras1 and Ras2 both have guanine nucleotide exchange factors, Cdc25 and Sdc25
are also known to regulate adenylate cyclase (Versele et al.) and Ras 1 and Ras 2 are necessary
to produce basal levels of cAMP production in Saccharomyces cerevisiae, but are not used in
glucose-dependant cAMP production (Versele et al, Colombo et al.). The glucose-sensing
pathway must be initiated by the presence of fermentable sugars in the cells’ environment or by
intracellular acidification (Versele et al, Thevelein et al.). The glucose sensed by the cell must
be phosphorylated in order to produce a rapid increase in cAMP (Lemaire et al.). The activation
of adenylate cyclase causes cAMP to accumulate within the cell, usually within seconds to
minutes (Lemaire et al.). Adenylate cyclase is the enzyme within the cell responsible for cAMP
production and can be regulated by several other proteins. Glucose-dependant stimulation of
cAMP production is regulated by a pathway that involves the surface receptor Gpr1p, its
Hodgson 7
associated G protein (Gpa2) and a signaling cascade that activates adenylate cyclase (Figure 3)
(Versele et al, Colombo et al., Kraakman et al.).
Figure 3. Activation of adenylate cyclase (Mol. Biology of the Cell, 3rdEd.)
Most G protein surface receptors have agonist and antagonist ligands that can bind to
them and induce or prevent function. Without an agonist, basal receptor activity is determined
by the equilibrium between the active and inactive states. Ligands affect this equilibrium,
possibly inducing a conformational change in the receptor, thereby changing its state (Dosil et
al.). As with most GPCRs studied to date, Gpr1p is thought to have seven trans-membrane
domains. The sixth trans-membrane domain plays a significant role in ligand binding and the
Hodgson 8
resultant conformational change in Gpr1p after binding (Lemaire et al.). To date, the ligands
glucose and sucrose have been shown to produce agonistic effects upon Gpr1p, and mannose has
been shown to produce antagonistic effects (Lemaire et. al). Several sugars have already been
tested for their agonistic and antagonistic activity upon Gpr1p. Among the ones tested to have
antagonistic function for Gpr1p include mannose, fructose, galactose, trehalose, turanose, and
palatinose (Lemaire et al.). Mannose is a strong antagonist and differs from the previous sugars
in a hydroxyl group at its second carbon, which is in an axial, instead of equatorial position. Due
to this isomeric change, mannose binds but does not activate Gpr1p. 2-deoxyglucose shares this
inability to activate gpr1p. Thus, Gpr1p has a high degree of specificity for its ligands (Lemaire
et al.). Further experimental evidence suggests that other sugars do not stimulate cAMP
production (Figure 4).
Ligand Antagonist
Function
Neither Agonist
nor Antagonist
Agonist
Function
Glucose *
Sucrose *
Mannose *
2-deoxyglucose *
Trehalose *
Turanose *
Palatinose *
Galactose *
Maltose *
6-deoxyglucose *
Hodgson 9
Figure 4. Agonistic and antagonistic effects of various sugars on Gpr1p (Lemaire et
al.)
Gpr1p has been shown to have substantially different affinities for sucrose and glucose
(Lemaire et al.). The effective concentration that affects 50% of cells (EC-50) ranges from about
20 to 75 mM for glucose as a ligand. However, the EC-50 for sucrose is around .5mM (Lemaire
et al.). One possible explanation that has been offered for this is based upon the glucose rich
environment that Saccharomyces cerevisiae usually resides in. However, when glucose is in
short supply, Gpr1p also serves to detect low concentrations of sucrose in order to ensure
viability, thus explaining the affinities for each sugar (Lemaire et al.)
In addition to cAMP production, cell size is also related to stimulation of Gpr1p.
Saccharomyces cerevisiae possesses regulatory mechanisms that coordinate its growth during the
cell division cycle (Johnston et al.). Before the initiation of budding, the cells must attain a
critical size required for different growth conditions (Johnston et al., Lorincz). Generation times
for growth of cells to critical size range from 2.1 to 3 hours (Johnston et al.). Additionally, cell
size is related to the kind of carbon source present in the cells’ medium (Tamaki et al.). The
surface receptor protein Gpr1p and its cognitive G protein Gpa2 have been speculated to be
responsible for modulation of the changes in cell size (Tamaki et al.). This coincides with the
findings that cells with mutant cAMP pathways have smaller volumes (Tamaki et al.), indicating
that the cAMP pathway is related to cell size modulation. In previous studies, GPR1 and GPA2
delete cells have also displayed smaller cell sizes than wild type cells when grown in the
presence of glucose (Tamaki et al.) Glucose is known to increase intracellular cAMP levels,
causing a rapid initial spike upon glucose exposure. (Beullens et al.) The cAMP pathway
Hodgson 10
regulates cell size through the activity of G1 cyclins (Tamaki et al.) Furthermore, Gpr1p and
Gpa2 are required to maintain a large cell size, and cells grown in the presence of glucose
display a larger volume (Tamaki et al.).
