The percentage of cell viability in cultures with amino acid supplementation was higher than in the culture without amino acid supplementation, and reached almost the same value as the control sample (90%) 12 h after application of the shock conditions. Yeast responds to osmotic stress by regulation of the uptake of amino acids in protein synthesis [319].
Previous work [27] (Chapter 4) has shown that the expression of Hsp70p, Hsp60p and
Hsp12p decreased in response to VHG conditions, with these observation also being in agreement with transcriptomic data observed by Varel et al. [319].
In this study, as seen in Table 5.2, the relative expression of Hsp12p, Hsp24p, and Hsp26p decreased at 2 and 10 h after application of the glucose shock. Beyond that time, however, the expression level of these proteins recovered, and their relative abundances surpassed those of the non-shocked control samples (see Table 5.2 for details). Furthermore, high concentrations of most intracellular amino acids were also found at this time (refer to Figure 5.3 for details), suggesting that the addition of these amino acids might induce protein synthesis in S. cerevisiae in response to high glucose stress. Data observed here are also in agreement with data observed for immobilized cells under VHG conditions with amino acid supplementation (when compared to free cells or immobilized cells under VHG without amino acid supplementation) [284]. Furthermore, the expression of other heat- shock proteins (such as Ssa1p, Ssa2p, Ssa4p, Ssb1p, Ssb2p, Ssc1p and Sse1p) showed a slight decrease after 2 h of shock conditions, however, their expression levels recovered at 10 h and 12 h (when compared to the control sample). Further detail is available elsewhere [27, 284] (Section 4.4 and 7.4). These data suggest that heat-shock proteins may play a role in protecting the protein biosynthesis machinery. Together with the differential regulation of these heat-shock proteins, the expression of Gpd1p (glycerol-3-phosphate dehydrogenase) was also up-regulated during sampling time. This finding is in agreement with previous work [27] (Chapter 4), since high levels of glycerol was formed at the beginning of fermentation under VHG conditions [27] (Chapter 4).
The cell viabilities were observed to decrease by 42%, and 33% for cells grown in VHG conditions both without and with amino acid supplementation compared to control samples (15%) during the first few hours. However, the cell viability recovered faster for cells grown in these stress conditions with amino acid supplementation (data not shown). The high concentrations of intracellular leucine, methionine, and alanine might have a
relationship to overall protein synthesis. Therefore, data generated here suggests that the survival and growth of cells in response to VHG conditions might relate to the intracellular amino acids uptake and protein synthesis (this was seen at 10 h after stress conditions were applied). Of course, further work is required in the future to validate this.
Figure 5.6. A representation of the response of yeast to osmotic stress. Bold arrows are representative for flux of molecules (such as water, ions, trehalose, glycogen), as well as physical forces (such as turgor pressure). Dashed and plain arrows are representative for putative pathways, interactions, chains of events that may be triggered, enhanced (+) or negatively affected (-) by osmotic stress. This picture and legends are reproduced from [268].
In response to stress conditions, yeast regulates the expression of its proteins in order to mitigate the stress. The general effects of osmotic stress conditions on protein expression in yeast are shown in Figure 5.6. Membrane proteins may play an important role for yeast to sense the changes in its environment. Indeed, the signaling of high osmolar concentrations are sensed by the plasma membrane which then might transmit this signal through post- translational protein modifications. This process might be carried out by proton- translocating plasma membrane ATPases relating to the maintenance of the intracellular pH
(pHi) homeostasis [320]. It has been shown previously that these proteins are involved with stress conditions because of the stress-induced intracellular acidification resulting from exposure of yeast to adverse conditions, as a consequence of dissipation of the electrochemical gradient [268]. A sudden increase in external osmolarity is sensed mainly by the membrane (see Figure 5.6), subsequently, as a result the loss of turgor pressure would occur that would stimulate and modify the activity of protein kinases, changing protein expressions [268]. Most of this response is to protect and recover processes, for example trehalose synthesis, and the recovery of expressions of heat-shock proteins [268].
5.4.6. The expression of proteins related to translation processes (aminoacyl-tRNA biosynthesis) and yeast growth
Most detected transfer RNA (tRNA) synthetase proteins were significantly increased in relative abundance at 10 h following glucose shock. This might be a result of the stimulation of high intracellular acid concentration observed at this time. Although all tRNA synthase proteins relating to amino acids processes were not detected in this study, but most tRNA synthase proteins detected here were significantly changed. Each tRNA is aminoacylated with specific amino acids by an aminoacyl tRNA synthetase as follows:
Amino acid + ATP → aminoacyl-AMP + PPi
and then: Aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP [321].
Furthermore, aminoacyl-tRNA synthetases catalyse the esterifications of specific amino acids to one of their compatible cognate tRNAs to form aminoacyl-tRNA, required for protein biosynthesis. tRNA proteins detected here included aspartyl-tRNA synthetase (Dps1p), leucyl-tRNA synthetase (Cdc60p), Glycyl-tRNA synthetase (Grs1p), lysine-tRNA synthetase (Krs1p), asparaginyl-tRNA synthetase (Ded81p), and phenylalanyl-tRNA synthetase (Frs1p). A summary of the expression changes for these proteins is depicted in Table 5.2. Briefly, these synthetases are essential, as well as being related to the regulation of amino acids biosynthesis and amino acids transport [322].
The expression of most ribosomal proteins here increased significantly at 12 h, especially for, Rpl19ap, Rpl19bp. Obviously, the high expression of these proteins at 10 h and then 12 h resulted in an increase of yeast growth found especially at 12 h. Moreover, the expressions of ribosomal proteins were up-regulated at the early of shock conditions applied. These proteins, including Rpl8ap, Rp18bp, Rpl15ap, Rpl15bp, Rpl19ap, and Rpl19bp are components of the large 60S ribosomal subunits, while Rps0ap, Rps0bp, Rps6ap, Rps24ap, and Rps24bp are components of the small 40S ribosomal subunits. In yeast, the biogenesis of the ribosome is an evolutionarily conserved process that starts in the nucleolus with transcription of rRNA precursors and that ends in the cytoplasm with the formation of the mature 40S and 60S ribosomal subunits [323]. Ribosomes are known as the core of the translation machinery, they are large ribonucleoprotein particles comprised of two unequally-sized subunits (large subunit - 60S and small subunit - 40S), that perform the protein synthesis. Indeed, ribosomes have two main functions, decoding the message and the formation of peptide bonds [324]. The synthesis of the ribosome is almost performed in a specialized nuclear compartment, the nucleolus [325]. Therefore, the up- regulation of these proteins at 10 h (with amino acid supplemented cultures) may have led to an increase in overall protein synthesis rates, resulting in the increased cell growth seen at this time. Indeed, for example, the deletion of RPS0 reduces cell growth rate, moreover, RPS0 is required for maturation of 18 rRNA, and the deletion of both genes is lethal [325, 326].