Given our findings that Cas5 governs cell cycle dynamics and cell wall stress responses, we next explored the mechanisms by which it is regulated. Initially, we monitored the subcellular localization of Cas5 by indirect immunofluorescence in a strain in which both alleles of CAS5 were HA-tagged. The Cas5-HA protein was functional and sufficient to confer wild-type tolerance to caspofungin (Supplementary Fig. 3a). Under basal conditions, Cas5 appeared diffuse throughout the cytoplasm, however following treatment with caspofungin, Cas5 translocated to the nucleus (Fig. 5a). Owing to the moderate expression of Cas5 under its native promoter, we employed a strain in which we could overexpress Cas5 to facilitate its localization in the absence of stress. Overexpression of HA-tagged Cas5 confirmed that Cas5 was mostly cytoplasmic, as observed when under its native promoter (Fig. 5b). Overexpression also enabled us to observe a subpopulation of cells in which Cas5 was localized to the nucleus even in the absence of cell wall stress (Fig. 5b). Next, we assessed changes in Cas5 abundance in response to caspofungin. CAS5 transcript and protein levels were induced in response to cell wall stress, as measured by qRT-PCR and western blot analysis (Fig. 5c, d and Supplementary Fig. 2b). Thus, in response to cell wall perturbation by caspofungin, Cas5 is induced and translocates to the nucleus.Fig. 5Cas5 becomes activated by dephosphorylation in response to cell wall stress. a Cas5 localizes to the nucleus in response to caspofungin. Cells were fixed and Cas5 (red) was detected by indirect immunofluorescence using α-HA antibody and α-mouse IgG-Cy3. Nuclei (green) were visualized by DAPI staining. Scale bar represents 5 μm. b Cells were subcultured in rich medium to log phase and fixed. HA-tagged Cas5 (red) and nuclei (green) were visualized as in a. Scale bar represents 5 μm. c CAS5 is induced by caspofungin. The transcript level of CAS5 was monitored by qRT-PCR and normalized to GPD1. Error bars represent standard deviation (s.d.) from the mean of triplicate samples. Significance was measured with an unpaired t test in GraphPad Prism (****P < 0.0001). d Levels of Cas5 were monitored by western blot and detected with an α-HA antibody. Actin was detected with an α-β-actin antibody as a loading control. Full blots are shown in Supplementary Fig. 2b. e Cas5 is post-translationally modified upon cell wall stress treatment. Cells were grown to log phase and subsequently treated with 125 ng/ml of caspofungin or 50 μg/ml of calcofluor white for 1 h. Total protein was resolved by SDS-PAGE and the blot was hybridized with an α-HA to monitor Cas5 migration. Full blots are shown in Supplementary Fig. 2c. f Cas5 migration and actin detection were monitored as part d. Full blots are shown in Supplementary Fig. 2d. g Protein lysates were subjected to two-dimensional gel electrophoresis coupled with western blotting. Cas5 was detected with an α-HA antibody. h Cas5 is phosphorylated in the absence of stress. Cas5 migration was monitored by western blot and detected with an α-HA antibody. Treatment of protein lysate with lambda phosphatase resulted in a faster migrating band. Full blots are shown in Supplementary Fig. 2e. i Cas5 is required for caspofungin (CF)-induction of cell wall genes. Transcript levels of ECM331 and PGA13 were monitored by qRT-PCR and normalized to GPD1. Error bars represent s.d. from the mean of triplicate samples. Significance was measured with a Tukey’s multiple comparisons test in GraphPad Prism (****P < 0.0001, **P < 0.01)