The purpose of this project is to develop a reliable assay that quantifies cAMP production
in Saccharomyces cerevisiae in hopes of examining the effects of various sugars as either agonist
or antagonist ligands to Gpr1p. Additionally, the data produced by a cell size analysis in
conjunction with stimulation by sugars gives further evidence for the activity of Gpr1p. The
sugars used are glucose, mannose, galactose, sucrose, raffinose, and fructose. Measuring the
production of cAMP provides a direct way of examining the activity of Gpr1p. The methods and
results from this project give insight into developing a more efficient way to accomplish
successful cAMP extraction procedures. The cell size analysis also provides further evidence for
the activity of Gpr1p.
Materials and methods
Cells and media
The cells used in this study included Saccharomyces cerevisiae BY4741 wild type,
BY4741 GPR1 delete, BY4741 GPA2 delete, BY4741 GPR1 delete /Gpa2 constitutively active.
All cells were streaked on plates with YPD media (10g Yeast extract, 20 g Peptone, 20g
Dextrose, 2% agar). Prior to stimulation and cAMP extraction, cells were incubated in liquid
YPEG media (10g Yeast extract, 20g Peptone, 2% ethanol (20 g), and 2% glycerol (20g).
cAMP extraction protocol
The following cells were incubated in liquid YPEG media for 48 hours: Saccharomyces
cerevisiae BY4741 wild type, BY4741 GPR1 delete, BY4741 GPA2 delete, and BY4741 GPR1
delete /Gpa2 constitutively active. The cells were then separated into tubes containing 24
Hodgson 11
million cells per tube by measuring the OD600 of 150 µL from each tube and determining the
appropriate volume to extract from each tube to produce 24 million cells. The cells were then
centrifuged at 1700 x g in a refrigerated centrifuge at 5 °C for 5 minutes and re-suspended in
1260 µL of cold 25 mM MES buffer for 10 minutes. Cells and buffer (315 µL) were extracted
and placed in 5 ml of cold, 60% methanol and placed in a -20 °C freezer for 48 hours. The
appropriate sugar (95 µL) was then added to each tube. For tubes with multiple sugars, 95 µL of
each sugar was added to the tube. Cells were stimulated for four different time points. Initially,
the cells were left in buffer with sugar for 1, 2, 3, and 4 minutes. The cells were stimulated for
30 seconds, 1 minute, and 1 minute 30 seconds for the rest of the extraction procedures. After
the appropriate amount of stimulation, 315 µL of cells and buffer were transferred to cold 60%
methanol and stored for 48 hours. The cells were then centrifuged at 1700 x g for 5 minutes in a
refrigerated centrifuge at 5 °C for 5 minutes and the supernatant was removed. Trichloroacetic
acid (500 µL) was added to the tubes and the pellets were disrupted using vortexing. The
mixtures were then transferred to eppendorf tubes containing .5 ml of .5 mm glass beads. The
tubes were vortexed for 1 minute and put on ice, and this was repeated 8 times. The tubes were
then centrifuged at 3000 x g in a refrigerated centrifuge at 5 °C for 3 minutes and the supernatant
was removed to tubes on ice. Potassium carbonate (5M) was added to each tube until the pH
was 8, and tubes were tested with pHydrion pH paper. Each tube was left open until the mixture
stopped producing gas. The tubes were centrifuged at 3000 x g in a refrigerated centrifuge at 5
°C for 3 minutes and the supernatant was transferred to new eppendorf tubes. Concentrated
hydrochloric acid was then added to each tube until the pH was 6; each mixture was also tested
using pHydrion pH paper. Tris buffer (20 µL pH 7.5) was then added to each tube and the tubes
Hodgson 12
were centrifuged at 3000 x g in a refrigerated centrifuge at 5 °C for 3 minutes and the
supernatant was frozen in a -20 °C freezer and kept for analysis.
Cell size analysis protocol
Saccharomyces cerevisiae BY4741 wild type, BY4741 GPR1 delete, BY4741 GPA2
delete, BY4741 GPR1 delete /GPA2 constitutively active were used. All cells were streaked on
plates with YPD media (10g Yeast extract, 20 g Peptone, 20g Dextrose, 2% agar). Prior to
stimulation, cAMP extraction, and cell size analysis, cells were incubated in liquid YPEG media
(10g Yeast extract, 20g Peptone, 2% ethanol (20 g), and 2% glycerol (20g) for 48 hours.
