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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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Analysis of the Crz1p NLS#1. (A) Localization of fragments of the Crz1p NLS#1 (residues 394–422) fused to three tandem copies of GFP in strain ASY472 as determined by fluorescence microscopy. Localization was observed in untreated log-phase cells growing at 21°C and scored for nuclear localization. (B) Hydrophobic and basic residues are required for NLS function. Untreated living cells of strain ASY472 containing pRSP40 (NLS#1), pOM9-1 (NLS#1-3), pRSP136 (NLS#1-2), or pRSP153 (mutNLS#1-5) were grown at 21°C. GFP localization was observed using fluorescence microscopy. Bar, 20 μm.
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fig2: Analysis of the Crz1p NLS#1. (A) Localization of fragments of the Crz1p NLS#1 (residues 394–422) fused to three tandem copies of GFP in strain ASY472 as determined by fluorescence microscopy. Localization was observed in untreated log-phase cells growing at 21°C and scored for nuclear localization. (B) Hydrophobic and basic residues are required for NLS function. Untreated living cells of strain ASY472 containing pRSP40 (NLS#1), pOM9-1 (NLS#1-3), pRSP136 (NLS#1-2), or pRSP153 (mutNLS#1-5) were grown at 21°C. GFP localization was observed using fluorescence microscopy. Bar, 20 μm.

Mentions: Clearly, residues 394–422 are sufficient to target GFP to the nucleus, but it is unclear which residues are necessary for nuclear localization; therefore, a variety of deletions and mutations were made in this region and analyzed for their ability to target GFP to the nucleus. NLSs can be of two types, either monopartite, consisting of a single cluster of basic residues, or bipartite, consisting of two basic clusters separated by a spacer region (for review see Jans and Hubner, 1996). The Crz1p NLS could be of either type, thus the Crz1p NLS was initially divided into two fragments to separate the two basic clusters and fused to GFP to test their competence for nuclear targeting. The COOH-terminal half (NLS#1-1, amino acids 410–422) was not able to target GFP to the nucleus, whereas the NH2-terminal half (NLS#1-2, amino acids 394–410) displayed partial nuclear localization, suggesting that this region was sufficient to drive nuclear import (Fig. 2) . Deletion of the first two isoleucines (NLS#1-3, amino acids 396–410) completely abolished nuclear localization, indicating that these hydrophobic residues play a critical role in Crz1p NLS function (Fig. 2). Significant inhibition of nuclear localization was also observed by mutagenesis of the lysine (mutNLS#1-6) or arginine (mutNLS#1-5) clusters to alanine (Fig. 2 A), suggesting that the basic residues along with the hydrophobic residues are crucial for Crz1p NLS function. Finally, to determine if complete nuclear localization could be restored, clusters of residues were added back to NLS#1-2 until the complete NLS sequence was obtained (Fig. 2 A, NLS#1-4, NLS#1-5, NLS#1). NLS#1-4 and NLS#1-5 also exhibited partial nuclear localization, and thus the last two arginines are required for full activity of the NLS. Mutagenesis of the basic clusters in the full-length protein showed similar results (unpublished data). Substitution of either the NH2-terminal lysine (mutNLS#1-6) or arginine (mutNLS#1-5) clusters to alanine significantly inhibited Crz1p nuclear localization (Fig. 2 A), whereas mutagenesis of both clusters together completely eliminated nuclear localization (Fig. 1 A, mutNLS#1). However, mutagenesis of the COOH-terminal arginines to alanine had no effect on localization (unpublished data), indicating that these residues enhance nuclear localization but are not absolutely required.


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

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

Analysis of the Crz1p NLS#1. (A) Localization of fragments of the Crz1p NLS#1 (residues 394–422) fused to three tandem copies of GFP in strain ASY472 as determined by fluorescence microscopy. Localization was observed in untreated log-phase cells growing at 21°C and scored for nuclear localization. (B) Hydrophobic and basic residues are required for NLS function. Untreated living cells of strain ASY472 containing pRSP40 (NLS#1), pOM9-1 (NLS#1-3), pRSP136 (NLS#1-2), or pRSP153 (mutNLS#1-5) were grown at 21°C. GFP localization was observed using fluorescence microscopy. Bar, 20 μm.
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Related In: Results  -  Collection

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fig2: Analysis of the Crz1p NLS#1. (A) Localization of fragments of the Crz1p NLS#1 (residues 394–422) fused to three tandem copies of GFP in strain ASY472 as determined by fluorescence microscopy. Localization was observed in untreated log-phase cells growing at 21°C and scored for nuclear localization. (B) Hydrophobic and basic residues are required for NLS function. Untreated living cells of strain ASY472 containing pRSP40 (NLS#1), pOM9-1 (NLS#1-3), pRSP136 (NLS#1-2), or pRSP153 (mutNLS#1-5) were grown at 21°C. GFP localization was observed using fluorescence microscopy. Bar, 20 μm.
Mentions: Clearly, residues 394–422 are sufficient to target GFP to the nucleus, but it is unclear which residues are necessary for nuclear localization; therefore, a variety of deletions and mutations were made in this region and analyzed for their ability to target GFP to the nucleus. NLSs can be of two types, either monopartite, consisting of a single cluster of basic residues, or bipartite, consisting of two basic clusters separated by a spacer region (for review see Jans and Hubner, 1996). The Crz1p NLS could be of either type, thus the Crz1p NLS was initially divided into two fragments to separate the two basic clusters and fused to GFP to test their competence for nuclear targeting. The COOH-terminal half (NLS#1-1, amino acids 410–422) was not able to target GFP to the nucleus, whereas the NH2-terminal half (NLS#1-2, amino acids 394–410) displayed partial nuclear localization, suggesting that this region was sufficient to drive nuclear import (Fig. 2) . Deletion of the first two isoleucines (NLS#1-3, amino acids 396–410) completely abolished nuclear localization, indicating that these hydrophobic residues play a critical role in Crz1p NLS function (Fig. 2). Significant inhibition of nuclear localization was also observed by mutagenesis of the lysine (mutNLS#1-6) or arginine (mutNLS#1-5) clusters to alanine (Fig. 2 A), suggesting that the basic residues along with the hydrophobic residues are crucial for Crz1p NLS function. Finally, to determine if complete nuclear localization could be restored, clusters of residues were added back to NLS#1-2 until the complete NLS sequence was obtained (Fig. 2 A, NLS#1-4, NLS#1-5, NLS#1). NLS#1-4 and NLS#1-5 also exhibited partial nuclear localization, and thus the last two arginines are required for full activity of the NLS. Mutagenesis of the basic clusters in the full-length protein showed similar results (unpublished data). Substitution of either the NH2-terminal lysine (mutNLS#1-6) or arginine (mutNLS#1-5) clusters to alanine significantly inhibited Crz1p nuclear localization (Fig. 2 A), whereas mutagenesis of both clusters together completely eliminated nuclear localization (Fig. 1 A, mutNLS#1). However, mutagenesis of the COOH-terminal arginines to alanine had no effect on localization (unpublished data), indicating that these residues enhance nuclear localization but are not absolutely required.

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