FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023177616> ?p ?o ?g. }

• W2023177616 endingPage "11" @default.
• W2023177616 startingPage "1" @default.
• W2023177616 abstract "The use of chemostat culturing enables investigation of steady-state physiological characteristics and adaptations to nutrient-limited growth, while all other relevant growth conditions are kept constant. We examined and compared the proteomic response of wild-type Saccharomyces cerevisiae CEN.PK113-7D to growth in aerobic chemostat cultures limited for carbon sources being either glucose or ethanol. To obtain a global overview of changes in the proteome, we performed triplicate analyses using two-dimensional gel electrophoresis and identified proteins of interest using MS. Relative quantities of about 400 proteins were obtained and analyzed statistically to determine which protein steady-state expression levels changed significantly under glucose- or ethanol-limited conditions. Interestingly, only enzymes involved in central carbon metabolism showed a significant change in steady-state expression, whereas expression was only detected in one of both carbon source-limiting conditions for 15 of these enzymes. Side effects that were previously reported for batch cultivation conditions, such as responses to continuous variation of specific growth rate, to carbon-catabolite repression, and to accumulation of toxic substrates, were not observed. Moreover, by comparing our proteome data with corresponding mRNA data, we were able to unravel which processes in the central carbon metabolism were regulated at the level of the proteome, and which processes at the level of transcriptome. Importantly, we show here that the combined approach of chemostat cultivation and comprehensive proteome analysis allowed us to study the primary effect of single limiting conditions on the yeast proteome. The use of chemostat culturing enables investigation of steady-state physiological characteristics and adaptations to nutrient-limited growth, while all other relevant growth conditions are kept constant. We examined and compared the proteomic response of wild-type Saccharomyces cerevisiae CEN.PK113-7D to growth in aerobic chemostat cultures limited for carbon sources being either glucose or ethanol. To obtain a global overview of changes in the proteome, we performed triplicate analyses using two-dimensional gel electrophoresis and identified proteins of interest using MS. Relative quantities of about 400 proteins were obtained and analyzed statistically to determine which protein steady-state expression levels changed significantly under glucose- or ethanol-limited conditions. Interestingly, only enzymes involved in central carbon metabolism showed a significant change in steady-state expression, whereas expression was only detected in one of both carbon source-limiting conditions for 15 of these enzymes. Side effects that were previously reported for batch cultivation conditions, such as responses to continuous variation of specific growth rate, to carbon-catabolite repression, and to accumulation of toxic substrates, were not observed. Moreover, by comparing our proteome data with corresponding mRNA data, we were able to unravel which processes in the central carbon metabolism were regulated at the level of the proteome, and which processes at the level of transcriptome. Importantly, we show here that the combined approach of chemostat cultivation and comprehensive proteome analysis allowed us to study the primary effect of single limiting conditions on the yeast proteome. Two-dimensional (2D) 1The abbreviation used is: 2Dtwo-dimensional. gel electrophoresis is a powerful tool to visualize hundreds of proteins at a time, which in combination with MS leads to their identification. This technology has been applied to the yeast Saccharomyces cerevisiae for the large-scale identification of more than 400 proteins, resulting in yeast reference maps (1Boucherie H. Sagliocco F. Joubert R. Maillet I. Labarre J. Perrot M. Two-dimensional gel protein database of Saccharomyces cerevisiae..Electrophoresis. 1996; 17: 1683-1699Google Scholar, 2Norbeck J. Blomberg A. Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3–10) immobilized pH gradient in the first dimension: Reproducibility and evidence for isoelectric focusing of alkaline (pI > 7) proteins..Yeast. 