Examining the agonistic and antagonistic effects of various sugars on the surface receptor. protein Gpr1p in Saccharomyces cerevisiae.

Transcription

1 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 (423) in partial fulfillment for the degree Bachelor of Science with Honors April 27, 211 Project Advisor: Dr. Stephen Wright 211 William D. Hodgson

2 Hodgson 2 Table of Contents Page Abstract 3 Introduction 4 Materials and Methods 1 Results 14 Discussion 27 Acknowledgements 34 References 35

3 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.

4 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.

5 Hodgson 5 Figure 1. Overview of glucose and pheromone sensing in S. cerevisiae (Versele et al., 21). 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

6 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

7 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

8 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 Neither Agonist Agonist Function nor Antagonist Function Glucose * Sucrose * Mannose * 2-deoxyglucose * Trehalose * Turanose * Palatinose * Galactose * Maltose * 6-deoxyglucose *

9 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 5% of cells (EC-5) ranges from about 2 to 75 mm for glucose as a ligand. However, the EC-5 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

10 Hodgson 1 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 (1g Yeast extract, 2 g Peptone, 2g Dextrose, 2% agar). Prior to stimulation and camp extraction, cells were incubated in liquid YPEG media (1g Yeast extract, 2g Peptone, 2% ethanol (2 g), and 2% glycerol (2g). 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

11 Hodgson 11 million cells per tube by measuring the OD 6 of 15 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 17 x g in a refrigerated centrifuge at 5 C for 5 minutes and re-suspended in 126 µl of cold 25 mm MES buffer for 1 minutes. Cells and buffer (315 µl) were extracted and placed in 5 ml of cold, 6% methanol and placed in a -2 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 3 seconds, 1 minute, and 1 minute 3 seconds for the rest of the extraction procedures. After the appropriate amount of stimulation, 315 µl of cells and buffer were transferred to cold 6% methanol and stored for 48 hours. The cells were then centrifuged at 17 x g for 5 minutes in a refrigerated centrifuge at 5 C for 5 minutes and the supernatant was removed. Trichloroacetic acid (5 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 3 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 3 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 (2 µl ph 7.5) was then added to each tube and the tubes

12 Hodgson 12 were centrifuged at 3 x g in a refrigerated centrifuge at 5 C for 3 minutes and the supernatant was frozen in a -2 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 (1g Yeast extract, 2 g Peptone, 2g Dextrose, 2% agar). Prior to stimulation, camp extraction, and cell size analysis, cells were incubated in liquid YPEG media (1g Yeast extract, 2g Peptone, 2% ethanol (2 g), and 2% glycerol (2g) 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 OD 6 of 15 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 17 x g in a refrigerated centrifuge at 5 C for 5 minutes and resuspended in 126 µl of cold 25 mm MES buffer for 1 minutes. Cells and buffer (252 µl) was extracted and placed in cold 9% methanol and stored at -2 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 9% methanol after 2 hours 15 minutes, 2 hours 45 minutes, and 3 hours 15 minutes. The cells and methanol were then stored at -2 C for further analysis. To analyze the changes in cell size, the cells in 9% methanol were vortexed for 1 minute to break up pellets and clumps. Cells in methanol (1 µl) was then extracted and placed on a hemocytometer and examined at 4X under a microscope. Each slide was then photographed

13 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 Parameter TM camp: primary antibody solution (5 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 5 rpm. Each well was washed using 3 µl of wash buffer (1X concentrate) provided in the Parameter TM camp kit. This was process was performed 4 times, and the plate was inverted and blotted between each wash. Standard, control, or sample ( µl) was added to appropriate wells. Calibrator diluent ( µl) RD5-55 (provided in the Parameter TM camp kit) was added to the zero standard wells. camp conjugate (5 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 Parameter TM camp kit), and 2 µl of solution was added to each well. The plate was incubated for 3 minutes at room temperature and protected from light. Stop solution ( µl, provided by Parameter TM camp kit) was added to each well. Optical density was determined within 3 minutes using a microplate reader set to 495 nm. Protein Determination Cell extract (1 µl) was removed from each sample. A Bradford reagent dye was added (2 µl) and the absorbance was measured at 6 nm after 2 minutes. Serial dilutions of Bovine

14 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

15 camp concentration (pmol/ml) 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 time (minutes) 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 (>3 pmol/ml) which decreased over the course of three minutes. GPR1 delete cells showed an initial lower camp concentration (<25 pmol/ml) which decreased initially and then increased to >35 pmol/ml after three minutes.

