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A novel nuclear import pathway for the transcription factor TFIIS.

Albertini M, Pemberton LF, Rosenblum JS, Blobel G - J. Cell Biol. (1998)

Bottom Line: Kap119p also bound directly to a number of peptide repeat containing nucleoporins in overlay assays.In wild-type cells, TFIIS was primarily localized to the nucleus.Hence Kap119p is a novel karyopherin that is responsible for the import of the transcription elongation factor TFIIS.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.

ABSTRACT
We have identified a novel pathway for protein import into the nucleus. We have shown that the previously identified but uncharacterized yeast protein Nmd5p functions as a karyopherin. It was therefore designated Kap119p (karyopherin with Mr of 119 kD). We localized Kap119p to both the nucleus and the cytoplasm. We identified the transcription elongation factor TFIIS as its major cognate import substrate. The cytoplasmic Kap119p exists as an approximately stoichiometric complex with TFIIS. RanGTP, not RanGDP, dissociated the isolated Kap119p/TFIIS complex and bound to Kap119p. Kap119p also bound directly to a number of peptide repeat containing nucleoporins in overlay assays. In wild-type cells, TFIIS was primarily localized to the nucleus. In a strain where KAP119 has been deleted, TFIIS was mislocalized to the cytoplasm indicating that TFIIS is imported into the nucleus by Kap119p. The transport of various substrates that use other karyopherin-mediated import or export pathways was not affected in a kap119Delta strain. Hence Kap119p is a novel karyopherin that is responsible for the import of the transcription elongation factor TFIIS.

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Deletion of KAP119 causes slower growth and leads to  mislocalization of TFIIS. (A) Comparison of growth rates of  DF5α (wt) and of the KAP119 deletion strain, kap119Δ. Cultures  were inoculated at OD 0.1 into rich media, grown at 30°C and  samples were taken at the indicated time points. (B) Localization  by immunofluorescence in wild-type (wt), kap119Δ, and kap108Δ  strains in which the endogenous TFIIS had been replaced by  TFIIS–PrA. Whereas TFIIS–PrA was primarily localized to the  nucleus in the wt and kap108Δ strains, it was diffusely localized  throughout the cell in the kap119Δ strain. (C) A homogenate (T)  of kap119Δ and wt cells was fractionated into a cytosolic (C) and  a nuclear (N) fraction. Cell equivalent portions were analyzed by  SDS-PAGE and TFIIS–PrA, 3-phosphoglycerate kinase (3-PGK)  and Nop1p were detected with either rabbit anti–mouse IgG,  monoclonal mouse antiserum D66 or monoclonal mouse antiserum 22C5-D8. TFIIS was predominantly localized in the cytoplasm in kap119Δ cells.
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Figure 3: Deletion of KAP119 causes slower growth and leads to mislocalization of TFIIS. (A) Comparison of growth rates of DF5α (wt) and of the KAP119 deletion strain, kap119Δ. Cultures were inoculated at OD 0.1 into rich media, grown at 30°C and samples were taken at the indicated time points. (B) Localization by immunofluorescence in wild-type (wt), kap119Δ, and kap108Δ strains in which the endogenous TFIIS had been replaced by TFIIS–PrA. Whereas TFIIS–PrA was primarily localized to the nucleus in the wt and kap108Δ strains, it was diffusely localized throughout the cell in the kap119Δ strain. (C) A homogenate (T) of kap119Δ and wt cells was fractionated into a cytosolic (C) and a nuclear (N) fraction. Cell equivalent portions were analyzed by SDS-PAGE and TFIIS–PrA, 3-phosphoglycerate kinase (3-PGK) and Nop1p were detected with either rabbit anti–mouse IgG, monoclonal mouse antiserum D66 or monoclonal mouse antiserum 22C5-D8. TFIIS was predominantly localized in the cytoplasm in kap119Δ cells.

Mentions: To investigate whether TFIIS was a nuclear import substrate of Kap119p we prepared a strain lacking KAP119. The kap119Δ strain was viable and grew on YPD plates but was characterized by a slightly extended doubling time compared with the wild-type haploid strain (Fig. 3 A). As TFIIS is primarily localized to the nucleus (Nakanishi et al., 1995) it should be mislocalized to the cytoplasm in the kap119Δ strain if Kap119p functions as its principal cognate Kap. To facilitate localization of TFIIS we protein A–tagged the endogenous TFIIS gene in a wild-type and a kap119Δ strain and investigated its cellular distribution by immunofluorescence and cell fractionation.