1997; 13: 1519-1534Google Scholar, 3Perrot M. Sagliocco F. Mini T. Monribot C. Schneider U. Shevchenko A. Mann M. Jeno P. Boucherie H. Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999)..Electrophoresis. 1999; 20: 2280-2298Google Scholar, 4Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Boucherie H. Mann M. Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels..Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Google Scholar, 5Boucherie H. Dujardin G. Kermorgant M. Monribot C. Slonimski P. Perrot M. Two-dimensional protein map of Saccharomyces cerevisiae: Construction of a gene-protein index..Yeast. 1995; 11: 601-613Google Scholar, 6Wildgruber R. Reil G. Drews O. Parlar H. Gorg A. Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients from pH 6 to pH 12 and matrix-assisted laser desorption/ionization-time of flight mass spectrometry..Proteomics. 2002; 2: 727-732Google Scholar, 7Maillet I. Lagniel G. Perrot M. Boucherie H. Labarre J. Rapid identification of yeast proteins on two-dimensional gels..J. Biol. Chem. 1996; 271: 10263-10270Google Scholar). Other S. cerevisiae studies used 2D gel electrophoresis to obtain an overview of global changes in the yeast proteome as function of stimuli such as cadmium (8Vido K. Spector D. Lagniel G. Lopez S. Toledano M.B. Labarre J. A proteome analysis of the cadmium response in Saccharomyces cerevisiae..J. Biol. Chem. 2001; 276: 8469-8474Google Scholar), lithium (9Bro C. Regenberg B. Lagniel G. Labarre J. Montero-Lomeli M. Nielsen J. Transcriptional, proteomic, and metabolic responses to lithium in galactose-grown yeast cells..J. Biol. Chem. 2003; 278: 32141-32149Google Scholar), H2O2 (10Godon C. Lagniel G. Lee J. Buhler J.M. Kieffer S. Perrot M. Boucherie H. Toledano M.B. Labarre J. The H2O2 stimulon in Saccharomyces cerevisiae..J. Biol. Chem. 1998; 273: 22480-22489Google Scholar), or sorbic acid (11De Nobel H. Lawrie L. Brul S. Klis F. Davis M. Alloush H. Coote P. Parallel and comparative analysis of the proteome and transcriptome of sorbic acid-stressed Saccharomyces cerevisiae..Yeast. 2001; 18: 1413-1428Google Scholar). two-dimensional. Most of these differential proteome studies on S. cerevisiae were performed on cells cultured in batch mode, i.e. in shake flasks or in reactors. Batch cultivation makes use of a closed system, in which all nutrients are in excess at the start of the cultivation. In terms of microbial physiology, such batch cultivation is relatively poorly controlled, because the composition of the growth medium and consequently the growth rate changes continuously. The yeast cells take up nutrients from the media while metabolites are excreted in the culture system, and their growth arrests when one of the nutrients is depleted or when too many toxic substrates are accumulated. The high concentration of carbon source in the culture, which is essential for this type of cultures, may lead to carbon-catabolite repression (12Gancedo J.M. Yeast carbon catabolite repression..Microbiol. Mol. Biol. Rev. 1998; 62: 334-361Google Scholar). The specific growth rate μ is directly affected by these continuous changes and is only constant during the exponential growth phase. Changes in specific growth rate are known to have a high impact on gene expression in S. cerevisiae (13Diderich J.A. Schepper M. Van Hoek P. Luttik M.A. Van Dijken J.P. Pronk J.T. Klaassen P. Boelens H.F. De Mattos M.J. Van Dam K. Kruckeberg A.L. Glucose uptake kinetics and transcription of HXT genes in chemostat cultures of Saccharomyces cerevisiae..J. Biol. Chem. 1999; 274: 15350-15359Google Scholar, 14Hayes A. Zhang N. Wu J. Butler P.R. Hauser N.C. Hoheisel J.D. Lim F.L. Sharrocks A.D. Oliver S.G. Hybridization array technology coupled with chemostat culture: Tools to interrogate gene expression in Saccharomyces cerevisiae..Methods. 2002; 26: 281-290Google Scholar, 15Verwaal R. Paalman J.W. Hogenkamp A. Verkleij A.J. Verrips C.T. Boonstra J. HXT5 expression is determined by growth rates in Saccharomyces cerevisiae..Yeast. 2002; 19: 1029-1038Google Scholar) and thus also on the yeast proteome. Although most studies concern batch-cultured yeast cells that are collected from the exponential growth phase, their proteome analyses may not only reveal the primary effect of interest, but may additionally reflect the aforementioned effects. A few research groups used chemostat cultivation for yeast proteome analysis (see for example Refs. 16Pratt J.M. Petty J. Riba-Garcia I. Robertson D.H. Gaskell S.J. Oliver S.G. Beynon R.J. Dynamics of protein turnover, a missing dimension in proteomics..Mol. Cell. Proteomics. 