16 concentration (pmol/ml) Hodgson 16 GPA2 delete and GPR1 delete/gpa2 constitutively active cells stimulated with glucose GPA2 delete GPR1 delete/gpa2 contitutively active clone A GPR1 delete/gpa2 contitutively active clone B time (minutes) 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.

17 camp concentration (pmol/ml) Hodgson 17 Figure 8. camp concentration curve obtained from an ELISA using known camp concentrations of 12 pmol/ml, 3 pmol/ml, and 7.5 pmol/ml. 25 camp concentration in yeast mutants 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 (>2 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 (<6

18 camp concentration (pmol/ml) 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 (>2 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 1-12). camp levels in wild type cells are known to increase after 3 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 3 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 time (seconds) Glucose Mannose Glucose and Mannose Figure 1. Wild type Saccharomyces cerevisiae cells were stimulated with the sugars glucose, mannose, and glucose and mannose with camp levels measured before stimulation and after 3 seconds.

19 camp concentration (pmol/ml) camp concentration (pmol/ml) Hodgson 19 GPR1 delete cells stimulated with glucose and mannose time (seconds) 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 3 seconds. GPR1 delete/gpa2 contitutively active cells stimulated with glucose and mannose time (seconds) 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 3 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

20 camp (µg camp/µg total protein) Hodgson 2 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 time (seconds) Trial 1 Figure 13. Wild type cells were stimulated with glucose and samples were extracted every 15 seconds for camp analysis.

21 camp (µg camp/ µg total protein) camp (µg camp/ µg total protein) Hodgson 21 camp concentration in wild type cells stimulated with glucose time (seconds) 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 time (seconds) 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 3 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, 1 µl of cell extract was removed from each sample. A color reagent dye was added (2 µl) and the absorbance was measured at 6 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

22 area (mm^2) area (mm^2) area (mm^2) area (mm^2) 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 Galactose Glu wt Glu del Gal wt Gal del time (minutes) time (minutes) Mannose Sucrose Man wt Man del Suc wt Suc del time (minutes) time (minutes)

23 area (mm^2) Hodgson 23 Fructose time (minutes) 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.

24 area (mm^2) area (mm^2) area (mm^2) area (mm^2) area (mm^2) Hodgson 24 Glucose Mannose Glu wt Glu del Man wt Man del time (minutes) time (minutes) Galactose Raffinose Gal wt Gal del Raf wt Raf del time (minutes) time (minutes) Sucrose Suc wt Suc del time (minutes) 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 15 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 18 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 18 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 2 and

25 area (mm^2) area (mm^2) Hodgson 25 22). All GPR1 delete cells exposed to sugar also showed a decrease in area at 18 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 Glu Man Gal Raf Suc time (minutes) Figure 19. Changes in cell area as a function of time for all wild type cells with sugars added. GPR1 delete cell size time (minutes) Glu Man Gal Raf Suc Figure 2. Changes in cell area as a function of time for all GPR1 delete cells with sugars added.

26 area (mm^2) area (mm^2) Hodgson 26 Wild type cell size time (minutes) 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 time (minutes) 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

27 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 5 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, 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

28 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 (<5 pmol/ml) and GPR1 delete/gpa2 constitutively active show high camp concentrations (>2 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 3 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 3 seconds. The expected result was an increase in camp production with glucose and a decrease in camp production when stimulated with mannose. However, figure 1 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

29 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 3 seconds. Only one of the trials indicated an increase in camp production in wild type cells after stimulation with glucose. Additionally during this trial, 1µL 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.

30 Hodgson 3 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 15 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 15 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.

31 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 19 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

32 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 18 minutes, and a further decrease in size after 18 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 18 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

33 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.

34 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.

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37 Hodgson 37 Wheals, A Size Control Models of Saccharomyces cerevisiae Cell Proliferation. Microbiology Group, School of Biological Sciences, University of Bath, Bath, United Kingdom