The following cells were incubated in liquid YPEG media for 48 hours: Saccharomyces
cerevisiae BY4741 wild type, and BY4741 GPR1 delete. The cells were then separated into
tubes containing 12 million cells per tube by measuring the OD600 of 150 µl from each tube and
determining the appropriate volume to extract from each tube to produce 12 million cells. The
cells were then centrifuged at 1700 x g in a refrigerated centrifuge at 5 °C for 5 minutes and re-
suspended in 1260 µl of cold 25 mM MES buffer for 10 minutes. Cells and buffer (252 µl) was
extracted and placed in cold 90% methanol and stored at -20 °C for further analysis. The cells
were then stimulated with 95 µl of the appropriate sugar. For tubes with multiple sugars, 95 µl
of each sugar was added to each tube. Cells were then left in a shaker at room temperature, and
252 µl of cells and buffer mixture was extracted and transferred to cold 90% methanol after 2
hours 15 minutes, 2 hours 45 minutes, and 3 hours 15 minutes. The cells and methanol were
then stored at -20 °C for further analysis.
To analyze the changes in cell size, the cells in 90% methanol were vortexed for 1 minute
to break up pellets and clumps. Cells in methanol (10 µl) was then extracted and placed on a
hemocytometer and examined at 400X under a microscope. Each slide was then photographed
Hodgson 13
for analysis. To measure changes in cell size, each photograph was enlarged a fixed amount and
the diameter in mm of each individual cell in the photograph was measured. The average cell
diameter in mm as well as the average area of in mm² of each cell type was obtained and
compared to the previous sizes.
ELISA assay
The following is taken from R&D Systems ParameterTM
cAMP:
primary antibody solution (50 µL) was added to all wells except zero standard well. The wells
were covered and incubated at room temperature for 1 hour on a horizontal orbital microplate
shaker set at 500 rpm. Each well was washed using 300 µL of wash buffer (1X concentrate)
provided in the ParameterTM
cAMP kit. This was process was performed 4 times, and the plate
was inverted and blotted between each wash. Standard, control, or sample (100 µL) was added
to appropriate wells. Calibrator diluent (100 µL) RD5-55 (provided in the ParameterTM
cAMP
kit) was added to the zero standard wells. cAMP conjugate (50 µL) was added to all wells. The
wells were then covered and incubated for 2 hours on a horizontal shaker. The wells were then
washed 4 times using the same wash process mentioned above. The substrate solution was
prepared by mixing equal amounts of color reagents A and B (provided by ParameterTM
cAMP
kit), and 200 µL of solution was added to each well. The plate was incubated for 30 minutes at
room temperature and protected from light. Stop solution (100 µL, provided by ParameterTM
cAMP kit) was added to each well. Optical density was determined within 30 minutes using a
microplate reader set to 495 nm.
Protein Determination
Cell extract (10 µL) was removed from each sample. A Bradford reagent dye was added
(200 µL) and the absorbance was measured at 600 nm after 2 minutes. Serial dilutions of Bovine
Hodgson 14
Serum Albumin were treated similarly and used to generate a standard curve. The amount of
cAMP compared to the amount of total protein was then determined to provide a means for
future standardization procedures.
Results
cAMP extraction/ELISA
Activation of the glucose sensing pathway produces a transient increase in cAMP
production (Lemaire et al.), so an efficient and quantitative assay for this nucleotide is needed.
This project used a cAMP extraction protocol and an ELISA assay to quantify cAMP, to indicate
the activity of Gpr1p.
To examine the effectiveness of the ELISA assay, an ELISA was run using a series of
known cAMP concentrations to generate a standard curve. The cAMP concentrations used,
along with the standard curve can be seen below in (Figure 5). This assay exhibited a linear
dose-response relationship with a high correlation coefficient.
Figure 5. Known cAMP concentrations were tested using the ELISA protocol to determine
its validity and generate a standard curve
Hodgson 15
An initial stimulation and cAMP extraction protocol was run to examine the effects of
glucose, a known GPR1p agonist, on cAMP levels in Saccharomyces cerevisiae cells. In this
experiment, we tested extracts of the samples of cells stimulated with sugar for cAMP
concentration. GPR1 delete and GPA2 delete cells were used as negative controls while cells
with constitutively active GPA2 were used as a positive control. All cells were stimulated with
glucose and cell samples were extracted at one minute intervals.
Wild type and GPR1 delete cells stimulated with glucose
0
50
100
150
200
250
300
350
400
0 1 2 3
time (minutes)
cA
MP
co
ncen
trati
on
(pm
ol/
mL
)
wild type
GPR1 delete
Figure 6. Wild type and GPR1 delete cells were stimulated with glucose and then frozen
for cAMP extraction at 1 minute intervals.