2002; 1: 579-591Google Scholar and 17Salusjarvi L. Poutanen M. Pitkanen J.P. Koivistoinen H. Aristidou A. Kalkkinen N. Ruohonen L. Penttila M. Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae..Yeast. 2003; 20: 295-314Google Scholar), an approach that avoids growth rate-dependent changes and thus allows investigation of the effect of e.g. a single nutrient limitation at a fixed growth rate. A chemostat culture is continuously fed with fresh media at a constant rate, and the volume in the chemostat vessel is kept constant by continuous, constant-rate removal of culture fluid that contains yeast cells, spent media, and metabolites. As a result, the specific growth rate μ of the culture can be fixed and the dilution rate can be controlled accurately. A steady-state condition is achieved when the total number of cells and the total volume in the chemostat vessel remain constant (18Harder W. Kuenen J.G. A review. Microbial selection in continuous culture..J. Appl. Bacteriol. 1977; 43: 1-24Google Scholar). In a sugar-limited chemostat culture, the carbon source is almost completely consumed, resulting in very low residual concentrations and therefore avoiding carbon-catabolite repression. Moreover, the very low residual concentrations avoid accumulation of toxic substrates. The growth medium in a chemostat is designed such that only one single nutrient limits the growth, while all other nutrients are present in excess. Because growth of microorganisms like S. cerevisiae in their natural environment and also in many industrial applications (e.g. industrial production of bakers’ yeast) is generally limited by nutrient availability, the effect of nutrient limitation on the yeast proteome can be optimally studied using chemostat cultures. Thus chemostat cultivation enables proteome-wide investigation of the effect of one particular nutrient limitation at a fixed growth rate, while all other growth parameters are kept constant. The aim of the present study is to investigate the carbon source-dependent response on the proteome of S. cerevisiae. Aerobic chemostat cultures were used, which were limited for the carbon sources glucose or ethanol, allowing analysis of the protein expression levels under glycolytic and gluconeogenic conditions, respectively. It has been shown by Piper et al. (19Piper M.D.W. Daran-Lapujade P. Bro C. Regenberg B. Knudsen S. Nielsen J. Pronk J.T. Reproducibility of oligonucleotide microarray transcriptome analyses—An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae..J. Biol. Chem. 2002; 277: 37001-37008Google Scholar) that this culturing approach provides highly reproducible results at the level of transcriptome analyses, not only between independent chemostat cultures at one laboratory, but also between two different laboratories. However, as has been suggested by Daran-Lapujade et al. (20Daran-Lapujade P. Jansen M.L. Daran J.M. Van Gulik W. De Winde J.H. Pronk J.T. Role of transcriptional regulation in controlling fluxes in central carbon metabolism of Saccharomyces cerevisiae. A chemostat culture study..J. Biol. Chem. 2004; 279: 9125-9138Google Scholar), who compared genome-wide transcript levels with in vivo fluxes for glucose- and ethanol-limited chemostat cultures, control of the central carbon pathways takes place to a large extent via post-transcriptional mechanisms. Therefore, we performed a comparative proteome analysis of the wild-type S. cerevisiae CEN.PK113-7D, using 2D gel electrophoresis followed by MS for protein identification. Triplicate analyses allowed adequate statistical analysis, resulting in appropriate relative quantitative analysis of protein expression of glucose- versus ethanol-limited conditions. To investigate which of the genes involved in the central carbon metabolism are regulated at the level of the transcriptome and which at the level of the proteome, we compared the corresponding expression ratios for equal growth conditions. Our results indicate the importance of using chemostat cultures, as we observed significant effects in steady-state expression levels solely of enzymes involved in the central carbon metabolism. By using the combination of chemostat cultivation and comprehensive proteome analysis, we show that we have a unique possibility to study the effect of single limiting conditions on the yeast proteome. Wild-type Saccharomyces cerevisiae strain CEN.PK113-7D (MATa) (21Pronk J.T. Wenzel T.J. Luttik M.A.H. Klaassen C.C.M. Scheffers W.A. Steensma H.Y. Vandijken J.P. Energetic aspects of glucose-metabolism in a pyruvate-dehydrogenase-negative mutant of Saccharomyces cerevisiae..Microbiology. 