## 3 Results  
gpp2 \(\Delta\), cnal \(\Delta\), and cnb1 \(\Delta\) Mutants Are Sensitive to Cationic Stress and Fail to Accumulate Glycerol  
Reports from our group have shown that cationic homeostasis depends upon Gpp2 phosphatase and that glycerol supplementation of the medium remediates cationic stresssensitivity in the \(gpp2\Delta\) strain [21]. Also, our previous work and others have shown that Gpp2 is a target of the calcineurin complex, and it physically interacts with Cna1\(\Delta\)C and Cnb1 [17; 19]. Based on these observations, we hypothesized that the calcineurin complex controls cationic stress, at least partially, by activating the Gpp2 protein and glycerol biosynthesis. In order to challenge this hypothesis, first, we tested whether \(cna1\Delta\) and \(cnb1\Delta\) strains would show growth deficiency under cationic stress (NaCl, KCl, and LiCl). Also, we added \(gpp2\Delta\) and \(met3\Delta\) (ATP sulfurylase) mutants to this experiment [20; 21]. The first was a positive control since we know it is hypersensitive to NaCl and KCl, and the latter was added to the analysis because ATP sulfurylase physically interacts with Cna1. Therefore, the mutants' growth may also be affected by cationic stress. Figure 1A shows that all strains tested are sensitive to 1 M NaCl and KCl and 90 mM LiCl compared to the wild type to different degrees. Notably, the \(gpp2\Delta\) and \(met3\Delta\) strains are hypersensitive to NaCl/KCl and LiCl, respectively, whereas both calcineurin mutants are sensitive to all salts used. This result suggests that calcineurin is involved in cationic homeostasis, probably by, directly or indirectly, causing post-translational modifications in the Gpp2 protein. Figure 1: Sensitivity to cationic stress in \(cnb1\Delta\), \(cna1\Delta\), \(met3\Delta\), and \(gpp2\Delta\) mutant strains compared to the wild type (H99). (**A**) The growth phenotype of wild-type and mutant strains cultures in YEPD medium containing NaCl, KCl (1 M each), and LiCl (0.09 M). The plates were incubated at 30 \({}^{\circ}\)C for 48 h. (**B**) The quantification of intracellular glycerol in \(cna1\Delta\), \(cnb1\Delta\), and \(gpp2\Delta\) mutant and wild-type H99 strains. The growth of wild-type and mutant strains occurred in YPD medium with (gray bars) and without (black bars) 1 M NaCl supplementation at 30 \({}^{\circ}\)C overnight at 150 rpm. A two-way ANOVA multiple-comparison test was used to find relevant statistical differences (\(p\) value \(<\) 0.0001 ***). Since Gpp2 is responsible for the last step in glycerol biosynthesis and, theoretically, its deletion would lead to low intracellular glycerol, we carried out glycerol quantification in the wild type, \(cna1\Delta\), \(cnb1\Delta\), and \(gpp2\Delta\) in the presence or absence of NaCl. Figure 1B shows that in the wild-type strain (H99), the glycerol content increases significantly in the presence of NaCl; however, all three mutants fail to accumulate glycerol in the presence of NaCl. Taken all together, these results indicate that glycerol is an important osmolyte during cationic stress, and its biosynthesis and accumulation are dependent on Gpp2, a glycerol phosphate phosphatase, which may be activated by the calcineurin signaling complex. Cna1 and Cub1 Subunits Are Required for Proper Gpp2 Protein Integrity and Localization to Cytoplasmic Puncta  
Since calcineurin is a serine/threonine phosphatase, we checked whether a Gfp-Gpp2 fusion protein would be modified by deleting the genes encoding the regulatory (\(CNB1\)) and catalytic (\(CNA1\)) calcineurin subunits. First, Western blotting was carried out in the absence or presence of NaCl in the wild-type (CNU151) strain containing the _GFP-GPP2_ allele. As shown in Figure 2A, a 75 KDa band was observed, as expected from the theoretical molecular weight calculated for the fusion protein. In the presence of 1.5 M NaCl, besides the main band at 75 KDa, a lower-molecular-weight band was detected in response to NaCl added to the medium (Figure 2A).