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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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Related in: MedlinePlus

Cellular localization of Kap119p. A strain where endogenous Kap119p was replaced by protein A-tagged Kap119p  (Kap119-PrA) was used. (A) Yeast cells were visualized by  Nomarski (left) and Kap119–PrA was detected by indirect immunofluorescence (middle). Nuclei were visualized by DAPI staining (right). (B) A cell homogenate (T) was fractionated into a cytosolic (C) and a nuclear (N) fraction. Cell equivalent amounts  were analyzed by SDS-PAGE and Kap119–PrA, cytoplasmic  3-phosphoglycerate kinase (3-PGK), and nucleolar Nop1p were  detected immunologically using either rabbit anti–mouse IgG,  monoclonal mouse antiserum D66, or monoclonal mouse antiserum 22C5-D8.
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Figure 1: Cellular localization of Kap119p. A strain where endogenous Kap119p was replaced by protein A-tagged Kap119p (Kap119-PrA) was used. (A) Yeast cells were visualized by Nomarski (left) and Kap119–PrA was detected by indirect immunofluorescence (middle). Nuclei were visualized by DAPI staining (right). (B) A cell homogenate (T) was fractionated into a cytosolic (C) and a nuclear (N) fraction. Cell equivalent amounts were analyzed by SDS-PAGE and Kap119–PrA, cytoplasmic 3-phosphoglycerate kinase (3-PGK), and nucleolar Nop1p were detected immunologically using either rabbit anti–mouse IgG, monoclonal mouse antiserum D66, or monoclonal mouse antiserum 22C5-D8.

Mentions: The KAP119 gene was replaced by KAP119-PrA coding for a chimeric Kap119p that is COOH terminally fused to IgG-binding domains of Staphylococcus aureus protein A. The resulting Kap119–PrA strain showed no difference in its growth rate compared with wild-type cells. By immunofluorescence (Fig. 1 A) and by cell fractionation (Fig. 1 B) the Kap119–PrA was localized to the cytoplasm and the nucleus indicating that the protein might shuttle between these two compartments. Cell fraction analyses (Fig. 1 B) indicated that about one-third of the total Kap119–PrA was localized to the nucleus. Considering that the nucleus occupies less than one-third of the cellular volume, Kap119p appears to be slightly more concentrated in the nucleus than in the cytoplasm.


A novel nuclear import pathway for the transcription factor TFIIS.

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

Cellular localization of Kap119p. A strain where endogenous Kap119p was replaced by protein A-tagged Kap119p  (Kap119-PrA) was used. (A) Yeast cells were visualized by  Nomarski (left) and Kap119–PrA was detected by indirect immunofluorescence (middle). Nuclei were visualized by DAPI staining (right). (B) A cell homogenate (T) was fractionated into a cytosolic (C) and a nuclear (N) fraction. Cell equivalent amounts  were analyzed by SDS-PAGE and Kap119–PrA, cytoplasmic  3-phosphoglycerate kinase (3-PGK), and nucleolar Nop1p were  detected immunologically using either rabbit anti–mouse IgG,  monoclonal mouse antiserum D66, or monoclonal mouse antiserum 22C5-D8.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132971&req=5

Figure 1: Cellular localization of Kap119p. A strain where endogenous Kap119p was replaced by protein A-tagged Kap119p (Kap119-PrA) was used. (A) Yeast cells were visualized by Nomarski (left) and Kap119–PrA was detected by indirect immunofluorescence (middle). Nuclei were visualized by DAPI staining (right). (B) A cell homogenate (T) was fractionated into a cytosolic (C) and a nuclear (N) fraction. Cell equivalent amounts were analyzed by SDS-PAGE and Kap119–PrA, cytoplasmic 3-phosphoglycerate kinase (3-PGK), and nucleolar Nop1p were detected immunologically using either rabbit anti–mouse IgG, monoclonal mouse antiserum D66, or monoclonal mouse antiserum 22C5-D8.
Mentions: The KAP119 gene was replaced by KAP119-PrA coding for a chimeric Kap119p that is COOH terminally fused to IgG-binding domains of Staphylococcus aureus protein A. The resulting Kap119–PrA strain showed no difference in its growth rate compared with wild-type cells. By immunofluorescence (Fig. 1 A) and by cell fractionation (Fig. 1 B) the Kap119–PrA was localized to the cytoplasm and the nucleus indicating that the protein might shuttle between these two compartments. Cell fraction analyses (Fig. 1 B) indicated that about one-third of the total Kap119–PrA was localized to the nucleus. Considering that the nucleus occupies less than one-third of the cellular volume, Kap119p appears to be slightly more concentrated in the nucleus than in the cytoplasm.

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