The fermentation conditions as well as the industrial strain (EC1118) used in this study are identical to those reported in (Rossignol et al. 2003) for transcriptome analysis and the sampling stages for proteome analysis have also been chosen according to those previously described, making these data directly comparable. Culture samples from two stages of yeast growth showing strong differences in transcript regulation were subjected to two-dimensional gel electrophoresis. The first sampling stage (reference stage) is located at the start of the exponential phase (Rossignol et al. 2003). The second sampling stage (stationary stage) consisted of cells in the stationary phase, which face 6% ethanol (Rossignol et al. 2003). We also sampled a third intermediate stage (Rossignol et al. 2003), which is located at the end of the growth phase. Preliminary results indicated few differences between this later stage and the stationary stage (data not shown). Thus, we focused our analysis on the reference and stationary stage. Figure 1a shows the two-dimensional gel proteome map at the start of the growth (reference stage) and Fig. 1b shows the two-dimensional gel proteome map at the stationary stage. We detected up to 744 spots, present in the two stages analysed. Our analysis detected 89 spots for which the intensity was at least twofold stronger in stationary phase than in reference stage and 124 spots for which it was at least twofold weaker. Typical examples of regulated spots are shown in Fig. 1c. Protein identification was performed by selecting the spots with the highest or most variable abundance at the stages analysed. Two methods were used for identification: mass spectrometry and gel matching. Interestingly, the two-dimensional gel map obtained was much more similar to industrial brewery yeast gel map (Kobi et al. 2004), and to a lower extent to distilling strain gel map (Hansen et al. 2006), than those from laboratory strains (Boucherie et al. 1995) in term of spot intensity and distribution. Eighty-four spots were identified among the most intense and the most regulated spots. Thirty-four spots were identified by mass spectrometry and 50 by gel matching. Among the spots identified by mass spectrometry, 10 were first identified by gel matching and confirmed by mass spectrometry (Hxk2, Eno1, Pdc1, Adh1, Tps1, Asn1, Met17, Met6, Yef3, Vma2). Overall, spots identified correspond to 59 proteins, as several spots corresponded to the same protein. The proteins were identified and listed by function (Table 1). The functional category most represented is glycolysis and ethanol production, with 10 proteins identified corresponding to 21 spots. Enolase 2 and alcohol dehydrogrenase 1 were the two most expressed proteins in our strain and under our experimental conditions. These proteins represent the major part of the proteins detectable at the reference and stationary stages, with 28% and 36% of the overall spot intensity, respectively. Most of these proteins are induced between the reference and stationary stage (Hxk2, Glk1, Fba1, Tpi1, Eno1, Adh1) and only Pdc1 is repressed. The proteins involved in glycerol production (Rhr2, Hor2, Gpd1) and other carbohydrate metabolic pathways (Tps1, Ino1, Zwf1) all show a similar induction pattern.
The amino acid metabolism category had the highest number of proteins identified (12 proteins). Most of these proteins were more expressed in the stationary stage than in the reference stage (Leu2, His4, Asn1, Cys3, Gdh1, Met17, Sam2) and a few were repressed (Lys20, Ilv5). Met6, Met17 and Sam2, involved in sulphate assimilation, were highly expressed under our fermentation conditions. They represent 7% of the total number of spots quantified on the gels in the stationary stage. Quantitatively, this is the second most important metabolic pathway after glycolysis. This metabolic pathway is important in wine yeast because its function determines the production of H2S, which is an undesirable by-product given its negative sensorial impact. Interestingly, proteins of the sulfur pathway are among the most expressed. They are involved in steps located downstream of H2S production and are thought to be potentially critical for H2S release. A high activity of these enzymes may be one condition enabling yeast to produce only low amounts of H2S, thus leading to its selection. All of the proteins involved in protein synthesis identified are down-regulated in the stationary phase during the fermentation, whereas Prb1, a protein which participated in protein degradation, is induced. This correlates with the decrease in protein synthesis observed at this stage of fermentation (Salmon 1989) and with the decrease of mRNA abundance of ribosomal proteins previously described (Rossignol et al. 2003). At the stationary stage, assimilable nitrogen is depleted from the medium (Rossignol et al. 2003) and cells probably undergo amino acid recycling. Moreover, induction of Prb1p during grain fermentation in industrial distilling fermentors has already been described (Hansen et al. 2006). This is also consistent with the idea that most protein degradation during starvation depends on vacuolar proteases (Egner et al. 1993).
Several proteins involved in stress response have been identified and show various types of regulation. Most are repressed (Ssa2, Sti1, Sse1, Hsp78, Tom70) and two are induced: Hsp26 (heat shock protein) and Ahp1 (thioredoxin peroxidase). Most chaperones involved in protein folding (cytoplasmic or mitochondrial) are down-regulated, probably in relation to the decrease in protein synthesis, whereas heat-shock proteins with specific functions are induced. Trabalzini et al. (2003) reported a similar pattern with an enological yeast in YPD modified medium at a later time point. Several Hsp proteins were down-regulated, whereas Hsp26 and oxidative stress proteins were induced. Hsp26 was more highly expressed in a strain with a good fermentation profile than in a strain leading to sluggish fermentation at the entry into stationary phase in industrial conditions (Zuzuarregui et al. 2006). The authors have suggested that this protein is important for adaptation to the stationary phase in alcoholic fermentation. Authors ended with the same conclusion for the product of YPR127w (Zuzuarregui et al. 2006), which is also induced in the present work (Table 1).