## 3 Results
The same behavior can be observed for the Gfp-Gpp2 fusion protein in the \(cnb1\Delta\) strain (Figure 2B). However, in the \(cna1\Delta\) strain, the 75 KDa band was abolished, and only the lower-molecular-weight band was detected (Figure 2B). This result suggests that the Cna1 catalytic subunit is required to maintain the full-length Gpp2. Its absence seems to lead to Gfp-Gpp2 being processed to a lower-molecular-weight band, estimated at 60 KDa. Also, the wild-type and mutant strains (\(cna1\Delta\) and \(cnb1\Delta\)) containing the _GFP-GPP2_ allele were exposed to 100 \(\mu\)g/mL of cyclosporine A, which was previously described as a calcineurin inhibitor [30], and Western blotting was performed. Figure 2C shows that the inactivation of calcineurin in the wild-type strain leads to a progressive increase in Gfp-Gpp2 accumulation after 2 and 4 h post-induction. The same can be seen for the \(cnb1\Delta\) strain, where the main 75 KDa band accumulates in larger amounts after 2 and 4 h; in addition, the \(cnb1\Delta\) strain has the 60 KDa band in higher amounts as well. CsA had no observable effect on the Gfp-Gpp2 band in the \(cna1\Delta\) background strain. This result suggests that Gpp2 is a target of calcineurin-mediated post-translational modifications. Fluorescence microscopy was performed to detect the subcellular localization of Gfp-Gpp2 in the same conditions as the Western blots. In the wild type, Gfp-Gpp2 is distributed homogeneously in the cytoplasm. However, after 2 h, in the presence of NaCl, the Gfp-Gpp2 protein accumulated in distinct punctual dots in the cytoplasm (Figure 3A). These cytoplasm puncta increased from an average of one to more than four puncta per yeast cell in the presence of NaCl (Figure 3B). Fluorescence microscopy of the mutant strains \(cna1\Delta\) and \(cnb1\Delta\) carrying the _GFP-GPP2_ allele showed no particular organization as cytoplasmic puncta, either in the absence or in the presence of NaCl (Figure 3C). The effect of cyclosporine A was analyzed, and after 4 h in the presence of NaCl and 100 \(\mu\)g/mL of CsA, the Gfp-Gpp2 puncta localization was lost (Figure 3D). The data suggest that the Gfp-GPP2 protein undergoes post-translational modifications and distinct localization during cationic stress; both processes depend on calcineurin. Figure 2: The Western blotting expression pattern of the Gfp-Gpp2 protein. (**A**) A Western blot of total proteins extracted from the wild-type strain (CNU151) expressing the _GFP-GPP2_ allele cultivated for 2 h in SD + N (30 \({}^{\circ}\)C) in the presence or absence of 1.5 M NaCl. (**B**) A Western blot of total proteins extracted from the wild-type strain (CNU151) and calcineurin mutant strains (CNU189, _cnb1\(\Delta\)_, and CNU193 _cna1\(\Delta\)_) expressing the _GFP-GPP2_ allele cultivated for 2 h in SD + N (30 \({}^{\circ}\)C) in the presence or absence of 1.5 M NaCl. (**C**) A Western blot of total proteins extracted from the wild-type strain (CNU151) and calcineurin mutant strains (CNU189, _cnb1\(\Delta\)_, and CNU193 _cna1\(\Delta\)_) created with 100 \(\mu\)g/mL of cyclosporin A (CsA) for 0, 2, or 4 h in SD + N (30 \({}^{\circ}\)C). The mouse anti-GFP primary antibody was used at a 1:7000 dilution, and the anti-mouse horseradish peroxidase-linked secondary antibody was applied at a 1:2000 dilution. Loading control was performed by the detection of the Histone H3 protein with the rabbit anti-His3 primary antibody (1:2000) and anti-rabbit horseradish peroxidase-linked secondary antibody (1:2000). Figure 3: _Cont_. Global Transcriptional Profiles of the gpp2\(\Delta\), \(cna1\Delta\), and \(cnb1\Delta\) Strains under Cationic Stress  
Our data support that calcineurin contributes to the cationic stress response by causing Gpp2 protein modifications and altering its spatial localization, culminating in glycerol biosynthesis.