## 4 Discussion
Our data do not allow us to conclude whether the modifications caused by calcineurin to the Gpp2 protein are direct or indirect or to deduce the nature of the modification. The fact that Gpp2 interacts physically with Cna1 suggests a direct interaction [19]. Similarly, the Tor pathway plays an important role in the cationic stress response in yeast [38]. One important post-translational mechanism is the regulation of Ypk1-Torc2-dependent kinase, which, in turn, leads to the dephosphorylation of Gpd1 during osmotic stress, rendering this protein active and, therefore, triggering glycerol accumulation [41]. Apparently, _S. cerevisiae_ and _C. neoformans_ use different signaling apparatuses to post-translationally regulate the glycerol biosyntheticpathway at different steps (Figure 5A) to guarantee efficient and quick regulation during cationic stress. Calcineurin and Crz1 localize to stress granules or P-bodies during heat stress [18], a localization pattern very similar to the one observed here for Gfp-Gpp2 (Figure 3). While our data do not allow us to conclude that Gpp2 is a resident protein of P-bodies, we can speculate that once it physically interacts with Cna1 and is concentrated in cytoplasmic puncta during cationic stress, it may migrate to stress granules in a calcineurin-dependent fashion. However, this hypothesis remains to be further explored. The compilation of previous and present data indicates that Gpp2 is a target of calcineurin, which is modified downstream of this signaling complex at the post-translational level, triggering glycerol biosynthesis [19; 21; 22]. In addition, C. _neoformans_ calcineurin does not seem to activate the main transcriptional targets like in _S. cerevisiae_, as mentioned before, suggesting that it may cause a different transcriptional response that has not been described. Under these circumstances, we aimed to learn more about the global transcriptional response generated during cationic stress when cells are unable to undergo glycerol biosynthesis. The results highlight that cationic stress causes a general carbohydrate metabolism adaptation in wild-type and glycerol-deficient mutant strains, highlighting changes in cell wall maintenance and chitin metabolism in all strains, which is in line with required modifications on the cell wall surface that are important for maintaining cell wall integrity and turgor during stress [42; 43]. However, carbohydrate metabolism is still very divergent between wild-type and glycerol-deficient strains (Figure 6A); the calcineurin and _gpp2\(\Delta\)_ mutants have a metabolic flow toward glyconeogenesis, glycosyl hydrolys, pentose phosphate pathway, and amino sugar and glycogen mobilization, highlighting the need for extra energy derived from alternative carbon sources during glycerol deficiency. This reinforces the link between cell wall integrity and carbon metabolism, which seem to be key factors when glycerol biosynthesis fails, probably by channeling the rescued alternative carbon to \(\beta\)-glucan biosynthesis. This model is supported by our observation that glycerol-deficient mutants have higher expression levels of glycogen phosphorylase (_S. cerevisiae__GPH_1 gene, CNAG_06666) and UDP-glucose pyrophosphylase (_S. cerevisiae__UGP_1, CNAG_02748), which are key steps in \(\beta\)-glucan metabolism [44; 45]. Furthermore, it is extremely important to raise the point that during cationic stress, the wild type's metabolism flows toward glycerol biosynthesis, with _GPD_ and _GPP_ genes being induced (Figure 5); on the contrary, in the glycerol-deficient mutants, metabolism flows to glycerol-3-phosphate, which probably serves as an intermediate metabolite to both glycerolipid biosynthesis and glycolysis, with _GPT_2/_SCT_1 and _GUT_2 being highly induced (respectively), suggesting that glycerolipid biosynthesis is favored, probably to contribute to plasma membrane dynamics during stress.