1994; 140: 601-610Google Scholar) was grown at 30 °C in 2-liter chemostats (Applikon, Schiedam, The Netherlands), with a working volume of 1.0 liter as described (22Van Den Berg M.A. De Jong-Gubbels P. Kortland C.J. Van Dijken J.P. Pronk J.T. Steensma H.Y. The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation..J. Biol. Chem. 1996; 271: 28953-28959Google Scholar). Cultures were fed with a defined mineral medium that limited growth by glucose or ethanol with all other growth requirements in excess. The dilution rate was set at 0.10 h−1. The pH was measured online and kept constant at 5.0 by the automatic addition of 2 m KOH with the use of an Applikon ADI 1030 biocontroller. Stirrer speed was 800 rpm, and the airflow was 0.5 liters·min−1. Dissolved oxygen tension was measured online with an Ingold model 34-100-3002 probe (Mettler Toledo, Greifensee, Switzerland) and was between 60 and 75% of air saturation. The off-gas was cooled by a condenser connected to a cryostat set at 2 °C and analyzed as previously described (23Van Maris A.J. Luttik M.A. Winkler A.A. Van Dijken J.P. Pronk J.T. Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae..Appl. Environ. Microbiol. 2003; 69: 2094-2099Google Scholar). Steady-state samples were taken after ∼10–14 volume changes to avoid strain adaptation due to long-term cultivation (24Ferea T.L. Botstein D. Brown P.O. Rosenzweig R.F. Systematic changes in gene expression patterns following adaptive evolution in yeast..Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9721-9726Google Scholar). Dry-weight, metabolite, dissolved oxygen, and off-gas profiles had to be constant over at least 5 volume changes prior to sampling for protein extraction. Culture dry weights were determined via filtration as described by Postma et al. (25Postma E. Verduyn C. Scheffers W.A. Van Dijken J.P. Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae..Appl. Environ. Microbiol. 1989; 55: 468-477Google Scholar) The defined mineral medium composition was based on that described by Verduyn et al. (26Verduyn C. Postma E. Scheffers W.A. Van Dijken J.P. Effect of benzoic acid on metabolic fluxes in yeasts: A continuous-culture study on the regulation of respiration and alcoholic fermentation..Yeast. 1992; 8: 501-517Google Scholar). The carbon source was 256 ± 19 mmol of carbon per liter. S. cerevisiae protein extracts were prepared for analysis with 2D gel electrophoresis using a combined approach of Boucherie et al. (5Boucherie H. Dujardin G. Kermorgant M. Monribot C. Slonimski P. Perrot M. Two-dimensional protein map of Saccharomyces cerevisiae: Construction of a gene-protein index..Yeast. 1995; 11: 601-613Google Scholar) and Harder et al. (27Harder A. Wildgruber R. Nawrocki A. Fey S.J. Larsen P.M. Gorg A. Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis..Electrophoresis. 1999; 20: 826-829Google Scholar). In brief, yeast cells were lyophilized prior to protein extraction. Between 65 and 75 mg dry-weight of yeast cells was used as starting material for protein extraction. Glass beads (acid washed, 425–600 μm; Sigma, St. Louis, MO) were added to the lyophilized yeast cells. Cells were disrupted by vortexing six times 60 s. The samples were cooled on ice for 30 s in between the vortex steps. After cell lysis, the yeast cells were resuspended in 650 μl of hot (95 °C) SDS sample buffer (0.1 m Tris-HCl, pH 7.0, 1.0% (w/v) SDS supplemented with protease inhibitors (Complete Protease Inhibitor Mixture Tablets; Roche Diagnostics, Somerville, NJ). The sample was boiled for 10 min and subsequently cooled on ice. Subsequently, 75 μl of a DNase and RNase solution (1% (w/v) DNase I, 0.25% (w/v) RNase A, 50 mm MgCl2, 0.5 m Tris-HCl, pH 7.0) was added and incubated on ice. The sample was diluted by adding 2.0 