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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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RanGTP but not RanGDP dissociates TFIIS from and  binds to Kap119p. Incubation of Sepharose-bound Kap119–PrA/ TFIIS complex with recombinant RanGTP results in dissociation  of TFIIS. Three times the previously used amount of Kap119– PrA cytosol was used for immunoisolation. Equal amounts of  IgG-Sepharose–bound Kap119–PrA/TFIIS were incubated for 60  min at 21°C with either buffer (−), with RanGDP (5 μM) or with  RanGTP (5 μM). The bound and unbound fractions from a subsequent two step elution with 250 and 4,500 mM MgCl2 were analyzed by SDS-PAGE and Coomassie blue staining. Lanes 1 and 2  represent equivalent amounts of purified RanGDP and RanGTP  that were used for incubation. Note that the recovery of  RanGDP in the unbound fraction was less than 100%. Numbers  on the left indicate position of Mr markers.
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Figure 6: RanGTP but not RanGDP dissociates TFIIS from and binds to Kap119p. Incubation of Sepharose-bound Kap119–PrA/ TFIIS complex with recombinant RanGTP results in dissociation of TFIIS. Three times the previously used amount of Kap119– PrA cytosol was used for immunoisolation. Equal amounts of IgG-Sepharose–bound Kap119–PrA/TFIIS were incubated for 60 min at 21°C with either buffer (−), with RanGDP (5 μM) or with RanGTP (5 μM). The bound and unbound fractions from a subsequent two step elution with 250 and 4,500 mM MgCl2 were analyzed by SDS-PAGE and Coomassie blue staining. Lanes 1 and 2 represent equivalent amounts of purified RanGDP and RanGTP that were used for incubation. Note that the recovery of RanGDP in the unbound fraction was less than 100%. Numbers on the left indicate position of Mr markers.

Mentions: RanGTP has been reported to dissociate several import substrate/Kap complexes (Rexach and Blobel, 1995; Izaurralde et al., 1997; Siomi et al., 1997). To test whether RanGTP would dissociate TFIIS from Kap119p, we incubated the Sepharose-bound Kap119–PrA/TFIIS complex with recombinant RanGTP. After extensive washing, the Sepharose was split into three equal pools. One pool was further incubated with buffer alone while the two others were incubated with either RanGDP or RanGTP. Nearly all of the bound TFIIS (Fig. 6, lanes 3–5) was released from Kap119-PrA (Fig. 6, lanes 9–11) after incubation with RanGTP (Fig. 6, compare lane 5 with 11), but not after incubation with buffer (Fig. 6, compare lane 3 with 9) or with RanGDP (Fig. 6, compare lane 4 with 10). A portion of the added RanGTP remained bound to Kap119– PrA (Fig. 6, lane 5) whereas RanGDP did not bind (Fig. 6, lane 4). Hence, incubation of the complex with RanGTP resulted in dissociation of TFIIS and binding of RanGTP to Kap119–PrA. Importantly, identical amounts of Kap119–PrA were eluted from each pool.


A novel nuclear import pathway for the transcription factor TFIIS.

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

RanGTP but not RanGDP dissociates TFIIS from and  binds to Kap119p. Incubation of Sepharose-bound Kap119–PrA/ TFIIS complex with recombinant RanGTP results in dissociation  of TFIIS. Three times the previously used amount of Kap119– PrA cytosol was used for immunoisolation. Equal amounts of  IgG-Sepharose–bound Kap119–PrA/TFIIS were incubated for 60  min at 21°C with either buffer (−), with RanGDP (5 μM) or with  RanGTP (5 μM). The bound and unbound fractions from a subsequent two step elution with 250 and 4,500 mM MgCl2 were analyzed by SDS-PAGE and Coomassie blue staining. Lanes 1 and 2  represent equivalent amounts of purified RanGDP and RanGTP  that were used for incubation. Note that the recovery of  RanGDP in the unbound fraction was less than 100%. Numbers  on the left indicate position of Mr markers.
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Figure 6: RanGTP but not RanGDP dissociates TFIIS from and binds to Kap119p. Incubation of Sepharose-bound Kap119–PrA/ TFIIS complex with recombinant RanGTP results in dissociation of TFIIS. Three times the previously used amount of Kap119– PrA cytosol was used for immunoisolation. Equal amounts of IgG-Sepharose–bound Kap119–PrA/TFIIS were incubated for 60 min at 21°C with either buffer (−), with RanGDP (5 μM) or with RanGTP (5 μM). The bound and unbound fractions from a subsequent two step elution with 250 and 4,500 mM MgCl2 were analyzed by SDS-PAGE and Coomassie blue staining. Lanes 1 and 2 represent equivalent amounts of purified RanGDP and RanGTP that were used for incubation. Note that the recovery of RanGDP in the unbound fraction was less than 100%. Numbers on the left indicate position of Mr markers.
Mentions: RanGTP has been reported to dissociate several import substrate/Kap complexes (Rexach and Blobel, 1995; Izaurralde et al., 1997; Siomi et al., 1997). To test whether RanGTP would dissociate TFIIS from Kap119p, we incubated the Sepharose-bound Kap119–PrA/TFIIS complex with recombinant RanGTP. After extensive washing, the Sepharose was split into three equal pools. One pool was further incubated with buffer alone while the two others were incubated with either RanGDP or RanGTP. Nearly all of the bound TFIIS (Fig. 6, lanes 3–5) was released from Kap119-PrA (Fig. 6, lanes 9–11) after incubation with RanGTP (Fig. 6, compare lane 5 with 11), but not after incubation with buffer (Fig. 6, compare lane 3 with 9) or with RanGDP (Fig. 6, compare lane 4 with 10). A portion of the added RanGTP remained bound to Kap119– PrA (Fig. 6, lane 5) whereas RanGDP did not bind (Fig. 6, lane 4). Hence, incubation of the complex with RanGTP resulted in dissociation of TFIIS and binding of RanGTP to Kap119–PrA. Importantly, identical amounts of Kap119–PrA were eluted from each pool.

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