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. 2002 Mar 1;16(5):608–619. doi: 10.1101/gad.967602

Figure 4.

Figure 4

Identification of the Crz1p NES. (A) WT (ASY472) cells expressing 3xGFP–SV40NLS–Crz1p186–279 (LMB162), 3xGFP–SV40NLS–Crz1p186–250 (LMB207), or 3xGFP–SV40NLS–mSRR186–279 (LMB 211) were grown to log phase at 21°C. Cells were treated with 200 mM CaCl2 and visualized using fluorescence microscopy. Bar, 20 μm. (B) Summary of export data for 3xGFP–SV40NLS–Crz1p constructs. (+) Cytosolic localization (export); (−) nuclear localization (no export). (C) The Crz1p NES interacts with Msn5p in vivo. A two-hybrid strain containing a HIS3 reporter (PJ69-4A) expressing AD–CRZ1186–279 (LMB193), AD–mSRR186–279 (LMB218), or GAL4BD–MSN5 (BM3694) alone or in combination was spotted onto media with or without histidine and grown at 21°C for 5 d. Cells were spotted using fivefold serial dilutions beginning at an OD600 of 0.05. (D) The Crz1p NES is similarly phosphorylated in WT and cnb1Δ cells. Whole cell extracts from WT (ASY472) or cnb1Δ (ASY475) strains containing LMB162 or LMB211 were analyzed by Western blot using anti-GFP antibody. Where indicated, extracts were treated with or without (mock) 200 units of λ phosphatase and incubated at 30° for 30 min.