According to the results in figure 6, wild type cells stimulated with glucose showed an
initial higher cAMP concentration (>300 pmol/mL) which decreased over the course of three
minutes. GPR1 delete cells showed an initial lower cAMP concentration (<250 pmol/mL) which
decreased initially and then increased to >350 pmol/mL after three minutes.
Hodgson 16
GPA2 delete and GPR1 delete/GPA2 constitutively active cells
stimulated with glucose
0
100
200
300
400
500
0 1 2 3
time (minutes)
co
ncen
trati
on
(p
mo
l/m
L)
GPA2 delete
GPR1 delete/GPA2
contitutively active clone A
GPR1 delete/GPA2
contitutively active clone B
Figure 7. GPA2 delete and GPR1 delete/GPA2 constitutively active cells were stimulated
with glucose and then frozen for cAMP extraction at 1 minute intervals.
According to the results in figure 7, GPA2 delete cells showed little variation in cAMP
concentration over the course of 2 minutes and then showed a decrease in cAMP concentration
after 3 minutes. Both GPR1 delete/GPA2 constitutively active clones showed an initial increase
in cAMP concentration follwed by a decrease after 1 minute.
Another stimulation, cAMP extraction procedure, and ELISA assay was run to further
test the effects of glucose on wild type, GPR1 delete, and GPA2 constitutively active
Saccharomyces cerevisiae cells. Figures 8 and 9 show the standard curve generated from an
ELISA assay using known cAMP concentrations along with the results from the cell extract
samples.
Hodgson 17
Figure 8. cAMP concentration curve obtained from an ELISA using known cAMP
concentrations of 120 pmol/mL, 30 pmol/mL, and 7.5 pmol/mL.
cAMP concentration in yeast mutants
0
50
100
150
200
250
1 2 3
cAM
P c
on
cen
trat
ion
(p
mo
l/m
L)
wt
GPR1 del
GPR1 del/GPA2 ca
Time (minutes)
Figure 9. Wild type, GPR1 delete, and GPR1 delete/GPA2 constitutively active were
stimulated with glucose and cells were extracted and frozen for cAMP measurement after 1
and 2 minutes.
In this experiment, wild type cells showed a high concentration of cAMP (>200
pmol/mL) initially without glucose, followed by a decrease in cAMP when stimulated with
glucose after 1 and 2 minutes. GPR1 delete cells showed lower cAMP concentrations (<60
Hodgson 18
pmol/mL) along with a decrease in cAMP concentrations after 1 and 2 minutes. GPR1
delete/GPA2 constitutively active cells showed a high cAMP concentration (>200 pmol/mL)
over the course of the experiment.
A third extraction and ELISA procedure was run in order to test wild type, GPR1 delete,
and GPA2 constitutively active Saccharomyces cerevisiae cells after stimulation with glucose, a
known Gpr1p agonist, mannose, a known Gpr1p antagonist, and a mixture of both glucose and
mannose (Figures 10-12). cAMP levels in wild type cells are known to increase after 30 seconds
(Lemaire et al.). Therefore, cAMP concentrations in wild type, GPR1 delete, and GPA2
constitutively active cells were tested without sugar and then cells were extracted and frozen 30
seconds later after stimulation with glucose, mannose, or glucose and mannose to determine the
difference between the effects of glucose and mannose stimulation.
Wild type cells stimulated with glucose and mannose
110
111
112
113
114
115
116
117
0 30
time (seconds)
cA
MP
co
ncen
trati
on
(pm
ol/
mL
)
Glucose
Mannose
Glucose and Mannose
Figure 10. Wild type Saccharomyces cerevisiae cells were stimulated with the sugars
glucose, mannose, and glucose and mannose with cAMP levels measured before stimulation
and after 30 seconds.
Hodgson 19
GPR1 delete cells stimulated with glucose and mannose
90
95
100
105
110
115
120
125
130
0 30
time (seconds)
cA
MP
co
ncen
trati
on
(pm
ol/
mL
)
Glucose
Mannose
Glucose and Mannose
Figure 11. GPR1 delete Saccharomyces cerevisiae cells were stimulated with the sugars
glucose, mannose, and glucose and mannose with cAMP levels measured before stimulation
and after 30 seconds.
GPR1 delete/GPA2 contitutively active cells stimulated with
glucose and mannose
100105110115120125130135140145150
0 30
time (seconds)
cA
MP
co
ncen
trati
on
(pm
ol/
mL
)
Glucose
Mannose
Glucose and Mannose
Figure 12. GPR1 delete/GPA2 constitutively active Saccharomyces cerevisiae cells were
stimulated with the sugars glucose, mannose, and glucose and mannose with cAMP levels
measured before stimulation and after 30 seconds.
