## 4 Discussion  
Calcineurin signaling is a key mechanism of adaptation in _C. neoformans_; it coordinates the thermal stress response through transcription factor dephosphorylation, spatial localization changes, and global transcriptional responses, leading to changes in cell wall dynamics, growth, virulence, and drug resistance, among other stress responses. The best-known calcineurin target is the Zinc-Finger protein Crz1, which triggers a calcineurin-dependent transcriptional response [6]. However, phenotypic analysis indicates that calcineurin acts in a Crz1-dependent and Crz1-independent manner in _C. neoformans_, suggesting that the complex has diverse means of signaling and different targets [15]. Indeed, in _C. neoformans_, calcineurin targets go beyond transcription factors. Our previous work also showed that Cna1 physically interacts with the Cys3 transcription factor, ATP sulfurylase, and Gpp2, a phosphatase involved in glycerol biosynthesis and osmoregulation [19; 20; 21]. In this paper, we show that calcineurin mutants are mildly sensitive to cationic stress compared to _gpr2\(\Delta\)_, which is highly sensitive to hyperosmotic shock. This difference is likely because glycerol biosynthesis can be induced at the transcriptional level by Hog1, which is presumably still active in the calcineurin mutants, allowing at least a partial cationic stress response at the transcriptional level [34], whereas glycerol biosynthesis cannot be accomplished without Gpp2 activity. However, _cna1\(\Delta\)_ and _cnb1\(\Delta\)_ mutants undergo low glycerol accumulation during cationic stress to the same degree as _gpp2\(\Delta\)_, suggesting that one reason why calcineurin mutants are sensitive to cationic stress is the inability to accumulate glycerol, as shown in Figure 1A,B, due to post-translational modification in the Gpp2 protein, which leads to its accumulation as a lower-molecular-weight protein compared to the wild type and the lack of proper subcellular localization (Figures 2 and 3). In _S. cerevisiae_, Hog1, calcineurin, and Torc2-Ypk1 signaling pathways share the role of promoting the cationic stress response [35; 36; 37; 38]; however, to our knowledge, the direct link between calcineurin and glycerol biosynthesis has not been documented in _C. neoformans_, despite earlier reports on the osmosensitivity of calcineurin mutants in _C. neoformans_, which is also dependent on the serotype [10]. While having multiple signaling systems controlling a specific osmotic stress response is not without precedent, corroborating our findings as mentioned above, the mechanisms through which calcineurin takes part in the osmotic stress response are diverse in _C. neoformans_ and _S. cerevisiae_, according to our data. In _S. cerevisiae_, part of the ion homeostasis response is calcineurin-regulated, since _ENA1_ transcription (Na\({}^{+}\) and K\({}^{+}\) influx pump), among others, is calcineurin-dependent [35; 39]. On the contrary, in _C. neoformans_, _ENA1_ and _NHA1_ (Na\({}^{+}\) efflux pump) expression have been reported as Hog1-dependent [34; 40]. We searched our transcriptomes under cationic stress for these genes, and the data indicated that calcineurin does not regulate these genes in _C. neoformans_ as it does in _S. cerevisiae_. In spite of the lack of evidence on the transcriptional control of calcineurin over cationic stress, similar to what has been found in _S. cerevisiae_, calcineurin mutants in _C. neoformans_ are sensitive to cationic stress (Figure 1). Moreover, physical and functional links between calcineurin and Gpp2 have been found [17; 19]. In the present work, we showed two important modifications of the Gpp2 protein linked to calcineurin activity: lower-molecular-weight Gpp2 protein (Figure 2) and abnormal localization (Figure 3) were observed in calcineurin mutant background strains. Also, our data suggest that the interaction between calcineurin and Gpp2 is important to glycerol accumulation, which, once broken, leads to growth arrest in response to cationic stress.