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Calcineurin-dependent nuclear import of the transcription factor Crz1p requires Nmd5p.

Polizotto RS, Cyert MS - J. Cell Biol. (2001)

Bottom Line: We describe the identification and characterization of a novel nuclear localization sequence (NLS) in Crz1p, which requires both basic and hydrophobic residues for activity, and show that the karyopherin Nmd5p is required for Crz1p nuclear import.We also demonstrate that the binding of Crz1p to Nmd5p is dependent upon its phosphorylation state, indicating that nuclear import of Crz1p is regulated by calcineurin.Finally, we demonstrate that residues in both the NH2- and COOH-terminal portions of Crz1p are required for regulated Crz1p binding to Nmd5p, supporting a model of NLS masking for regulating Crz1p nuclear import.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

ABSTRACT
Calcineurin is a conserved Ca2+/calmodulin-specific serine-threonine protein phosphatase that mediates many Ca2+-dependent signaling events. In yeast, calcineurin dephosphorylates Crz1p, a transcription factor that binds to the calcineurin-dependent response element, a 24-bp promoter element. Calcineurin-dependent dephosphorylation of Crz1p alters Crz1p nuclear localization. This study examines the mechanism by which calcineurin regulates the nuclear localization of Crz1p in more detail. We describe the identification and characterization of a novel nuclear localization sequence (NLS) in Crz1p, which requires both basic and hydrophobic residues for activity, and show that the karyopherin Nmd5p is required for Crz1p nuclear import. We also demonstrate that the binding of Crz1p to Nmd5p is dependent upon its phosphorylation state, indicating that nuclear import of Crz1p is regulated by calcineurin. Finally, we demonstrate that residues in both the NH2- and COOH-terminal portions of Crz1p are required for regulated Crz1p binding to Nmd5p, supporting a model of NLS masking for regulating Crz1p nuclear import.

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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.
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fig1: 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.

Mentions: To identify the location of the Crz1p NLS, a series of green fluorescent protein (GFP)–Crz1p fusions were analyzed for calcineurin-dependent regulation of localization (Fig. 1 A). In wild-type cells, full-length GFP–Crz1p localizes to the cytoplasm until the addition of Ca2+, after which it rapidly localizes to the nucleus. This localization is dependent on calcineurin because GFP–Crz1p remains cytoplasmic in cnb1Δ cells even in the presence of Ca2+ (Fig. 1 B; Stathopoulos-Gerontides et al., 1999). A fusion of the NH2-terminal 425 amino acids of Crz1p to GFP exhibits a localization pattern identical to that of full-length GFP–Crz1p (Fig. 1, N-term-425); however, a fusion including only the first 388 residues displays constitutively cytoplasmic localization, suggesting that residues 388–425 contain an NLS (Fig. 1 A, N-term-388). Furthermore, full-length Crz1p lacking amino acids 394–425 is constitutively localized to the cytoplasm, demonstrating that this region is necessary for nuclear localization (Fig. 1 A, NLS#1Δ).


Calcineurin-dependent nuclear import of the transcription factor Crz1p requires Nmd5p.

Polizotto RS, Cyert MS - J. Cell Biol. (2001)

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.
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fig1: 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.
Mentions: To identify the location of the Crz1p NLS, a series of green fluorescent protein (GFP)–Crz1p fusions were analyzed for calcineurin-dependent regulation of localization (Fig. 1 A). In wild-type cells, full-length GFP–Crz1p localizes to the cytoplasm until the addition of Ca2+, after which it rapidly localizes to the nucleus. This localization is dependent on calcineurin because GFP–Crz1p remains cytoplasmic in cnb1Δ cells even in the presence of Ca2+ (Fig. 1 B; Stathopoulos-Gerontides et al., 1999). A fusion of the NH2-terminal 425 amino acids of Crz1p to GFP exhibits a localization pattern identical to that of full-length GFP–Crz1p (Fig. 1, N-term-425); however, a fusion including only the first 388 residues displays constitutively cytoplasmic localization, suggesting that residues 388–425 contain an NLS (Fig. 1 A, N-term-388). Furthermore, full-length Crz1p lacking amino acids 394–425 is constitutively localized to the cytoplasm, demonstrating that this region is necessary for nuclear localization (Fig. 1 A, NLS#1Δ).

Bottom Line: We describe the identification and characterization of a novel nuclear localization sequence (NLS) in Crz1p, which requires both basic and hydrophobic residues for activity, and show that the karyopherin Nmd5p is required for Crz1p nuclear import.We also demonstrate that the binding of Crz1p to Nmd5p is dependent upon its phosphorylation state, indicating that nuclear import of Crz1p is regulated by calcineurin.Finally, we demonstrate that residues in both the NH2- and COOH-terminal portions of Crz1p are required for regulated Crz1p binding to Nmd5p, supporting a model of NLS masking for regulating Crz1p nuclear import.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

ABSTRACT
Calcineurin is a conserved Ca2+/calmodulin-specific serine-threonine protein phosphatase that mediates many Ca2+-dependent signaling events. In yeast, calcineurin dephosphorylates Crz1p, a transcription factor that binds to the calcineurin-dependent response element, a 24-bp promoter element. Calcineurin-dependent dephosphorylation of Crz1p alters Crz1p nuclear localization. This study examines the mechanism by which calcineurin regulates the nuclear localization of Crz1p in more detail. We describe the identification and characterization of a novel nuclear localization sequence (NLS) in Crz1p, which requires both basic and hydrophobic residues for activity, and show that the karyopherin Nmd5p is required for Crz1p nuclear import. We also demonstrate that the binding of Crz1p to Nmd5p is dependent upon its phosphorylation state, indicating that nuclear import of Crz1p is regulated by calcineurin. Finally, we demonstrate that residues in both the NH2- and COOH-terminal portions of Crz1p are required for regulated Crz1p binding to Nmd5p, supporting a model of NLS masking for regulating Crz1p nuclear import.

Show MeSH
Related in: MedlinePlus