## 4 Discussion  
PolyP is widely recognized as an anionic polymer that binds and de-toxifies heavy metal ions [41]. However, in yeast, particular mechanisms of polyP-involving detoxification are understudied. Manganese-adapted yeast cells contain longer polyP chains [42], but this could be a side effect of VTC4 activation by manganese ions [28]. PolyP content increases during the elongated lag phase in manganese excess conditions and decreases at the active growth stage [42]. It is known that the excess of manganese ions results in oxidative stress, while manganese-phosphate complexes provide antioxidant activity [26]. Thus, phosphate released through polyP degradation in yeast cells could allow for manganese adaptation. In bacteria, polyP is known to be necessary for oxidative stress adaptation [2] due to its ability to act as a chaperone [24; 25]. In our study, we found evidence for polyP involvement in stress response in yeast: The reduced polyP content through Ppn1 polyphosphatase overexpression leads to a significant alteration in expression of genes responsible for manganese and oxidative stress adaptation. Manganese homeostasis in _S. cerevisiae_ involves numerous proteins [51; 52; 26; 53]. The adaptation of yeast cells to toxic concentrations of manganese ions involves--in particular, the transport systems responsible for manganese compartmentalization, the systems of phosphorus transport and polyphosphate accumulation, as well as other membrane proteins. In this study, we identified new proteins contributing to manganese adaptation in _S. cerevisiae_. The protein encoded by _PHM7_ is essential for this process. The Phm7 protein sequence contains several transmembrane domains [54], and the protein is localized in the plasma membrane [55]. Its ortholog CaPhm7 in _Candida albicans_ is also localized in the plasma membrane, and participates in drug resistance and filamentous growth [56; 57]. The cells lacking  
Figure 6: The micrographs of the cells of the parent BY4743 strain and \(\Delta\)_plm7_ and \(\Delta\)_smr1_ knockout strains of _S. cerevisiae_ cultivated in YPD medium with (Mn) and without 5 mM MnSO4 to the stationary growth stage (72 h). Top—phase contrast, bottom—LIVE/DEAD Fungal Light Yeast Viability Kit staining. _CaPHM_7 are sensitive to SDS and ketoconazole, but resistant to rapamycin and zinc [56]. This evidence suggests that Phn7 is a membrane transporter of divalent metal cations. Another important observation is the role of Pho84 in oxidative stress response, in addition to its known role in phosphate and manganese transport. Pho84 is the major high-affinity plasma membrane phosphate importer of _S. cerevisiae_ and an important element of phosphate homeostasis [58]. Pho84 is a phosphate transceptor: It transports phosphate and mediates rapid phosphate activation of the protein kinase A (PKA) pathway [59]. This protein also acts as a divalent cation transporter [52]. The knockout strain shows multiple phenotypic changes: Poor growth on a low-phosphate medium, constitutive up-regulation of the PHO pathway, and negligible levels of polyP, enhanced arsenate [60], and manganese resistance [61]. The overexpression of _PHO84_ induces enhanced heavy metal accumulation [62]. In our case, _PHO84_ is down-regulated in the CRN/PPN1 strain that exhibits manganese and resistance to peroxide. In _C. albicans_, Pho84 participates in oxidative stress response: The _Alpha84_ mutant accumulates intracellular ROS in the absence of extrinsic oxidative stress and hyperactivates oxidative stress signaling [63]. Thus, _PHO84_ down-regulation enhances resistance to manganese by decreasing metal ion uptake and mobilizing oxidative stress signaling. This down-regulation can also cause resistance to peroxide. The mechanisms of _PHO84_ down-regulation in CRN/PPN1 are still in question. The intracellular phosphate level may be increased as a result of polyP hydrolysis in the PPN1 overproducing strain CRN/PPN1, which leads both to _PHO84_ down-regulation and an increase of the ROS level.