According to these results, cAMP levels in wild type cells remained the same after
stimulation with glucose. However, when stimulated with mannose, and glucose and mannose
Hodgson 20
simultaneously, the cAMP concentration increased slightly. GPR1 delete cells showed a slight
decrease in cAMP concentration after stimulation with glucose, a decrease when stimulated with
mannose, and a slight increase in cAMP concentration after stimulation with glucose and
mannose simultaneously. GPR1 delete/GPA2 constitutively active cells showed higher cAMP
concentrations (125pmol/mL). They also showed a slight decrease in cAMP concentration when
stimulated with mannose, a slight decrease when stimulated with glucose and mannose
simultaneously, and an increase in cAMP production when stimulated with glucose.
To further test the validity of the cAMP extraction procedure on Saccharomyces
cerevisiae wild type cells, three samples of wild type cells were stimulated using only glucose
and samples were extracted at 15 second intervals during stimulation and frozen for cAMP
analysis (Figures 13-15).
cAMP concentration in wild type cells stimulated with glucose
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
0 15 30 45 60
time (seconds)
cA
MP
(µ
g c
AM
P/µ
g t
ota
l
pro
tein
)
Trial 1
Figure 13. Wild type cells were stimulated with glucose and samples were extracted every
15 seconds for cAMP analysis.
Hodgson 21
cAMP concentration in wild type cells stimulated with glucose
0.0017
0.0018
0.0019
0.002
0.0021
0.0022
0.0023
0.0024
0 15 30
time (seconds)
cA
MP
(µ
g
cA
MP
/ µ
g t
ota
l
pro
tein
)
Trial 2
Figure 14. Wild type cells were stimulated with glucose and samples were extracted every
15 seconds for cAMP analysis.
cAMP concentration in wild type cells stimulated with glucose
0
0.0005
0.001
0.0015
0.002
0.0025
0.003
0.0035
0 15 30 60
time (seconds)
cA
MP
(µ
g
cA
MP
/ µ
g t
ota
l
pro
tein
)
Trial 3
Figure 15. Wild type cells were stimulated with glucose and samples were extracted every
15 seconds for cAMP analysis.
Trials 1 and 2 showed a decrease in cAMP concentrations after 30 seconds. However,
trial 2 showed an increase in cAMP concentration in wild type cells when stimulated with
glucose. As a further standardization procedure during this experiment, 10 µL of cell extract was
removed from each sample. A color reagent dye was added (200 µL) and the absorbance was
measured at 600 nm, allowing us to determine the total amount of protein in the extract. Serial
dilutions of Bovine Serum Albumin were also examined using spectroscopy and used to generate
Hodgson 22
a standard curve to compare to the amount of protein extracted from the cells. The µg cAMP per
µg of total protein was then determined to provide a means of standardization for future
procedures.
Cell Size Analysis
At this point in the project cells were further evalutated using a cell size analysis to
prrovide additional evidence for Gpr1p activity. Gpr1p is known to be responsible for regulating
cell size (Tamaki et al., Johnston et al., Lorincz et al.); therefore Gpr1p activity was tested by
stimulating with various sugars and evaluating cell size over a course of time points.
Glucose
0
20
40
60
80
100
120
140
0 135 165 195
time (minutes)
are
a (
mm
^2)
Glu wt
Glu del
Galactose
0
50
100
150
200
250
0 135 165 195
time (minutes)
are
a (
mm
^2)
Gal wt
Gal del
Mannose
0
20
40
60
80
100
120
140
160
0 135 165 195
time (minutes)
are
a (
mm
^2)
Man wt
Man del
Sucrose
0
20
40
60
80
100
120
140
160
180
200
0 135 165 195
time (minutes)
are
a (
mm
^2)
Suc wt
Suc del
Hodgson 23
Fructose
0
20
40
60
80
100
120
140
160
180
0 135 165 195
time (minutes)
are
a (
mm
^2)
Fruc wt
Fruc del
Figure 17. Changes in cell area as a function of time for wild type and GPR1 delete cells for
each individual sugar added
According to Figure 17, all wild type cells displayed an initial increase in area upon the
addition of a sugar. Wild type cells exposed to galactose, sucrose, and mannose all peaked in
size after 165 minutes, then displayed a decrease in area. Wild type cells exposed to fructose
peaked in size after 135 minutes, and then steadily decreased in area. Wild type cells exposed to
glucose steadily increased in area. All GPR1 delete cells displayed an initial decrease in area
upon exposure to sugars. GPR1 delete cells exposed to glucose, galactose, mannose, and sucrose
all has the smallest area after 135 minutes, peaked in area after 165 minutes, and then decreased
in area with the exception of cells exposed to glucose, which increased slightly in area. GPR1
delete cells exposed to fructose steadily decreased in area.
