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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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Nmd5p is required for cation resistance and CDRE::lacZ activity. (A) nmd5Δ and crz1Δ strains share similar cation sensitivities. Yeast strains were plated onto YPD alone or containing 150 mM LiCl, 400 mM CaCl2, or 6 mM MnCl2 and grown at 30°C for 3 d. WT, strain YPH499; crz1Δ, strain ASY472; nmd5Δ, strain RPY176. (B) Nmd5p is required for CDRE::lacZ activity. Strains YPH499 (WT), ASY472 (crz1Δ), and RPY176 (nmd5Δ) containing the CDRE::lacZ reporter (pAMS366) were grown in synthetic medium at 21°C for 6 h as follows: untreated (white bars), with 200 mM CaCl2 (black bars), with 200 mM CaCl2 and 2 μg/ml FK520 (gray bars), and β-galactosidase activity was assayed and done in triplicate. The SD is representative of the error between the samples.
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fig3: Nmd5p is required for cation resistance and CDRE::lacZ activity. (A) nmd5Δ and crz1Δ strains share similar cation sensitivities. Yeast strains were plated onto YPD alone or containing 150 mM LiCl, 400 mM CaCl2, or 6 mM MnCl2 and grown at 30°C for 3 d. WT, strain YPH499; crz1Δ, strain ASY472; nmd5Δ, strain RPY176. (B) Nmd5p is required for CDRE::lacZ activity. Strains YPH499 (WT), ASY472 (crz1Δ), and RPY176 (nmd5Δ) containing the CDRE::lacZ reporter (pAMS366) were grown in synthetic medium at 21°C for 6 h as follows: untreated (white bars), with 200 mM CaCl2 (black bars), with 200 mM CaCl2 and 2 μg/ml FK520 (gray bars), and β-galactosidase activity was assayed and done in triplicate. The SD is representative of the error between the samples.

Mentions: Next, we examined the physiological consequences of disrupting Crz1p nuclear localization in the nmd5Δ strain. Crz1p-dependent transcriptional activation occurs through binding to the CDRE promoter element (Stathopoulos and Cyert, 1997); thus, to determine if Nmd5p affects Crz1p-dependent transcription an nmd5Δ strain was transformed with a plasmid containing four tandem copies of the CDRE fused to lacZ. Cells were treated with Ca2+ or Ca2+ plus the calcineurin inhibitor FK520, an isomer of FK506, and assayed for β-galactosidase activity (Fig. 3 B). Wild-type cells treated with Ca2+ displayed a 15-fold stimulation of β-galactosidase activity, which was abrogated by the calcineurin inhibitor FK520. This activation was absent in cells lacking calcineurin (cnb1Δ) and significantly reduced in nmd5Δ cells. Phenotypic analysis of nmd5Δ cells also reveals a defect in Crz1p-dependent transcriptional activation. crz1Δ and nmd5Δ cells were plated on YPD containing high concentrations of Li+, Mn2+, or Ca2+, ions to which crz1Δ cells are sensitive (Stathopoulos and Cyert, 1997). Both nmd5Δ and crz1Δ strains display similar phenotypes (Fig. 3 A) consistent with Nmd5p being required for calcineurin/Crz1p-dependent transcriptional activation in vivo.


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

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

Nmd5p is required for cation resistance and CDRE::lacZ activity. (A) nmd5Δ and crz1Δ strains share similar cation sensitivities. Yeast strains were plated onto YPD alone or containing 150 mM LiCl, 400 mM CaCl2, or 6 mM MnCl2 and grown at 30°C for 3 d. WT, strain YPH499; crz1Δ, strain ASY472; nmd5Δ, strain RPY176. (B) Nmd5p is required for CDRE::lacZ activity. Strains YPH499 (WT), ASY472 (crz1Δ), and RPY176 (nmd5Δ) containing the CDRE::lacZ reporter (pAMS366) were grown in synthetic medium at 21°C for 6 h as follows: untreated (white bars), with 200 mM CaCl2 (black bars), with 200 mM CaCl2 and 2 μg/ml FK520 (gray bars), and β-galactosidase activity was assayed and done in triplicate. The SD is representative of the error between the samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196176&req=5

fig3: Nmd5p is required for cation resistance and CDRE::lacZ activity. (A) nmd5Δ and crz1Δ strains share similar cation sensitivities. Yeast strains were plated onto YPD alone or containing 150 mM LiCl, 400 mM CaCl2, or 6 mM MnCl2 and grown at 30°C for 3 d. WT, strain YPH499; crz1Δ, strain ASY472; nmd5Δ, strain RPY176. (B) Nmd5p is required for CDRE::lacZ activity. Strains YPH499 (WT), ASY472 (crz1Δ), and RPY176 (nmd5Δ) containing the CDRE::lacZ reporter (pAMS366) were grown in synthetic medium at 21°C for 6 h as follows: untreated (white bars), with 200 mM CaCl2 (black bars), with 200 mM CaCl2 and 2 μg/ml FK520 (gray bars), and β-galactosidase activity was assayed and done in triplicate. The SD is representative of the error between the samples.
Mentions: Next, we examined the physiological consequences of disrupting Crz1p nuclear localization in the nmd5Δ strain. Crz1p-dependent transcriptional activation occurs through binding to the CDRE promoter element (Stathopoulos and Cyert, 1997); thus, to determine if Nmd5p affects Crz1p-dependent transcription an nmd5Δ strain was transformed with a plasmid containing four tandem copies of the CDRE fused to lacZ. Cells were treated with Ca2+ or Ca2+ plus the calcineurin inhibitor FK520, an isomer of FK506, and assayed for β-galactosidase activity (Fig. 3 B). Wild-type cells treated with Ca2+ displayed a 15-fold stimulation of β-galactosidase activity, which was abrogated by the calcineurin inhibitor FK520. This activation was absent in cells lacking calcineurin (cnb1Δ) and significantly reduced in nmd5Δ cells. Phenotypic analysis of nmd5Δ cells also reveals a defect in Crz1p-dependent transcriptional activation. crz1Δ and nmd5Δ cells were plated on YPD containing high concentrations of Li+, Mn2+, or Ca2+, ions to which crz1Δ cells are sensitive (Stathopoulos and Cyert, 1997). Both nmd5Δ and crz1Δ strains display similar phenotypes (Fig. 3 A) consistent with Nmd5p being required for calcineurin/Crz1p-dependent transcriptional activation in vivo.

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