Fig. 7
Cas5 activation in response to caspofungin is coupled with dephosphorylation by the protein phosphatase Glc7. a Depletion of GLC7 confers sensitivity to caspofungin. Caspofungin susceptibility assays were conducted in YPD medium in the presence or absence of 1 μg/ml of doxycycline (DOX). Growth was measured by absorbance at 600 nm after 48 h at 30 °C. Optical densities were averaged for duplicate measurements. Data were quantitatively displayed with color using Treeview (see color bar in Fig. 2). b Upregulation of Cas5 expression does not depend on Glc7. CAS5-HA/CAS5 and CAS5-HA/CAS5 tetO-GLC7/glc7Δ strains were cultured in the absence or presence of doxycycline and caspofungin, as indicated. Cas5 was monitored by western blot and detected with an α-HA antibody. Hsp90 protein levels served as a loading control. Full blots are shown in Supplementary Fig. 2g. c Post-translational modification of Cas5 is absent upon GLC7 depletion. The western blot was performed as described in b, except caspofungin treated samples were diluted fivefold to achieve equal loading of Cas5. Full blots are shown in Supplementary Fig. 2h. d Dephosphorylation of Cas5 is required for the induction of cell wall genes in response to caspofungin treatment. CAS5-HA/CAS5 and CAS5-HA/CAS5 tetO-GLC7/glc7Δ strains were grown in the absence or presence of doxycycline (DOX) or caspofungin, as indicated. The transcript levels of ECM331 and PGA13 were monitored by qRT-PCR and normalized to GPD1. Plotted are the fold-changes in transcript levels in the presence of caspofungin relative to untreated. Error bars represent standard deviation (s.d.) from the mean of triplicate samples. The treatment conditions were compared using a Tukey’s multiple comparisons test in GraphPad Prism (****P < 0.0001, ***P < 0.001)
