Figure 3.

GFP–Crz1 shuttles from the nucleus to the cytoplasm and nuclear localization requires calcineurin activity. Log-phase cultures of strain ASY472 containing GFP–CRZ1 were grown at 21°C and analyzed by fluorescence microscopy to determine GFP–Crz1p localization. (E–H) Cells incubated for 2 hr with cycloheximide prior to other manipulations described (+chx). (A–D) Cells not incubated with cycloheximide (−chx). (B,F) At t = 0 min, CaCl₂ was added to one aliquot of each culture (200 mm) and cells were observed at t = 30 min; (A,E) Equivalent cells not exposed to Ca²⁺. (D,H) At t = 60 min an aliquot of each Ca²⁺-treated culture was incubated with 3 μg/ml FK506 and observed at t = 90 min. (C,G) Cells incubated with Ca²⁺ for 90 min, not treated with FK506.