Hodgson 24
Glucose
0
20
40
60
80
100
120
0 150 165 180 195
time (minutes)
area (
mm
^2)
Glu wt
Glu del
Mannose
0
20
40
60
80
100
120
0 150 165 180 195
time (minutes)
are
a (
mm
^2)
Man wt
Man del
Galactose
0
20
40
60
80
100
120
0 150 165 180 195
time (minutes)
are
a (
mm
^2)
Gal wt
Gal del
Raffinose
0
20
40
60
80
100
120
140
0 150 165 180 195
time (minutes)
are
a (
mm
^2)
Raf wt
Raf del
Sucrose
0
20
40
60
80
100
120
140
160
0 150 165 180 195
time (minutes)
are
a (
mm
^2)
Suc wt
Suc del
Figure 18. Changes in cell area as a function of time for wild type and GPR1 delete cells
for each individual sugar added
The wild type cells exposed to glucose and mannose initially decreased in areas after 150
minutes, and wild type cells exposed to raffinose remained the same size. Wild type cells
exposed to glucose and raffinose both peaked in area after 180 minutes, whereas wild type cells
exposed to mannose peaked in area at 165 minutes and then gradually decreased in area. Wild
type cells exposed to galactose and sucrose initially increased in area, which decreased at 165
minutes, increased at 180 minutes, and then finally decreased after 195 minutes (Figures 19 and
21). GPR1 delete cells exposed to glucose initially slightly increased in area, whereas all other
GPR1 delete cells exposed to the other sugars showed an initial decrease in area (Figures 20 and
Hodgson 25
22). All GPR1 delete cells exposed to sugar also showed a decrease in area at 180 minutes and
then increased in area after 195 minutes except mannose which peaked in area after 165 minutes
and then steadily decreased in area.
Wild type cell size
0
20
40
60
80
100
120
0 150 165 180 195
time (minutes)
are
a (
mm
^2) Glu
Man
Gal
Raf
Suc
Figure 19. Changes in cell area as a function of time for all wild type cells with sugars
added.
GPR1 delete cell size
0
20
40
60
80
100
120
140
0 150 165 180 195
time (minutes)
are
a (
mm
^2) Glu
Man
Gal
Raf
Suc
Figure 20. Changes in cell area as a function of time for all GPR1 delete cells with sugars
added.
Hodgson 26
Wild type cell size
0
20
40
60
80
100
120
140
160
0 135 165 195
time (minutes)
area
(m
m^
2)
Glu
Gal
Man
Suc
Fruc
Figure 21. Changes in cell area as a function of time for all wild type cells with sugars
added.
GPR1 delete cell size
0
20
40
60
80
100
120
140
160
180
0 135 165 195
time (minutes)
area
(m
m^
2)
Glu
Gal
Man
Suc
Fruc
Figure 22. Changes in cell area as a function of time for all GPR1 delete cells with sugars
added.
Discussion
cAMP extraction/ ELISA
The goal of this project was to test the effects of various sugars on the surface receptor
protein Gpr1p in Saccharomyces cerevisiae. In order to test these effects, a reliable and
quantifiable method of determining Gpr1p activity must be developed. The methods used in this
project were designed to quantify the amount of cAMP produced after stimulation with sugar by
using a cAMP extraction procedure in conjunction with an ELISA assay. However, the results
Hodgson 27
from the ELISA assays used in this project did not turn out as expected. A re-occurring problem
was inconsistency with results obtained from the cAMP extraction and ELISA assays. The
standard curve generated from known cAMP concentrations turned out accurate with each assay,
indicating that the ELISA itself was working properly. Conversely, the results obtained from the
samples of cell extract did not follow the expected trends.
Figure 6 displays the results obtained from the first cAMP extraction/ELISA procedure.
According to this figure, cAMP concentration in wild type cells stimulated with glucose
decreased steadily over 3 minutes, which is contradictory to the findings that wild type cells
stimulated with glucose will produce an initial spike in cAMP concentration (Lemaire et al.).
Results from previous studies show a ~1 nmol/g WW increase in cAMP upon exposure to 50
mM glucose (Lemaire et al.). Furthermore, GPR1 delete cells stimulated with glucose produced
an initial decrease in cAMP concentration. This was the expected result since GPR1 is thought
to be responsible for cAMP production. Previous studies show that cAMP production does not
initially increase when exposed to glucose (Lemaire et al., Tamaki et al. 1998). In figure 7,
GPA2 delete cells lacking Gpr1p’s cognate G protein Gpa2 show a slight decrease in cAMP
production. With the exception of clone A at time 0, cells with a constitutively active Gpa2
protein also showed high levels of cAMP over the course of 3 minutes, indicating that the Gpa2
protein may be responsible in producing cAMP at high levels if constitutively active. These
trends have also been reproduced in other studies, confirming that GPA2 constitutively active
cells produce higher cAMP levels and GPR1 delete cells produce lower cAMP levels (Tamaki et
al 1998).
