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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