<p>The goal of this research was to use two-dimensional electrophoresis to examine changes in abundance of enzymes of the glycolytic pathway in the yeast Saccharomyces cerevisiae grown on carbon sources that support either fermentation to ethanol or oxidative metabolism. Large-scale profiling of protein abundances (expression proteomics) often detects changes in protein abundance between physiological states. Such changes in enzyme abundance are often interpreted as evidence of metabolic change although most textbooks emphasise control of enzyme activities not enzyme amount. Two-dimensional difference gel electrophoresis (2DDIGE) was therefore used to examine differences in protein abundance between S. cerevisiae strain BY4741 grown on either glucose (fermentation) or glycerol. Growth on 2% glucose, but not on glycerol, was accompanied by extensive production of ethanol. Doubling times for growth were 2 h 5 min in glucose and 9 h 41 min in glycerol. Conditions for extraction and two-dimensional electrophoresis of proteins were established. One hundred and seventy nine proteins were identified by MALDI mass spectrometry of tryptic digests of protein spots excised from Coomassie stained gels. All of the enzymes for conversion of glucose to ethanol, except for the second enzyme of glycolysis phosphoglucose isomerase, were identified using twodimensional electrophoresis of 100 μg of protein from cells grown on 2% glucose. Identification of proteins excised from the DIGE gels was more challenging, partly because of the lower amount of protein. Eight of the proteins that showed statistically significant differences in abundance (≥ 2-fold, p ≤ 0.01) between glucose and glycerol were identified by mass spectrometry of proteins excised from the 2DDIGE gels, and a further 18 varying proteins were matched to proteins identified from the Coomassie stained gels. Of these total 26 identified or matched proteins, subunits of five of the enzymes for conversion of glucose to ethanol were more abundant from the fermentative cells grown on glucose. The more abundant glycolytic enzymes were phosphofructokinase 2, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase and enolase, plus pyruvate decarboxylase that was required for conversion of the glycolytic product pyruvate to acetaldehyde. The alcohol dehydrogenases Adh1 and Adh4 that convert acetaldehyde to ethanol were detected but did not vary significantly between growth on glucose or glycerol. The results confirmed that in this case changes in abundance of some enzymes were consistent with the altered metabolic output. Future studies should examine whether changes in the abundance and activity of these enzymes are responsible for the differences in metabolism.</p>
Proteomic analysis of Saccharomyces cerevisiae grown on glucose or glycerol
