Figure 2. PxIxIT Motif Mutations in Crz1 Alter Transcriptional Activity, In Vivo Phosphorylation States, and Nuclear Localization.

(A) β-galactosidase activity of crzlΔ 4×-CDRE-lacZ strains expressing different Crz1 variants, as noted. All plasmids were constructed in the vector pRS315. CaCl₂ was added to the cell culture at the concentrations indicated, 1 hr before harvesting. FK506 was added 1 hr prior to CaCl₂, where indicated. “No added Ca²⁺” refers to cells grown in standard media, which contains 700 µM CaCl₂. Error bars indicate standard deviation (see Experimental Procedures for details).

(B) Immunoblot analysis of cell extracts from crz1Δ cells expressing vector or the indicated HA-tagged CRZ1 constructs, using anti-HA monoclonal antibody. Cells were grown either in the presence of FK506 (lanes 3,5, and 7) or solvent (lanes 1,2,4, and 6). CaCl₂ (50 mM) was added to the cultures for 1 hr prior to sample preparation. A nonspecific band that is recognized by the anti-HA antibody but is independent of Crz1 is marked with an asterisk (*).

(C) Representative fluorescent images of crz1 Δ strain expressing GFP-Crz1, GFP-Crz1^PVIAVN, or GFP-Crz1^PVIVIT from pUG36-based plasmids. Cells were visualized within 5–15 min of CaCl₂ addition as indicated. “No CaCl₂” refers to cells grown in standard media, which contains 700 µM CaCl₂. The panels represent examples of the localization category as indicated: N, nuclear; N/C, nuclear/cytoplasmic; and C, cytoplasmic.

(D) Graphical representation of percentage of cells scored as nuclear, nuclear/cytoplasmic, and cytoplasmic from fluorescent images. The data represent the same conditions shown in (C), i.e., no added CaCl₂, and 5 or 15 min after addition of 50 mM or 200 mM CaCl₂. At least 200 total cells were scored for each condition.