Amongst the amino acids supplied here, lysine is the only amino acid that is synthesised in yeast through a metabolic pathway entirely from the same pathway used in bacteria [305].
Moreover, lysine is also not consumed by yeast as a nitrogen source [306], but it plays a main role in the regulation of methionine, threonine, and arginine biosynthesis. This amino
acid interfaces with glutamate biosynthesis in yeast since the synthesis both of them originate from α–ketoglutarate [305].
The biosynthesis of valine and isoleucine are performed by sharing several proteins. Two proteins involving the biosynthesis of these amino acids are detected here including Ilv3p and Ilv5p. Surprisingly, the relative expression of these proteins were depressed under 2 h and 10 h compared to the control sample, but stimulated at 12 h. The main functions of these proteins are to generate and consume the α-aceto-α-hydroxy acids from the vicinal diketones, diacetyl and 2,3-pentadione [307]. Furthermore, these proteins are also required for maintenance of wild type mitochondrial DNA [308]. Moreover, the expressions of Leu4p and Met6p were also significantly up-regulated at 12h, while their expressions were down-regulated at 2 and 10 h (see Table 5.2 for detail). The observed expression changes for these proteins (Ilv3p, Ilv5p, Leu4p, Met6p) were in agreement with the observation of yeast growth, since high expressions of these proteins at 12 h led to an increase in growth from 12 to 24 h (data not shown). Levels of these were also agreement with the expression of Din7p (mitochondrial nuclease), since the function of this protein is in DNA repair and replication, as well as in regulation of the stability of the mitochondrial genome, moreover, this protein is induced during meiosis [309]. These proteomic data are also in agreement with the fluctuations of intracellular leucine and valine concentrations, since high concentrations were observed at 10 h and then 12 h (see Figure 5.3 for details).
Another protein of interest here was Bat1p, known as a mitochondrial branched-chain amino acid aminotransferase. This protein plays an important function in iron homeostasis by being related to the efficient transfer of a Fe-S cluster from the mitochondria (where the clusters are synthesised) to the cytosol, and this process involves the mitochondrial ABC transporter Atm1p [310] (see Figure 5.5). The process plays an important role in the maintenance of mitochondria function, since a disturbance has been shown elsewhere to lead to an accumulation of iron in the mitochondria, damaging and loss of mitochondrial
DNA [310]. In the maintenance of the mitochondria, protein Ilv5p has also been shown to play a function in enhancing stability of mitochondrial DNA [308]. Therefore, the increase in expression observed in Bat1p and Ilv5p under glucose shock stress might act to preserve the mitochondrial DNA.
Figure 5.5. The relationship of most amino acid metabolisms in relationship with proteins detected here (modified from [310]).
In S. cerevisiae, the Leu3p-α-isopropylmalate complex, which controls the rate of transcription of LEU1 and LEU2, is related to the regulation of ammonia assimilation by Gdh1p, and is induced to serve as a carrier function (bothat the plasma membrane and at the inner mitochondrial membrane),as well as a mitochondrial malic enzyme [310]. Protein Gdh1p was also up-regulated significantly at 12 h compared to the control sample. The expression of transketolase Tkl1p, a key enzyme in the pentose phosphate shunt [311, 312], was also up-regulated (see Table 5.2 for details).
Unlike the expressions of other proteins that were down-regulated at the beginning of the high glucose shock, the expression of Ura1p and Ura2p were up-regulated, with significantly increased expression at both 10 h and 12 h. Ura1p is a dihydroorotate dehydrogenase that catalyses the fourth step in the de novo biosynthesis of pyrimidines by converting dihydroorotic acid into orotic acid [313, 314]. It has been shown previously that a loss of Ura1p activity leads to a decrease in cell growth [315], and increased expression of this protein results from increased levels of the intermediate dihydoorotic acid [315]. A bifunctional carbamoylphosphate synthetase protein, Ura2p, catalyses the fourth step in this pathway, and this was up-regulated at both 10 h and 12 h. This protein contains two domains, an N-terminal carbamoyl phosphate synthetase domain (CPSase), and a C- terminal aspartate transcarbamoylase (ATCase) domain [316]. The CPSase domain converts glutamine to carbomoyl-phosphate, while the ATCase domain converts this intermediate (which is sequestered and channelled from the CPSase domain) to carbamyol- aspartate [317]. Moreover, Ura2p is integral in regulating the de novo synthesis of pyrimidine nucleotides [318]. Therefore, while Ura2p catalyses the first two steps of the de novo synthesis of pyrimidine ribonucleotides, Ura1p catalyses the fourth step in this pathway. Both Ura1p and Ura2p were significantly up-regulated at 10 h and then 12 h, suggesting that the de novo synthesis of pyrimidine ribonucleotides might be also activated at this time.