Identification and functional characterization of a novel nuclear localization signal present in the yeast Nab2 poly(A)+ RNA binding protein

Abstract

The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin alpha/importin alpha, which acts as the NLS receptor, and karyopherin beta1/importin beta, which binds karyopherin alpha and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin beta1, termed karyopherin beta2 or transportin, and does not require a karyopherin alpha-like adapter protein. A yeast homolog of karyopherin beta2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin beta1, but not the Kap104p homolog karyopherin beta2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin alpha. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

FIG. 1

Mapping of the Kap104p binding domain of Nab2p by using the yeast two-hybrid assay. Full-length Kap104p was expressed as a fusion protein linked to the GAL4 DNA binding domain, while wild-type and mutant forms of Nab2p were expressed as fusions with the VP16 transcription activation domain. The ability of Kap104p to interact with the indicated wild-type and mutant forms of Nab2p was then assayed in the yeast two-hybrid indicator strain Y190, which contains GAL4 DNA binding sites linked to the lacZ gene, and is given at right in milli-optical density units of β-Gal activity per milliliter of yeast extract. The indicated Nab2p missense mutants M1, M2, and M3 were constructed in the context of the boxed 161-to-271 Nab2p sequence which showed high-affinity binding to Kap104p.

FIG. 2

Subcellular localization in yeast cells of substrate proteins bearing wild-type and M3 mutant forms of the Kap104p binding domain of Nab2p. Yeast cells expressing wild-type GFP (D and H), a GFP fusion to Nab2p residues 161 to 271 (B and F), or a GFP fusion to the M3 mutant form of this same sequence (C and G) were fixed and stained with DAPI, a dye that binds DNA specifically. The subcellular localization of the GFP proteins was then identified in a fluorescence microscope (A through D) and compared to the localization of the DAPI-stained nucleus (E through H).

FIG. 3

Subcellular localization in human 293T cells of substrate proteins bearing wild-type and M3 mutant forms of the Kap104p binding domain of Nab2p. Human 293T cells were transfected with expression plasmids encoding wild-type β-Gal (C) or encoding β-Gal fused to the wild-type (A) or M3 mutant (B) form of the 161-to-271 Kap104p binding domain of Nab2p. Immunofluorescence analysis, using a β-Gal-specific monoclonal antibody, revealed that linkage to the wild-type Nab2p sequence resulted in the relocalization of β-Gal to the nucleus (A), whereas the β-Gal–M3 fusion showed the same cytoplasmic localization as that seen with wild-type β-Gal (B and C). No detectable fluorescence signal was observed with mock-transfected 293T cells (data not shown).

FIG. 4

Protein import into isolated mammalian nuclei mediated by yeast karyopherin β homologs. Isolated HeLa cell nuclei were incubated with fluorescein-labeled substrate proteins consisting of GST fused to the M9 NLS (A and B), the αNLS (C and D), or the SV40 TNLS (G and H) or of MBP fused to residues 161 to 271 of Nab2p (E and F). In addition, the nuclei were incubated with either purified recombinant yeast Kap104p or Kap95p, as well as with added human Ran, p10, and a source of energy. After incubation at 30°C for 15 min, the nuclei were fixed and analyzed for nuclear import of the labeled substrate proteins.

FIG. 5

Protein import into isolated mammalian nuclei mediated by human karyopherins β1 and β2. The in vitro nuclear import assays were performed as described for Fig. 4 except that recombinant human karyopherins β2 and β1 were substituted for their respective yeast homologs Kap104p and Kap94p. Panels are as described in the legend to Fig. 4.

FIG. 6

Mutations of Nab2p that block Kap104p binding also block in vitro nuclear import. Fluorescein-labeled substrate proteins, consisting of MBP fused to the wild-type or M1, M2, or M3 mutant form of Nab2p aa 161 to 271 (Fig. 1), were analyzed for in vitro import into isolated mammalian nuclei, as described for Fig. 4, by using either yeast Kap104p (A to D) or human karyopherin β1 (E to H) as an import factor.

FIG. 7

The Nab2p NLS binds both Kap104p and karyopherin β1 (Kar β1) in vitro. Microaffinity columns (15 μl) consisting of the indicated purified recombinant import factors linked to an Affi-Gel 10 matrix were loaded with purified recombinant MBP-Nab2p(161-271) in 50 μl of binding buffer. After being washed with 200 μl of binding buffer, bound proteins were eluted in 50 μl of 1 M MgCl₂. The flowthrough (FT) and eluate (E) samples (but not the wash) were then analyzed by SDS-PAGE and visualized by staining. A parallel experiment using purified MBP as a column load did not reveal binding to any of the four affinity columns (data not shown).

FIG. 8

The Nab2p NLS can specifically compete with the αNLS for karyopherin β1-mediated nuclear import. In vitro nuclear import assays, using human karyopherin β1 (Kar β1; A to C) or β2 (D) and fluorescein-labeled GST-αNLS (A to C) or GST-M9 (D), were performed as described for Fig. 5 except that unlabeled recombinant MBP (B) or recombinant MBP-Nab2p(161-271) (C and D) was added to the isolated nuclei at a 20-fold molar excess over the labeled substrate proteins prior to initiation of the import assay.

FIG. 9

Ran is required for both karyopherin β2 (Kar β2)- and Kap104p-mediated nuclear import. Nuclear import assays were performed as described for Fig. 4 and 5 except that the Ran mutant T24N was substituted for wild-type Ran (RAN wt) in the two incubations visualized at the right.