Figure 5.

Identification of the export regulatory region. (A) WT (ASY472) or cnb1Δ (ASY475) cells expressing 3xGFP–SV40NLS–Crz1p_186–340 (LMB160), 3xGFP–SV40NLS–Crz1p_186–310 (LMB180), or 3xGFP–SV40NLS–PIISIQΔ_186–340 (LMB228) were grown to log phase at 21°C. Cells were treated with 200 mM CaCl₂ and visualized using fluorescence microscopy. Bar, 20 μm. (B) Overexpression of activated calcineurin affects the localization of GFP–SV40NLS–Crz1p_186–340. ASY472 cells expressing CNA2 trunc or vector (pVT100-L) and LMB160, LMB180, or LMB228 were grown to log phase at 21°C and visualized using fluorescence microscopy. Bar, 20 μm. (C) Constructs containing the Crz1p export regulatory region interact with calcineurin in vivo. A two-hybrid strain (PJ69-4A) containing a HIS3 reporter and expressing GAL4AD–Crz1p fusions LMB189, LMB226, or LMB224 and GAL4BD–CNA1 (BJP2014) alone or in combination were spotted on plates with or without histidine plus 100 mM CaCl₂ and grown at 30°C for 2–3 d. Cells were spotted using fivefold serial dilutions beginning at an OD₆₀₀ of 1.0. (D) The Crz1p/Msn5p interaction is dependent on Crz1p phosphorylation state. WT and cnb1Δ two-hybrid strains (PJ69-4A and ASY3) containing a HIS3 reporter and expressing LMB189 and GAL4BD–MSN5 (BM3694) alone or in combination were spotted on media with or without histidine plus 100 mM CaCl₂ and grown at 21°C for 5 d. Cells were spotted using fivefold serial dilutions beginning at an OD₆₀₀ of 1.0.