A novel nuclear import pathway for the transcription factor TFIIS

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.

Figure 1

Cellular localization of Kap119p. A strain where endogenous Kap119p was replaced by protein A-tagged Kap119p (Kap119-PrA) was used. (A) Yeast cells were visualized by Nomarski (left) and Kap119–PrA was detected by indirect immunofluorescence (middle). Nuclei were visualized by DAPI staining (right). (B) A cell homogenate (T) was fractionated into a cytosolic (C) and a nuclear (N) fraction. Cell equivalent amounts were analyzed by SDS-PAGE and Kap119–PrA, cytoplasmic 3-phosphoglycerate kinase (3-PGK), and nucleolar Nop1p were detected immunologically using either rabbit anti–mouse IgG, monoclonal mouse antiserum D66, or monoclonal mouse antiserum 22C5-D8.

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.

Figure 3

Deletion of KAP119 causes slower growth and leads to mislocalization of TFIIS. (A) Comparison of growth rates of DF5α (wt) and of the KAP119 deletion strain, kap119Δ. Cultures were inoculated at OD 0.1 into rich media, grown at 30°C and samples were taken at the indicated time points. (B) Localization by immunofluorescence in wild-type (wt), kap119Δ, and kap108Δ strains in which the endogenous TFIIS had been replaced by TFIIS–PrA. Whereas TFIIS–PrA was primarily localized to the nucleus in the wt and kap108Δ strains, it was diffusely localized throughout the cell in the kap119Δ strain. (C) A homogenate (T) of kap119Δ and wt cells was fractionated into a cytosolic (C) and a nuclear (N) fraction. Cell equivalent portions were analyzed by SDS-PAGE and TFIIS–PrA, 3-phosphoglycerate kinase (3-PGK) and Nop1p were detected with either rabbit anti–mouse IgG, monoclonal mouse antiserum D66 or monoclonal mouse antiserum 22C5-D8. TFIIS was predominantly localized in the cytoplasm in kap119Δ cells.

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.

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.

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 M _r markers.

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. M _r 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.