Limits...
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.

Show MeSH
Crz1p and Nmd5p form an import complex. (A) Nmd5p binding to Crz1p is disrupted by Gsp1p–GTP. 5 μg GST–Crz1p was bound to glutathione resin and incubated with 5 μg thrombin-cleaved Nmd5p. After extensive washing, the resin was incubated with 10 μM Gsp1p(Q71L)–GTP, Gsp1p(Q71L)–GDP, or buffer alone. The supernatant was collected, and the washed resin was resuspended in Laemmli loading buffer. Equivalent amounts of the bound and unbound fractions were analyzed on a 6% SDS-PAGE followed by Western blotting using an anti-Nmd5p antibody that also recognizes GST (GST–Crz1p is shown as an internal control). (B) Nmd5p binds Crz1p in a calcineurin-dependent manner. 200 μg yeast cytosol from crz1Δ strains ASY472 (WT) and ASY475 (cnb1Δ) expressing HA-tagged Crz1p (pAMS451) were incubated with 50 μl Nmd5p–GST bound to glutathione resin. Equal amounts of the bound fractions were analyzed by Western blotting using an anti-HA antibody. The mobility of HA-Crz1p was compared with untreated yeast cytosol (Input Extract). As a control, WT yeast cytosol was incubated with GST-bound resin. The bottom displays a Ponceau stain analysis of the Western blot to demonstrate equivalent amounts of Nmd5p–GST.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196176&req=5

fig4: Crz1p and Nmd5p form an import complex. (A) Nmd5p binding to Crz1p is disrupted by Gsp1p–GTP. 5 μg GST–Crz1p was bound to glutathione resin and incubated with 5 μg thrombin-cleaved Nmd5p. After extensive washing, the resin was incubated with 10 μM Gsp1p(Q71L)–GTP, Gsp1p(Q71L)–GDP, or buffer alone. The supernatant was collected, and the washed resin was resuspended in Laemmli loading buffer. Equivalent amounts of the bound and unbound fractions were analyzed on a 6% SDS-PAGE followed by Western blotting using an anti-Nmd5p antibody that also recognizes GST (GST–Crz1p is shown as an internal control). (B) Nmd5p binds Crz1p in a calcineurin-dependent manner. 200 μg yeast cytosol from crz1Δ strains ASY472 (WT) and ASY475 (cnb1Δ) expressing HA-tagged Crz1p (pAMS451) were incubated with 50 μl Nmd5p–GST bound to glutathione resin. Equal amounts of the bound fractions were analyzed by Western blotting using an anti-HA antibody. The mobility of HA-Crz1p was compared with untreated yeast cytosol (Input Extract). As a control, WT yeast cytosol was incubated with GST-bound resin. The bottom displays a Ponceau stain analysis of the Western blot to demonstrate equivalent amounts of Nmd5p–GST.

Mentions: A Crz1p–Nmd5p complex was demonstrated biochemically using a glutathione S-transferase (GST) pull-down assay consisting of immobilized GST–Crz1p incubated with Nmd5p, both of which were produced in Escherichia coli. Analysis of the bound fractions by Western blotting revealed that Crz1p binds to Nmd5p (Fig. 4 A). Several labs have demonstrated that RanGTP dissociates the interaction between most import karyopherins and their target cargo proteins (Rexach and Blobel, 1995; Albertini et al., 1998; Kaffman et al., 1998). Incubation of immobilized Nmd5p–Crz1p complex with a mutant version of Gsp1p, the yeast homologue of Ran, that is locked in the GTP-bound state (Gsp1p(Q71L)–GTP), significantly disrupted the Nmd5p–Crz1p complex (Fig. 4 A), whereas addition of Gsp1p–GDP or buffer alone failed to disrupt Crz1p–Nmd5p binding.


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

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

Crz1p and Nmd5p form an import complex. (A) Nmd5p binding to Crz1p is disrupted by Gsp1p–GTP. 5 μg GST–Crz1p was bound to glutathione resin and incubated with 5 μg thrombin-cleaved Nmd5p. After extensive washing, the resin was incubated with 10 μM Gsp1p(Q71L)–GTP, Gsp1p(Q71L)–GDP, or buffer alone. The supernatant was collected, and the washed resin was resuspended in Laemmli loading buffer. Equivalent amounts of the bound and unbound fractions were analyzed on a 6% SDS-PAGE followed by Western blotting using an anti-Nmd5p antibody that also recognizes GST (GST–Crz1p is shown as an internal control). (B) Nmd5p binds Crz1p in a calcineurin-dependent manner. 200 μg yeast cytosol from crz1Δ strains ASY472 (WT) and ASY475 (cnb1Δ) expressing HA-tagged Crz1p (pAMS451) were incubated with 50 μl Nmd5p–GST bound to glutathione resin. Equal amounts of the bound fractions were analyzed by Western blotting using an anti-HA antibody. The mobility of HA-Crz1p was compared with untreated yeast cytosol (Input Extract). As a control, WT yeast cytosol was incubated with GST-bound resin. The bottom displays a Ponceau stain analysis of the Western blot to demonstrate equivalent amounts of Nmd5p–GST.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Crz1p and Nmd5p form an import complex. (A) Nmd5p binding to Crz1p is disrupted by Gsp1p–GTP. 5 μg GST–Crz1p was bound to glutathione resin and incubated with 5 μg thrombin-cleaved Nmd5p. After extensive washing, the resin was incubated with 10 μM Gsp1p(Q71L)–GTP, Gsp1p(Q71L)–GDP, or buffer alone. The supernatant was collected, and the washed resin was resuspended in Laemmli loading buffer. Equivalent amounts of the bound and unbound fractions were analyzed on a 6% SDS-PAGE followed by Western blotting using an anti-Nmd5p antibody that also recognizes GST (GST–Crz1p is shown as an internal control). (B) Nmd5p binds Crz1p in a calcineurin-dependent manner. 200 μg yeast cytosol from crz1Δ strains ASY472 (WT) and ASY475 (cnb1Δ) expressing HA-tagged Crz1p (pAMS451) were incubated with 50 μl Nmd5p–GST bound to glutathione resin. Equal amounts of the bound fractions were analyzed by Western blotting using an anti-HA antibody. The mobility of HA-Crz1p was compared with untreated yeast cytosol (Input Extract). As a control, WT yeast cytosol was incubated with GST-bound resin. The bottom displays a Ponceau stain analysis of the Western blot to demonstrate equivalent amounts of Nmd5p–GST.
Mentions: A Crz1p–Nmd5p complex was demonstrated biochemically using a glutathione S-transferase (GST) pull-down assay consisting of immobilized GST–Crz1p incubated with Nmd5p, both of which were produced in Escherichia coli. Analysis of the bound fractions by Western blotting revealed that Crz1p binds to Nmd5p (Fig. 4 A). Several labs have demonstrated that RanGTP dissociates the interaction between most import karyopherins and their target cargo proteins (Rexach and Blobel, 1995; Albertini et al., 1998; Kaffman et al., 1998). Incubation of immobilized Nmd5p–Crz1p complex with a mutant version of Gsp1p, the yeast homologue of Ran, that is locked in the GTP-bound state (Gsp1p(Q71L)–GTP), significantly disrupted the Nmd5p–Crz1p complex (Fig. 4 A), whereas addition of Gsp1p–GDP or buffer alone failed to disrupt Crz1p–Nmd5p binding.

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