ml of a solubilization buffer containing 2 m thiourea, 7 m urea, 4% (w/v) CHAPS, 2.5% (w/v) DTT, 2% (v/v) carrier ampholytes, pH 3–10 nonlinear, and protease inhibitors. The samples were shaken for 1 h on a Roller mixer SRT2 (Merck Eurolab B. V., Amsterdam, The Netherlands) at room temperature followed by clearing through centrifugation at 3,000 × g. Protein concentration was determined with a Bradford protein assay (Bio-Rad, Hercules, CA) using BSA as a standard. The cleared supernatants were stored in aliquots at −80 °C. For the first dimension, an amount of 150 μg of protein was loaded on a 13 cm Immobiline Dry-Strip pH 3–10 NL (Amersham Biosciences, Piscataway, NJ) in 250 μl sample buffer containing 7 m urea, 2 m thiourea, 4% (w/v) CHAPS, 2.5% (w/v) DTT, and 2% (v/v) carrier ampholytes, pH 3–10 nonlinear, and protease inhibitors. Rehydration and isoelectric focusing were carried out using an IPGphor (Amersham Biosciences). Strips were rehydrated for 13–15 h at 30 V, followed by IEF, with the current limited to 100 μA per strip, at 20 °C, for a total of 40–45 kVh (1 h at 500 V, 1 h at 1,500 V followed by 8,000 V for 38–43 kVh). Prior to the second dimension, the IPG strips were incubated for 15 min in equilibration buffer (50 mm Tris-HCl, pH 8.8, 6 m urea, 30% (v/v) glycerol, 2% (w/v) SDS) containing 1% (w/v) DTT, followed by 15 min incubation in equilibration buffer containing 2.5% (w/v) iodoacetamide. Second-dimension electrophoresis was performed on laboratory-cast 12.5% polyacrylamide gels in a Hoefer SE600 system (Amersham Biosciences). The IPG strips were placed on top of 12.5% polyacrylamide gels and sealed with a solution of 1% (w/v) agarose containing a trace of bromphenol blue. Gels were run at 10 mA per gel for 15 min followed by 20 mA per gel until the bromphenol blue had migrated to the bottom of the gel. Proteins were visualized using silver staining as described by Shevchenko et al. (28Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels..Anal. Chem. 1996; 68: 850-858Google Scholar). The silver-stained gels were scanned using a GS-710 Calibrated Imaging Densitometer (Bio-Rad). For each growth condition, namely for glucose- and ethanol-limitation, 2D gels were run in triplicate. Additionally, a master 2D gel was prepared, which contained a 1:1 mixture of the protein extract of the ethanol- and glucose-limited yeast cultures. In theory, this master 2D gel should contain all protein spots present on the ethanol- and the glucose-limited 2D gels and was used during image analysis as a master gel. Image analysis was performed using the PDQuest 7.1.0 software package (Bio-Rad). Normalization of spot volumes in a gel was performed using the “total density in valid spots” option. The quantitative and statistical analyses were performed using suitable functions within the PDQuest software and Excel software (Microsoft, Redmond, WA). The normalized intensity of spots on three replicate 2D gels was averaged and the standard deviation was calculated for each condition. The relative change in protein abundance for ethanol- versus glucose-limitation (indicated with “fold change E/G”) for each protein spot was calculated by dividing the averaged normalized spot quantity from the ethanol gels by the averaged normalized spot quantity from the glucose gels. A two-tailed nonpaired Student’s t-test was performed to determine if the relative change was statistically significant. The faintest spot that was detected had an intensity of 250 ppm, and if a spot was below this detection level for one growth condition while (far) above 250 ppm in the other condition, this was considered as a significant change in expression. In those situations the ratio “fold change E/G” could not be calculated and was thus indicated with E (i.e. only expression under ethanol limitation) or G (i.e. only expression under glucose limitation). Protein spots of interest were excised and in-gel digested with trypsin with a slightly modified protocol as described by Wilm et al. (29Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry..Nature. 