Figure 1.

Identification of the Crz1p NLS and requirement of Nmd5p activity for Crz1p nuclear localization. (A) Localization of GFP–CRZ1 fusions in strains ASY472 (crz1Δ) and ASY475 (crz1Δcnb1Δ) as determined by fluorescence microscopy. Localization was observed in cells incubated at 21°C for 10 min with or without 200 mM CaCl₂ and scored for both nuclear localization and Ca²⁺/calcineurin-dependent regulation. A protein was scored as positive for Ca²⁺/calcineurin-dependent regulation if its nuclear localization was dependent on the addition of Ca²⁺ and failed to localize in a cnb1Δ strain after Ca²⁺ addition. (B) Residues 394–422 are required for Ca²⁺-dependent nuclear localization of Crz1p, and this sequence is recognized by Nmd5p. Living cells of strain ASY472 (WT) or RPY176 (nmd5Δ) containing pRSP97 (full-length), pRSP114 (mutNLS#1), pRSP40 (GFP-NLS#1), or pRSP92 (GFP-NLS#2) were grown at 21°C and incubated with (+Ca²⁺) or without (−Ca²⁺) 200 mM CaCl₂ at 21°C for 10 min. GFP–Crz1p localization was observed using fluorescence microscopy. Bar, 20 μm.