Limits...
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.

Show MeSH

Related in: MedlinePlus

TFIIS–PrA pulls out Kap119p in a wt strain but does  not appear to pull out an alternative Kap in a kap119Δ strain. Cytosolic fractions were prepared from wt strain or from a kap119Δ  strain and analyzed as described in Fig. 2. The major band of  ∼120 kD that eluted from the IgG–Sepharose at 100 mM MgCl2  in the wt cytosol experiment (left) was identified by mass spectrometry as Kap119p. Note that no visible bands at the 100 mM  MgCl2 step were detected in the kap119Δ cytosol experiment  (right) in the region above 90 kD where most Kapβs migrate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132971&req=5

Figure 4: TFIIS–PrA pulls out Kap119p in a wt strain but does not appear to pull out an alternative Kap in a kap119Δ strain. Cytosolic fractions were prepared from wt strain or from a kap119Δ strain and analyzed as described in Fig. 2. The major band of ∼120 kD that eluted from the IgG–Sepharose at 100 mM MgCl2 in the wt cytosol experiment (left) was identified by mass spectrometry as Kap119p. Note that no visible bands at the 100 mM MgCl2 step were detected in the kap119Δ cytosol experiment (right) in the region above 90 kD where most Kapβs migrate.

Mentions: We tested whether cytosolic TFIIS binds only to Kap119p or whether it can also bind to other Kaps in a strain where Kap119p was deleted. Cytosol from a wild-type or a kap119Δ strain was incubated with IgG–Sepharose, bound protein eluted by MgCl2 step gradients, analyzed by SDS-PAGE, and then stained with Coomassie blue. In the wild-type strain a major band of ∼120 kD eluted a 100 mM MgCl2 (Fig. 4, left). Mass spectrometric analysis showed that this band was Kap119p. The TFIIS–PrA eluted between 1.0 and 4.5 M MgCl2. Judging from the staining intensity of the Kap119p and TFIIS–PrA bands, it appears that most of the cytosolic TFIIS–PrA was associated with Kap119p, suggesting that Kap119p is a major binding partner of TFIIS in the cytosol. Interestingly, in the cytosol prepared from the kap119Δ strain no clear candidates for additional Kaps were observed (Fig. 4, right) suggesting that TFIIS was not associated with another Kap in a strain deleted for Kap119p. This result is in agreement with the observed mislocalization of TFIIS reported above. We therefore conclude that Kap119p is the principal Kap for import of TFIIS.


A novel nuclear import pathway for the transcription factor TFIIS.

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

TFIIS–PrA pulls out Kap119p in a wt strain but does  not appear to pull out an alternative Kap in a kap119Δ strain. Cytosolic fractions were prepared from wt strain or from a kap119Δ  strain and analyzed as described in Fig. 2. The major band of  ∼120 kD that eluted from the IgG–Sepharose at 100 mM MgCl2  in the wt cytosol experiment (left) was identified by mass spectrometry as Kap119p. Note that no visible bands at the 100 mM  MgCl2 step were detected in the kap119Δ cytosol experiment  (right) in the region above 90 kD where most Kapβs migrate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: TFIIS–PrA pulls out Kap119p in a wt strain but does not appear to pull out an alternative Kap in a kap119Δ strain. Cytosolic fractions were prepared from wt strain or from a kap119Δ strain and analyzed as described in Fig. 2. The major band of ∼120 kD that eluted from the IgG–Sepharose at 100 mM MgCl2 in the wt cytosol experiment (left) was identified by mass spectrometry as Kap119p. Note that no visible bands at the 100 mM MgCl2 step were detected in the kap119Δ cytosol experiment (right) in the region above 90 kD where most Kapβs migrate.
Mentions: We tested whether cytosolic TFIIS binds only to Kap119p or whether it can also bind to other Kaps in a strain where Kap119p was deleted. Cytosol from a wild-type or a kap119Δ strain was incubated with IgG–Sepharose, bound protein eluted by MgCl2 step gradients, analyzed by SDS-PAGE, and then stained with Coomassie blue. In the wild-type strain a major band of ∼120 kD eluted a 100 mM MgCl2 (Fig. 4, left). Mass spectrometric analysis showed that this band was Kap119p. The TFIIS–PrA eluted between 1.0 and 4.5 M MgCl2. Judging from the staining intensity of the Kap119p and TFIIS–PrA bands, it appears that most of the cytosolic TFIIS–PrA was associated with Kap119p, suggesting that Kap119p is a major binding partner of TFIIS in the cytosol. Interestingly, in the cytosol prepared from the kap119Δ strain no clear candidates for additional Kaps were observed (Fig. 4, right) suggesting that TFIIS was not associated with another Kap in a strain deleted for Kap119p. This result is in agreement with the observed mislocalization of TFIIS reported above. We therefore conclude that Kap119p is the principal Kap for import of TFIIS.

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