Since the results from the wild type cells stimulated with glucose did not turn out as
expected after the first assay, the same assay was repeated using wild type, GPR1 delete, and
Hodgson 28
GPR1 delete/GPA2 constitutively active cells. According the results from this assay in figure 9,
wild type cells once again showed a decrease in cAMP concentration when stimulated with
glucose. This also contradicts the findings presented by Lemaire that cAMP production
increases when cells are stimulated with glucose. However, GPR1 delete cells show lower
cAMP concentrations (<50 pmol/mL) and GPR1 delete/GPA2 constitutively active show high
cAMP concentrations (>200 pmol/mL) throughout, which further affirms Gpr1p and Gpa2’s
relation to cAMP production. From these results, GPR1 delete cells appear to have low cAMP
concentrations when stimulated with glucose, and GPR1 delete/GPA2 constitutively active cells
show high cAMP concentrations when stimulated with glucose.
Following this assay, another cAMP extraction/ELISA assay was run using wild type,
GPR1 delete, and GPR1 delete/GPA2 constitutively active cells stimulated with glucose,
mannose, and glucose and mannose simultaneously. The largest change in cAMP production is
known to occur after 30 seconds (Lemaire et al.). Therefore the purpose of this assay was to
determine if stimulation with mannose, a known antagonist had an effect on cAMP production in
these cells after 30 seconds. The expected result was an increase in cAMP production with
glucose and a decrease in cAMP production when stimulated with mannose. However, figure 10
shows an increase in cAMP production when stimulated with mannose, and glucose and
mannose simultaneously, and neither an increase nor decrease in cAMP production when
stimulated with glucose. Previous studies have shown that mannose has a clear antagonistic
effect, producing a decrease in cAMP production when exposed to Saccharomyces cerevisiae
(Lemaire et al.) The expected result in our project was a decrease in cAMP production, which
was not obtained. GPR1 delete cells also showed a decrease in cAMP production when
stimulated with mannose, and GPR1 delete/GPA2 constitutively active cells showed an increase
Hodgson 29
in cAMP concentration when stimulated with mannose. However, at this point in the study, the
wild type cells had not yet responded as expected to stimulation with glucose or mannose.
After these results, the cAMP extraction procedure was modified to accompany large
changes in pH. The suspected reason for the inconsistent results was the possibility of adding
too much acid or base in the extraction procedure. It was assumed that large deviations from the
appropriate pH would have an effect on the concentration of cAMP. To compensate for this, a
precise amount of acid or base was added to each tube and the volume of acid or base was
recorded and accounted for in the determination of the final concentration. Additionally, more
care was taken in the method of determining the pH of each tube in the extraction procedure.
At this point in the project, only glucose and mannose had been used to stimulate Gpr1p.
Since this project was designed to test the effects of various sugars on this cell surface protein, a
cell size analysis was also employed to test its activity.
Since data from the cAMP extraction/ELISA tests was still producing inconsistent
results, three samples of wild type cells were stimulated using only glucose and samples were
extracted and frozen for later cAMP analysis every 15 seconds for more precise results regarding
cAMP concentrations in cells over the course of 1 minute. This extraction and assay was also
completed in three trials in order to produce more reliability. However, two out of three of the
trials showed an initial decrease in cAMP production after 30 seconds. Only one of the trials
indicated an increase in cAMP production in wild type cells after stimulation with glucose.
Additionally during this trial, 10µL of cell extract was extracted from each sample and Bovine
Serum Albumin was added to the extract and color change was observed to determine the protein
concentration. This standardization procedure can be used in the future to compare cAMP
concentration with total protein concentration.
Hodgson 30
Since the results of each assay produced unpredictable results conflicting with previous
findings about Gpr1p, it may be determined that the procedures used in this project did in fact
successfully extract cAMP from the cells. However the procedure itself was not a reliable
indicator to quantitatively analyze the cAMP extracted. Further modification to the extraction
procedure may produce more reliable results in the future. However, the cell size experiment
also incorporated in this project produced more reliable results to help indicate the activity of
Gpr1p when stimulated with various sugars.
Cell Size
Since the cAMP extraction procedure produced inconsistent results, a cell size analysis
was developed to provide further evidence for Gpr1p activity. Saccharomyces cerevisiae cells
have previously been shown to produce variations in size when exposed to different sugars
(Johnston et al., Tamaki et al.). This size analysis was designed to provide further evidence for
the activity of Gpr1p with glucose and other sugars as well. To reduce variability in the results,
the cell size experiment was conducted under the same conditions as the previous stimulation
and cAMP extraction procedure used in this project.
