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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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Various substrates  that are transported by other  Kaps are not mislocalized in  a kap119Δ strain. The cellular distribution in wt and  kap119Δ strains were directly  examined for an NLS–GFP  reporter (Shulga et al., 1996),  for an NLS–GFP–NES reporter (Stade et al., 1997)  and an Lhp1p–GFP reporter  (Rosenblum et al., 1998), in  fixed cells. Monospecific antibodies and indirect immunofluorescence were used to  detect Nlp3p (Wilson et al.,  1994). Nuclei were visualized by DAPI staining.
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Figure 5: Various substrates that are transported by other Kaps are not mislocalized in a kap119Δ strain. The cellular distribution in wt and kap119Δ strains were directly examined for an NLS–GFP reporter (Shulga et al., 1996), for an NLS–GFP–NES reporter (Stade et al., 1997) and an Lhp1p–GFP reporter (Rosenblum et al., 1998), in fixed cells. Monospecific antibodies and indirect immunofluorescence were used to detect Nlp3p (Wilson et al., 1994). Nuclei were visualized by DAPI staining.

Mentions: To determine whether deletion of KAP119 generally affected transport, we localized several substrates, whose transport is known to be mediated by a distinct cognate Kap, to see whether they were also mislocalized in a kap119Δ strain. We localized NLS–green fluorescent protein (GFP) (Shulga et al., 1996), NES–GFP–NLS (Stade et al., 1997), Lhp1p–GFP (Rosenblum et al., 1998), Npl3p (Wilson et al., 1994), and Nab2p (Aitchison et al., 1996) in both wild-type and kap119Δ strains. These substrates are transported by Kap60p/Kap95p, Kap124p (Crm1p), Kap108p, Kap111p, and Kap104p, respectively. As shown in Fig. 5, the localization of these substrates (data not shown for Nab2p) appeared similar in both strains. Hence deletion of Kap119p does not generally affect transport nor does Kap119p-mediated import specifically impact any of these other pathways.


A novel nuclear import pathway for the transcription factor TFIIS.

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

Various substrates  that are transported by other  Kaps are not mislocalized in  a kap119Δ strain. The cellular distribution in wt and  kap119Δ strains were directly  examined for an NLS–GFP  reporter (Shulga et al., 1996),  for an NLS–GFP–NES reporter (Stade et al., 1997)  and an Lhp1p–GFP reporter  (Rosenblum et al., 1998), in  fixed cells. Monospecific antibodies and indirect immunofluorescence were used to  detect Nlp3p (Wilson et al.,  1994). Nuclei were visualized by DAPI staining.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Various substrates that are transported by other Kaps are not mislocalized in a kap119Δ strain. The cellular distribution in wt and kap119Δ strains were directly examined for an NLS–GFP reporter (Shulga et al., 1996), for an NLS–GFP–NES reporter (Stade et al., 1997) and an Lhp1p–GFP reporter (Rosenblum et al., 1998), in fixed cells. Monospecific antibodies and indirect immunofluorescence were used to detect Nlp3p (Wilson et al., 1994). Nuclei were visualized by DAPI staining.
Mentions: To determine whether deletion of KAP119 generally affected transport, we localized several substrates, whose transport is known to be mediated by a distinct cognate Kap, to see whether they were also mislocalized in a kap119Δ strain. We localized NLS–green fluorescent protein (GFP) (Shulga et al., 1996), NES–GFP–NLS (Stade et al., 1997), Lhp1p–GFP (Rosenblum et al., 1998), Npl3p (Wilson et al., 1994), and Nab2p (Aitchison et al., 1996) in both wild-type and kap119Δ strains. These substrates are transported by Kap60p/Kap95p, Kap124p (Crm1p), Kap108p, Kap111p, and Kap104p, respectively. As shown in Fig. 5, the localization of these substrates (data not shown for Nab2p) appeared similar in both strains. Hence deletion of Kap119p does not generally affect transport nor does Kap119p-mediated import specifically impact any of these other pathways.

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