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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 pulls out TFIIS from the cytosol. A cytosolic fraction from a Kap119–PrA strain was incubated with IgG– Sepharose. The last wash fraction and fractions subsequently  eluted with a step gradient of 50–4,500 mM MgCl2 were analyzed  by SDS-PAGE and Coomassie blue staining. The Kap119–PrA  elutes between 1,000 and 4,500 mM MgCl2. The major band of  ∼35 kD eluting at 50 and 100 mM MgCl2 was identified by mass  spectrometry as the transcription factor TFIIS.
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Figure 2: Kap119–PrA pulls out TFIIS from the cytosol. A cytosolic fraction from a Kap119–PrA strain was incubated with IgG– Sepharose. The last wash fraction and fractions subsequently eluted with a step gradient of 50–4,500 mM MgCl2 were analyzed by SDS-PAGE and Coomassie blue staining. The Kap119–PrA elutes between 1,000 and 4,500 mM MgCl2. The major band of ∼35 kD eluting at 50 and 100 mM MgCl2 was identified by mass spectrometry as the transcription factor TFIIS.

Mentions: To coimmunoisolate potential transport substrate(s), a cytosol of the Kap119–PrA strain was incubated with IgG Sepharose. After extensive washing, the bound material was eluted with a MgCl2 step gradient ranging from 50 mM to 4.5 M MgCl2. The proteins in the eluted fractions were analyzed by SDS-PAGE and stained with Coomassie blue (Fig. 2). One major protein of ∼35 kD eluted at 50 and 100 mM MgCl2. In agreement with the previously observed elution behavior of transport substrates from PrA-tagged Kaps, the 35-kD band was a strong candidate for an Kap119p-bound transport substrate. As expected (Aitchison et al., 1996; Pemberton et al., 1997; Rosenblum et al., 1997; Rout et al., 1997), the Kap119–PrA eluted between 1.0 and 4.5 M MgCl2. By mass spectrometry, the 35-kD band was identified as TFIIS (also called PPR2, YSII, DST1, STALPHA, or YGL043) (Hubert et al., 1983; Davies et al., 1990; Clark et al., 1991; Nakanishi et al., 1992) by virtue of eight tryptic peptides between 1,000 and 1,915 D. The matching peptides, with 0.5 D tolerance, covered 29% of the TFIIS sequence. The TFIIS gene is not essential (Nakanishi et al., 1992) and codes for a protein of 309-amino acid residues with a calculated Mr of 34.8 kD. Homologous proteins have been identified in Drosophila, various mammalian genomes and in the vaccinia virus genome (for review see Kassavatis and Geiduschek, 1993). TFIIS binds to RNA polymerase II (Rappaport et al., 1988), and stimulates cleavage and elongation of nascent RNA in the transcription elongation complex (Izban and Luse, 1992; Nakanishi et al., 1992; Borukhov et al., 1993). As TFIIS contains a zinc-binding domain, it may directly interact with nucleic acid (Agarwal et al., 1991).


A novel nuclear import pathway for the transcription factor TFIIS.

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

Kap119–PrA pulls out TFIIS from the cytosol. A cytosolic fraction from a Kap119–PrA strain was incubated with IgG– Sepharose. The last wash fraction and fractions subsequently  eluted with a step gradient of 50–4,500 mM MgCl2 were analyzed  by SDS-PAGE and Coomassie blue staining. The Kap119–PrA  elutes between 1,000 and 4,500 mM MgCl2. The major band of  ∼35 kD eluting at 50 and 100 mM MgCl2 was identified by mass  spectrometry as the transcription factor TFIIS.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132971&req=5

Figure 2: Kap119–PrA pulls out TFIIS from the cytosol. A cytosolic fraction from a Kap119–PrA strain was incubated with IgG– Sepharose. The last wash fraction and fractions subsequently eluted with a step gradient of 50–4,500 mM MgCl2 were analyzed by SDS-PAGE and Coomassie blue staining. The Kap119–PrA elutes between 1,000 and 4,500 mM MgCl2. The major band of ∼35 kD eluting at 50 and 100 mM MgCl2 was identified by mass spectrometry as the transcription factor TFIIS.
Mentions: To coimmunoisolate potential transport substrate(s), a cytosol of the Kap119–PrA strain was incubated with IgG Sepharose. After extensive washing, the bound material was eluted with a MgCl2 step gradient ranging from 50 mM to 4.5 M MgCl2. The proteins in the eluted fractions were analyzed by SDS-PAGE and stained with Coomassie blue (Fig. 2). One major protein of ∼35 kD eluted at 50 and 100 mM MgCl2. In agreement with the previously observed elution behavior of transport substrates from PrA-tagged Kaps, the 35-kD band was a strong candidate for an Kap119p-bound transport substrate. As expected (Aitchison et al., 1996; Pemberton et al., 1997; Rosenblum et al., 1997; Rout et al., 1997), the Kap119–PrA eluted between 1.0 and 4.5 M MgCl2. By mass spectrometry, the 35-kD band was identified as TFIIS (also called PPR2, YSII, DST1, STALPHA, or YGL043) (Hubert et al., 1983; Davies et al., 1990; Clark et al., 1991; Nakanishi et al., 1992) by virtue of eight tryptic peptides between 1,000 and 1,915 D. The matching peptides, with 0.5 D tolerance, covered 29% of the TFIIS sequence. The TFIIS gene is not essential (Nakanishi et al., 1992) and codes for a protein of 309-amino acid residues with a calculated Mr of 34.8 kD. Homologous proteins have been identified in Drosophila, various mammalian genomes and in the vaccinia virus genome (for review see Kassavatis and Geiduschek, 1993). TFIIS binds to RNA polymerase II (Rappaport et al., 1988), and stimulates cleavage and elongation of nascent RNA in the transcription elongation complex (Izban and Luse, 1992; Nakanishi et al., 1992; Borukhov et al., 1993). As TFIIS contains a zinc-binding domain, it may directly interact with nucleic acid (Agarwal et al., 1991).

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