According to the results obtained from this analysis, it appears that the addition of sugars
to wild type Saccharomyces cerevisiae cells grown in YPEG media produces an initial increase
in cell area when measured between 135 to 150 minutes after stimulation. On two occasions,
wild type cells showed a slight initial decrease in area when exposed to glucose and mannose.
However, all other cells in the same experiment showed a decrease in area between 150 and 165
minutes. It is possible that the cells that showed an initial decrease in area had already peaked in
size and were in the process of growing smaller. Additionally, GPR1 delete cells showed an
average peak in cell size 165 minutes after stimulation, followed by a decrease in area.
Hodgson 31
According to previous studies, yeast cells must grow to a critical size before bud initiation
(Johnston et al). The experimentally established growth rates for these cells was from .33 to .23
h-1
(Johnston et al.). The increase in cell size over the course of 195 minutes found in this project
corresponds to these results.
Another noticeable trend is an initial decrease in cell size of GPR1 delete cells after
exposure to sugar. Out of all cells surveyed, only once did GPR1 delete cells show an initial
slight increase in area after exposure to sugar. These findings indicate that Gpr1p is responsible
for regulating cell size. Previous studies confirm this trend as well. Upon exposure to glucose,
GPR1 delete mutants showed smaller cell sizes than wild type cells (Tamaki et al.). In our study,
cells without Gpr1p decreased in size upon exposure to sugars, whereas wild type cells showed
an increase in size upon exposure to sugar, corresponding to the previous established trends.
After every experiment, wild type cells exposed to glucose showed an increase in cell
size from 165 to 190 minutes. Wild type cells exposed to mannose showed a decrease in cell
size between the same times. Glucose is a known agonist and mannose is a known antagonist of
Gpr1p (Lemaire et al). Additionally, cell volume has been shown to increase upon the exposure
to glucose (Tamaki et al.). The findings in our project could be a result of glucose’s agonistic
effects after 165 minutes and mannose’s antagonistic effects. Wild type cells exposed to all
other sugars displayed an increase in cell size, indicating the possibility that mannose in the only
antagonist of all the sugars. Furthermore, wild type cells exposed to glucose showed a continual
increase after 165 minutes, and wild type cells exposed to other sugars decreased in size after
this time point. One possible explanation for this phenomenon could be that glucose is an
agonist to Gpr1p, mannose is an antagonist, and all other sugars tested are neither agonists nor
antagonists. During previous studies, galactose, mannose, and fructose have been shown not to
Hodgson 32
have agonistic function (Lemaire et al.). Overall, it appears that cell size is a more reliable
indicator for Gpr1p function than the cAMP extraction procedure used in this project. Previous
research also supports the finding that Gpr1p and Gpa2 are responsible for cell size variation
(Tamaki et al.). In our experiment, wild type cells exposed to various sugars normally show an
increase in cell size, followed by a peak in cell size between 165 and 180 minutes, and a further
decrease in size after 180 minutes, with the exceptions of cells exposed to glucose and mannose.
Gpr1 delete cells exposed to sugars show an initial decrease in cell size, followed by a slight
increase, and another decrease around 180 minutes. With the exception of glucose and mannose,
none of the sugars studied in this experiment show a clear agonistic or antagonistic function;
however it is apparent that Gpr1p is a regulator of cell size when Saccharomyces cerevisiae cells
are exposed to sugars corresponding to previous research that also supports the finding that
Gpr1p and Gpa2 are responsible for cell size variation (Tamaki et al.). Overall, it appears that
cell size is a more reliable indicator for Gpr1p function than the cAMP extraction procedure used
in this project.
This project used two different methods to determine the activity of Gpr1p in
Saccharomyces cerevisiae. Although the cAMP extraction procedure was inconsistent, it did
indicate that GPR1 delete cells produce lower cAMP concentrations while GPR1 delete/GPA2
constitutively active cells produced higher cAMP concentrations. Since predictable results were
not obtained, only glucose and mannose were tested using the cAMP extraction and ELISA.
Additionally, the cell size experiments produced more reliable data that showed trends in cell
size when exposed to other sugars. Based on the results found here, with the exception of
glucose and mannose, the sugars tested could not be found to have a clear agonistic or
antagonistic function. Perhaps in the future, a more efficient cAMP extraction procedure could
Hodgson 33
be developed to be used in conjunction with another cell size experiment to further test the
activity of Gpr1p when exposed to these sugars.
Hodgson 34
Acknowledgements
I would like to thank the Carson-Newman College Biology and Chemistry departments,
especially Dr. Stephen Wright for his support and guidance with this project.
Hodgson 35
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