1996; 379: 466-469Google Scholar). In brief, the gel pieces were first destained using 30 mm potassium ferricyanide and 100 mm sodium thiosulphate solution, followed by washing and shrinking steps using 50 mm ammonium bicarbonate and ACN, respectively. Proteins were digested overnight at 37 °C by adding trypsin at a concentration of 10 ng/μl. After tryptic digestion, peptides were concentrated and desalted using a ZipTip μ-C18 (Millipore, Bedford, MA). Peptides were eluted directly on the MALDI-target with 1 μl of a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% ACN and 0.1% (v/v) TFA. Peptides were analyzed using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) using delayed extraction in positive reflectron mode at 20 kV accelerating voltage. Nano-LC-MS/MS was performed by coupling an Ultimate HPLC (LC Packings) to an ESI Q-TOF instrument (Micromass UK Ltd., Manchester, UK), operating in positive ion mode and equipped with a Z-spray nano-ESI source as described before (30Krijgsveld J. Ketting R.F. Mahmoudi T. Johansen J. Artal-Sanz M. Verrijzer C.P. Plasterk R.H.A. Heck A.J.R. Metabolic labeling of C-elegans and D-melanogaster for quantitative proteomics..Nat. Biotechnol. 2003; 21: 927-931Google Scholar). Briefly, peptides were delivered to a trap column (Aqua™ C18RP (Phenomenex, Torrance, CA); 15 mm × 100 μm inner diameter) at 5 μl/min by using a Famos autosampler (LC Packings, Amsterdam, The Netherlands) (31Meiring H.D. Van Der Heeft E. Ten Hove G.J. De Jong A.P.J.M. Nanoscale LC-MS(n): Technical design and applications to peptide and protein analysis..J. Sep. Sci. 2002; 25: 557-568Google Scholar). After reducing the flow to ∼150 nl/min by a splitter, the peptides were transferred to the analytical column (PepMap C18 (LC Packings); 20 cm × 50 μm inner diameter). The peptides were eluted with a linear gradient from 0–50% buffer B (0.1 m acetic acid in 80% (v/v) ACN) in 30 min. The column eluent was sprayed directly into the ESI source of the mass spectrometer via a butt-connected nano-ESI emitter (New Objectives, Woburn, MA). Peptides were fragmented in data-dependent mode. Proteins were identified using MASCOT software (www.matrixscience.com), and searches were performed using the Swiss-Prot or NCBInr database. The following search parameters were used: trypsin was used as enzyme, the peptide tolerance window was set to 100 ppm, one missed cleavage was allowed, and carbamidomethyl and oxidized methionine were set as fixed and variable modification, respectively. To investigate the effect of carbon source limitation at the level of Saccharomyces cerevisiae protein expression, a comparative proteome analysis was performed using aerobic chemostat cultures with glucose or ethanol as single growth-limiting nutrient, respectively. The dilution rate in both cultures was equal, namely 0.1 h−1. Under these conditions, the concentration of the carbon sources in the reservoir medium was ca. 250 mmol of carbon per liter, whereas their residual concentration in steady-state cultures were below their detection limits (i.e. less than 0.5 mm). In the glucose-limited culture, no ethanol was produced, and cells grew with a biomass yield on glucose of 0.5 g·g−1, reflecting complete respiratory catabolism. This is typical for steady-state growth of S. cerevisiae strain CEN.PK113-7D under glucose limitation at dilution rates below 0.3 h−1 (32Van Hoek P. Flikweert M.T. Van Der Aart Q.J. Steensma H.Y. Van Dijken J.P. Pronk J.T. Effects of pyruvate decarboxylase overproduction on flux distribution at the pyruvate branch point in Saccharomyces cerevisiae..Appl. Environ. Microbiol. 1998; 64: 2133-2140Google Scholar). In the ethanol-grown culture, over 95% of the substrate carbon was recovered, as either biomass or carbon dioxide and HPLC analysis of culture supernatants did not reveal the production of any low-molecular-mass metabolites. Yeast cells from both cultures were harvested and proteins were extracted using the extraction protocol developed by Harder et al. (27Harder A. Wildgruber R. Nawrocki A. Fey S.J. Larsen P.M. Gorg A. Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis..Electrophoresis. 1999; 20: 826-829Google Scholar), which we further optimized for our yeast samples. In Harders’ protocol, yeast cells are sonicated in buffer containing SDS to disrupt the cell walls and dissolve the proteins. In order to improve cell disruption and to minimize proteolysis, we performed an additional step. The yeast cells were lyophilized and subsequently vortexed with glass beads as described by Boucherie et al. (5Boucherie H. Dujardin G. Kermorgant M. Monribot C. Slonimski P. Perrot M. Two-dimensional protein map of Saccharomyces cerevisiae: Construction of a gene-protein index..Yeast. 