While the results reported were reproducibly obtained on all 2 D gels analyzed, it should be noted that some spots might have contained co migrating proteins that were not detected in the MALDI MS analysis. These proteins would have affected the relative quantification of the up regulated proteins. As MALDI MS identifies the prevalent proteins that are present in a gel sample, these errors are, however, considered to be negligible. Discussion The yeast transcription factor Yap1p is crucial for the normal response of yeast Inhibitors,Modulators,Libraries cells to a variety of stress con ditions including oxidative stress, drug induced stress, and heat shock. Previous studies indicated that most stress conditions induced the activity of Yap1p Inhibitors,Modulators,Libraries and, as a consequence, resulted in elevated expression of a number of genes encoding proteins that protect the cells against stress induced damage.

Although, Yap1p dependent expression of a diverse range of pro teins is essential for viability, a major unresolved ques tion concerns the complete pattern Drug_discovery of proteins expressed in a cell upon Yap1p overexpression. We re port here the first characterization of the proteome of Yap1p overexpressing yeast. The experimental approach enables the analysis of the relative protein levels under conditions that mimic stress. This resulted in many changes in the levels of proteins involved in crucial biological pathways. The glycolytic pathway plays a fundamental role in the provision of metabolic energy and intermediates during fermentative growth of the yeast S. cerevisiae.

The glycolytic enzymes, which are involved in the conversion of glucose to pyruvate, were significantly more abundant overexpressing yeast. Another isoenzyme, Pyk2p, was, however, not detected on the 2D gels. The regulation mode of pyruvate Inhibitors,Modulators,Libraries kinases Inhibitors,Modulators,Libraries is simi lar to that of hexokinases since Cdc19p is tightly regulated and activated by fructose 1,6 bisphophate, whereas Pyk2p is subject to glucose repression and appears to be insensitive to FBP levels. Relatively few of the identified proteins in the glycoly sis and pyruvate ethanol pathways exhibited more than two fold increment in the Yap1p overexpressing yeast. The response suggests that the levels are affected by Yap1p in different ways, and that other factors may also play a role in the regulation. Moreover, none of the enzymes in the citric acid cycle were found to be significantly up regulated upon Yap1p overexpres sion.

This is probably a result of the anaerobic cultiva tion conditions. During alcoholic fermentation of sugars, the glycolytic genes are the most efficiently expressed genes in yeast, and glycolytic enzymes comprise over 30% of the soluble cell protein. Moreover, two cru cial enzymes involved in the pyru vate ethanol pathway were significantly up regulated in the Yap1p overexpressing yeast, and that would probably result in a shortage of substrate for the TCA cycle.