A novel nuclear import pathway for the transcription factor TFIIS.

Albertini M, Pemberton LF, Rosenblum JS, Blobel G - J. Cell Biol. (1998)

Deletion of KAP119 causes slower growth and leads to  mislocalization of TFIIS. (A) Comparison of growth rates of  DF5α (wt) and of the KAP119 deletion strain, kap119Δ. Cultures  were inoculated at OD 0.1 into rich media, grown at 30°C and  samples were taken at the indicated time points. (B) Localization  by immunofluorescence in wild-type (wt), kap119Δ, and kap108Δ  strains in which the endogenous TFIIS had been replaced by  TFIIS–PrA. Whereas TFIIS–PrA was primarily localized to the  nucleus in the wt and kap108Δ strains, it was diffusely localized  throughout the cell in the kap119Δ strain. (C) A homogenate (T)  of kap119Δ and wt cells was fractionated into a cytosolic (C) and  a nuclear (N) fraction. Cell equivalent portions were analyzed by  SDS-PAGE and TFIIS–PrA, 3-phosphoglycerate kinase (3-PGK)  and Nop1p were detected with either rabbit anti–mouse IgG,  monoclonal mouse antiserum D66 or monoclonal mouse antiserum 22C5-D8. TFIIS was predominantly localized in the cytoplasm in kap119Δ cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132971&req=5

Figure 3: Deletion of KAP119 causes slower growth and leads to mislocalization of TFIIS. (A) Comparison of growth rates of DF5α (wt) and of the KAP119 deletion strain, kap119Δ. Cultures were inoculated at OD 0.1 into rich media, grown at 30°C and samples were taken at the indicated time points. (B) Localization by immunofluorescence in wild-type (wt), kap119Δ, and kap108Δ strains in which the endogenous TFIIS had been replaced by TFIIS–PrA. Whereas TFIIS–PrA was primarily localized to the nucleus in the wt and kap108Δ strains, it was diffusely localized throughout the cell in the kap119Δ strain. (C) A homogenate (T) of kap119Δ and wt cells was fractionated into a cytosolic (C) and a nuclear (N) fraction. Cell equivalent portions were analyzed by SDS-PAGE and TFIIS–PrA, 3-phosphoglycerate kinase (3-PGK) and Nop1p were detected with either rabbit anti–mouse IgG, monoclonal mouse antiserum D66 or monoclonal mouse antiserum 22C5-D8. TFIIS was predominantly localized in the cytoplasm in kap119Δ cells.
Mentions: To investigate whether TFIIS was a nuclear import substrate of Kap119p we prepared a strain lacking KAP119. The kap119Δ strain was viable and grew on YPD plates but was characterized by a slightly extended doubling time compared with the wild-type haploid strain (Fig. 3 A). As TFIIS is primarily localized to the nucleus (Nakanishi et al., 1995) it should be mislocalized to the cytoplasm in the kap119Δ strain if Kap119p functions as its principal cognate Kap. To facilitate localization of TFIIS we protein A–tagged the endogenous TFIIS gene in a wild-type and a kap119Δ strain and investigated its cellular distribution by immunofluorescence and cell fractionation.

Bottom Line: Kap119p also bound directly to a number of peptide repeat containing nucleoporins in overlay assays.In wild-type cells, TFIIS was primarily localized to the nucleus.Hence Kap119p is a novel karyopherin that is responsible for the import of the transcription elongation factor TFIIS.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.

ABSTRACT
We have identified a novel pathway for protein import into the nucleus. We have shown that the previously identified but uncharacterized yeast protein Nmd5p functions as a karyopherin. It was therefore designated Kap119p (karyopherin with Mr of 119 kD). We localized Kap119p to both the nucleus and the cytoplasm. We identified the transcription elongation factor TFIIS as its major cognate import substrate. The cytoplasmic Kap119p exists as an approximately stoichiometric complex with TFIIS. RanGTP, not RanGDP, dissociated the isolated Kap119p/TFIIS complex and bound to Kap119p. Kap119p also bound directly to a number of peptide repeat containing nucleoporins in overlay assays. In wild-type cells, TFIIS was primarily localized to the nucleus. In a strain where KAP119 has been deleted, TFIIS was mislocalized to the cytoplasm indicating that TFIIS is imported into the nucleus by Kap119p. The transport of various substrates that use other karyopherin-mediated import or export pathways was not affected in a kap119Delta strain. Hence Kap119p is a novel karyopherin that is responsible for the import of the transcription elongation factor TFIIS.

Show MeSH
Related in: MedlinePlus