1995; 11: 601-613Google Scholar), prior to SDS boiling. Furthermore, protease inhibitors were added to both the solubilization buffer and the hot SDS sample buffer for maximal reduction of endogenous proteolytic enzyme activity. More high-molecular-mass proteins (> 75 kDa) were observed on the 2D gels when this optimized protocol was used. In Fig. 1, typical 2D gel electrophoresis images of the ethanol-limited (Fig. 1A) and the glucose-limited (Fig. 1B) yeast cultures are shown. An average of ∼400 spots was detected on each 2D gel. The analyses were performed in triplicate to allow proper statistical analysis, i.e. a Student’s t-test was used to determine if the relative change in protein expression for glucose- versus ethanol-limitation was statistically significant. Based on this analysis we could sort differences in steady state protein abundances into three categories, i.e. proteins that were detected only in either glucose- or ethanol-limited conditions, proteins that were statistically significantly up- or down regulated, and proteins of which the steady state expression levels were unchanged. The first category consists of spots that were exclusively detected in one nutrient limitation group, e.g. a spot is observed in the glucose-limited gels but undetectable in the ethanol-limited gels, or vice versa, which is indicated with G or E in Table I, respectively. In total, 15 of these so-called on/off spots were detected on our 2D gels, typical examples of which are shown in Fig. 1C. These spots were further analyzed with MS, resulting in identification of 11 unique proteins, namely Hkx1p, Pda1p, Adh1p, Cit1p, Idp2p, Lsc2p, Sdh1p, Mdh2p, Icl1p, Mls1p, and Pck1p, whereas some of these spots were revealed to originate from the same protein (protein isoforms, Table I). Interestingly, all proteins from these on/off spots play a role in the central carbon metabolism. Furthermore, we searched for the other proteins that are known to play a role in the central carbon metabolism, thus we compared our 2D images with yeast proteome maps available on the internet (www.ibgc.-bordeaux2.fr/YPM/, www.expasy.org/cgi-bin/map2/def?Yeast) to spot the location of proteins of interest and confirmed the identities of these spots using MS (examples are presented in Fig. 1D). All identified and quantified central carbon metabolism proteins are listed in Table I, and their relative expression changes are depicted in Fig. 2, in which the central carbon metabolism proteins are put into four different categories, namely glycolytic enzymes (gray), enzymes that convert pyruvate to ethanol and acetyl-CoA and vice versa (white), enzymes from the TCA cycle (black), and enzymes from gluconeogenesis and glyoxylate cycle (hatched). This figure clearly shows the clustering of the enzymes involved in pathways of the central carbon metabolism, which are expected to change upon different carbon source limitation.Table IRelative changes in protein expression in ethanol- versus glucose-limited cultured yeastProteinDescriptionORFNormalized averaged spot quantityFold change E/Gp-valuemRNA fold change E/G" @default.
• W2023177616 created "2016-06-24" @default.
• W2023177616 creator A5002927365 @default.
• W2023177616 creator A5013116231 @default.
• W2023177616 creator A5037845155 @default.
• W2023177616 creator A5053603496 @default.
• W2023177616 creator A5080059357 @default.
• W2023177616 date "2005-01-01" @default.
• W2023177616 modified "2023-09-27" @default.
• W2023177616 title "Comparative Proteome Analysis of Saccharomyces cerevisiae Grown in Chemostat Cultures Limited for Glucose or Ethanol" @default.
• W2023177616 cites W1605514660 @default.
• W2023177616 cites W1966809971 @default.
• W2023177616 cites W1969416735 @default.
• W2023177616 cites W1972520547 @default.
• W2023177616 cites W1977664347 @default.
• W2023177616 cites W1984839760 @default.
• W2023177616 cites W1985447576 @default.
• W2023177616 cites W1987075204 @default.
• W2023177616 cites W1990744940 @default.
• W2023177616 cites W2008830511 @default.
• W2023177616 cites W2009188703 @default.
• W2023177616 cites W2023765770 @default.
• W2023177616 cites W2034121267 @default.
• W2023177616 cites W2043168428 @default.
