Karyopherin beta2 mediates nuclear import of a mRNA binding protein

Abstract

We have cloned and sequenced cDNA for human karyopherin beta2, also known as transportin. In a solution binding assay, recombinant beta2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1's previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin beta1, karyopherin beta2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both beta1 and beta2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, beta2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of beta2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of beta2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin beta2 inhibited karyopherin alpha/beta1-mediated import of a classical NLS containing substrate and, vice versa, beta1 inhibited beta2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of beta1 and beta2 to repeat nucleoporins.

Figure 1

Comparison of amino acid sequences of human karyopherin β2, yeast karyopherin β2 (Kap104p), and human karyopherin β1. Sequences were aligned using the clustalw v.1.6 program and analyzed with boxshade. Identical amino acids are indicated by black boxes and similar amino acids by gray boxes.

Figure 2

Karyopherin β2 binds to a fusion protein (GST-A1) containing the nuclear mRNA binding protein A1 via a specific sequence. (A) Purified recombinant karyopherin β2 analyzed by SDS/PAGE and visualized by Coomassie blue staining. (B) Immobilized GST-A1 was incubated with karyopherin β2 alone (lane 1) or with a 25× molar excess of a synthetic peptide representing the NLS of the A1 protein (lane 2) or with a 100× molar excess of the classical NLS peptide (lane 3). Bound and unbound fractions were analyzed by SDS/PAGE and Coomassie blue staining.

Figure 3

Karyopherin β2 binds to peptide repeat-containing nucleoporins. Proteins from purified rat liver nuclear envelopes were separated by SDS/PAGE, transferred to nitrocellulose, and incubated with ³⁵S-labeled karyopherin β2 (lane 1) or ³⁵S-labeled karyopherin β1 (lane 2). The arrows indicate positions of unidentified bands that interact with karyopherin β2.

Figure 4

Karyopherin β2 and β1 compete for binding to Nup98. Immobilized Nup98 was incubated with GST-β1 (lane 1), β2 (lane 2), GST-β1 and 10× molar excess of β2 (lane 3), or β2 and 10× molar excess of GST-β1 (lane 4). Bound and unbound fractions were analyzed by SDS/PAGE and Coomassie blue staining.

Figure 5

GST-A1 import into the nucleus requires karyopherin β2 and Ran. (A) Digitonin-permeabilized HeLa cells were incubated at 4°C with fluorescently labeled GST-A1, in the presence or absence of karyopherin β2 (2 μg/assay) as indicated. (B) Permeabilized cells were incubated at 20°C, with fluorescently labeled GST-A1 in the absence (panel 1) or in the presence of karyopherin β2 (2 μg/assay)

Figure 6

Karyopherin β1 and karyopherin β2 compete for nuclear import. (A) Digitonin-permeabilized HeLa cells were incubated at 20°C with fluorescently labeled NLS-human serum albumin, karyopherin α2, karyopherin β1, Ran, and p10 in the absence (panel 1) or in the presence of 10× molar excess of karyopherin β2 (panel 2). (B) Digitonin-permeabilized HeLa cells were incubated with fluorescently labeled GST-A1, karyopherin β2, and Ran in the absence (panel 1) or in the presence of 10× molar excess of karyopherin β1 (panel 2). (panels 2–6). Wild-type Ran (panels 3, 4, and 6) or a GTPase-deficient mutant Ran (panel 5) were added. Wheat germ agglutinin (WGA) was added in panel 6. (C) Permeabilized cells were incubated at 20°C with fluorescently labeled GST-A1 and karyopherin β2 (0.5 μg/assay) in the presence or absence of Ran as indicated.