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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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Kap119–PrA binds to various nucleoporins. Proteins of  lysates from E. coli strains expressing the peptide repeat regions  of Nup1p (amino acid residues 432–816) (Rexach and Blobel,  1995), Nup2p (amino acid residues 186–561) (Rexach and Blobel,  1995), or Nup159p (amino acid residues 441–876) (Kraemer et  al., 1995) were separated by SDS-PAGE, transferred to nitrocellulose, incubated with cytosol from either Kap95–PrA or  Kap119–PrA strains and then with HRP-labeled rabbit IgG. Mr  markers for the Nup1p and Nup2p experiments are shown to the  left of the Nup1p strips and for the Nup159p experiment to left of  the Nup159p strips. Note that Kap95–PrA interacts much more  strongly with Nup1p and Nup2p and their degradation products  than does Kap119–PrA.
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Figure 7: Kap119–PrA binds to various nucleoporins. Proteins of lysates from E. coli strains expressing the peptide repeat regions of Nup1p (amino acid residues 432–816) (Rexach and Blobel, 1995), Nup2p (amino acid residues 186–561) (Rexach and Blobel, 1995), or Nup159p (amino acid residues 441–876) (Kraemer et al., 1995) were separated by SDS-PAGE, transferred to nitrocellulose, incubated with cytosol from either Kap95–PrA or Kap119–PrA strains and then with HRP-labeled rabbit IgG. Mr markers for the Nup1p and Nup2p experiments are shown to the left of the Nup1p strips and for the Nup159p experiment to left of the Nup159p strips. Note that Kap95–PrA interacts much more strongly with Nup1p and Nup2p and their degradation products than does Kap119–PrA.

Mentions: During the course of nuclear import, the import complex needs to dock at the NPC. This docking is thought to be mediated by a subset of nucleoporins that bear peptide-repeat motifs, including Nup1p, Nup2p, and Nup159p (Radu et al., 1995b). Peptide repeat-containing domains of Nup1p, Nup2p, and Nup159p (Rexach and Blobel, 1995; Kraemer et al., 1995) were expressed in E. coli. Proteins of bacterial lysates were separated by SDS-PAGE, transferred to nitrocellulose, and then incubated with cytosol of the Kap119–PrA or Kap95–PrA strain and detected by luminol-based chemiluminescence (Fig. 7). As previously shown for Kap95–PrA (Iovine et al., 1995; Kraemer et al., 1995; Rexach and Blobel, 1995; Pemberton et al., 1997; Rosenblum et al., 1997), Kap119–PrA also bound to the tested nucleoporins: binding of Kap119–PrA to Nup1p and Nup2p was weaker than that of Kap95–PrA, whereas the extent of binding to Nup159p was similar for both PrA-tagged Kaps. These results indicate that Kap119p shares overlapping binding sites for peptide repeat containing Nups with other Kaps.


A novel nuclear import pathway for the transcription factor TFIIS.

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

Kap119–PrA binds to various nucleoporins. Proteins of  lysates from E. coli strains expressing the peptide repeat regions  of Nup1p (amino acid residues 432–816) (Rexach and Blobel,  1995), Nup2p (amino acid residues 186–561) (Rexach and Blobel,  1995), or Nup159p (amino acid residues 441–876) (Kraemer et  al., 1995) were separated by SDS-PAGE, transferred to nitrocellulose, incubated with cytosol from either Kap95–PrA or  Kap119–PrA strains and then with HRP-labeled rabbit IgG. Mr  markers for the Nup1p and Nup2p experiments are shown to the  left of the Nup1p strips and for the Nup159p experiment to left of  the Nup159p strips. Note that Kap95–PrA interacts much more  strongly with Nup1p and Nup2p and their degradation products  than does Kap119–PrA.
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Figure 7: Kap119–PrA binds to various nucleoporins. Proteins of lysates from E. coli strains expressing the peptide repeat regions of Nup1p (amino acid residues 432–816) (Rexach and Blobel, 1995), Nup2p (amino acid residues 186–561) (Rexach and Blobel, 1995), or Nup159p (amino acid residues 441–876) (Kraemer et al., 1995) were separated by SDS-PAGE, transferred to nitrocellulose, incubated with cytosol from either Kap95–PrA or Kap119–PrA strains and then with HRP-labeled rabbit IgG. Mr markers for the Nup1p and Nup2p experiments are shown to the left of the Nup1p strips and for the Nup159p experiment to left of the Nup159p strips. Note that Kap95–PrA interacts much more strongly with Nup1p and Nup2p and their degradation products than does Kap119–PrA.
Mentions: During the course of nuclear import, the import complex needs to dock at the NPC. This docking is thought to be mediated by a subset of nucleoporins that bear peptide-repeat motifs, including Nup1p, Nup2p, and Nup159p (Radu et al., 1995b). Peptide repeat-containing domains of Nup1p, Nup2p, and Nup159p (Rexach and Blobel, 1995; Kraemer et al., 1995) were expressed in E. coli. Proteins of bacterial lysates were separated by SDS-PAGE, transferred to nitrocellulose, and then incubated with cytosol of the Kap119–PrA or Kap95–PrA strain and detected by luminol-based chemiluminescence (Fig. 7). As previously shown for Kap95–PrA (Iovine et al., 1995; Kraemer et al., 1995; Rexach and Blobel, 1995; Pemberton et al., 1997; Rosenblum et al., 1997), Kap119–PrA also bound to the tested nucleoporins: binding of Kap119–PrA to Nup1p and Nup2p was weaker than that of Kap95–PrA, whereas the extent of binding to Nup159p was similar for both PrA-tagged Kaps. These results indicate that Kap119p shares overlapping binding sites for peptide repeat containing Nups with other Kaps.

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