• W2023177616 cites W2046385725 @default.
• W2023177616 cites W2078594804 @default.
• W2023177616 cites W2081663028 @default.
• W2023177616 cites W2084247229 @default.
• W2023177616 cites W2084338561 @default.
• W2023177616 cites W2084687433 @default.
• W2023177616 cites W2088933296 @default.
• W2023177616 cites W2092265987 @default.
• W2023177616 cites W2101348546 @default.
• W2023177616 cites W2101442503 @default.
• W2023177616 cites W2102878034 @default.
• W2023177616 cites W2113905862 @default.
• W2023177616 cites W2114071827 @default.
• W2023177616 cites W2114417330 @default.
• W2023177616 cites W2126024920 @default.
• W2023177616 cites W2129983577 @default.
• W2023177616 cites W2131603432 @default.
• W2023177616 cites W2135920982 @default.
• W2023177616 cites W2147892681 @default.
• W2023177616 cites W2150566147 @default.
• W2023177616 cites W2151258701 @default.
• W2023177616 cites W2162725033 @default.
• W2023177616 cites W2169875902 @default.
• W2023177616 cites W2170177782 @default.
• W2023177616 cites W2170924872 @default.
• W2023177616 doi "https://doi.org/10.1074/mcp.m400087-mcp200" @default.
• W2023177616 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15502163" @default.
• W2023177616 hasPublicationYear "2005" @default.
• W2023177616 type Work @default.
• W2023177616 sameAs 2023177616 @default.
• W2023177616 citedByCount "75" @default.
• W2023177616 countsByYear W20231776162012 @default.
• W2023177616 countsByYear W20231776162013 @default.
• W2023177616 countsByYear W20231776162014 @default.
• W2023177616 countsByYear W20231776162015 @default.
• W2023177616 countsByYear W20231776162017 @default.
• W2023177616 countsByYear W20231776162018 @default.
• W2023177616 countsByYear W20231776162019 @default.
• W2023177616 countsByYear W20231776162020 @default.
• W2023177616 countsByYear W20231776162021 @default.
• W2023177616 countsByYear W20231776162022 @default.
• W2023177616 countsByYear W20231776162023 @default.
• W2023177616 crossrefType "journal-article" @default.
• W2023177616 hasAuthorship W2023177616A5002927365 @default.
• W2023177616 hasAuthorship W2023177616A5013116231 @default.
• W2023177616 hasAuthorship W2023177616A5037845155 @default.
• W2023177616 hasAuthorship W2023177616A5053603496 @default.
• W2023177616 hasAuthorship W2023177616A5080059357 @default.
• W2023177616 hasBestOaLocation W20231776161 @default.
• W2023177616 hasConcept C104397665 @default.
• W2023177616 hasConcept C125111812 @default.
• W2023177616 hasConcept C185592680 @default.
• W2023177616 hasConcept C2777576037 @default.
• W2023177616 hasConcept C2779222958 @default.
• W2023177616 hasConcept C2780161600 @default.
• W2023177616 hasConcept C523546767 @default.
• W2023177616 hasConcept C54355233 @default.
• W2023177616 hasConcept C55493867 @default.
• W2023177616 hasConcept C86803240 @default.
• W2023177616 hasConceptScore W2023177616C104397665 @default.
• W2023177616 hasConceptScore W2023177616C125111812 @default.
• W2023177616 hasConceptScore W2023177616C185592680 @default.
• W2023177616 hasConceptScore W2023177616C2777576037 @default.
• W2023177616 hasConceptScore W2023177616C2779222958 @default.
• W2023177616 hasConceptScore W2023177616C2780161600 @default.
• W2023177616 hasConceptScore W2023177616C523546767 @default.
• W2023177616 hasConceptScore W2023177616C54355233 @default.
• W2023177616 hasConceptScore W2023177616C55493867 @default.
• W2023177616 hasConceptScore W2023177616C86803240 @default.
• W2023177616 hasIssue "1" @default.
• W2023177616 hasLocation W20231776161 @default.
• W2023177616 hasLocation W20231776162 @default.
• W2023177616 hasOpenAccess W2023177616 @default.
• W2023177616 hasPrimaryLocation W20231776161 @default.

• next