Recombinant karyopherin β3. (A) Full-length (lane 1) and C-terminal 183 amino acids (lane 2) of karyopherin β3 were subjected to SDS/PAGE on a 5–20% acrylamide gradient gel and stained with Coomassie blue. Places of molecular mass markers are indicated to the left. (B) Fifty nanograms of recombinant karyopherin β3 (lane 1) and 40 μg of HeLa cytosol S100 fraction (lane 2) were electrophoresed, transferred to nitrocellulose, and immunoblotted with anti-karyopherin β3 antibody 384AP, and the signal was detected by chemiluminescence.

Karyopherin β3 and karyopherin β1 compete for binding to repeat nucleoporins in overlay assay and in solution. (A) Nuclear envelope proteins were electrophoresed and transferred to nitrocellulose. Karyopherin β1 or β3 was added followed by the appropriate primary antibody and a secondary horseradish peroxidase-conjugated antibody. The signal was detected by chemiluminescence. The small arrows indicate a previously observed (3) unidentified band that interacts with both karyopherin β1 and β3. (B) Nuclear envelope protein blots were subjected to overlay assay with karyopherin β3 as in A but in the presence or absence of 10-fold molar excess of karyopherin β1 as indicated. The control lane (without karyopherin β1) contained glutathione elution buffer (see Materials and Methods) to control for the presence of this buffer in the karyopherin β1 preparation. The weak Nup98 signal in this lane may be due to the glutathione tripeptide competing with Nup98 for binding to karyopherin β3. (C) GST-karyopherin β3 fusion protein was bound to glutathione beads (lane 1), and Nup98 was added in the absence (lane 2) or presence (lane 3) of 5-fold molar excess of karyopherin β3. Lane 4 shows the Nup98 preparation in the absence of beads. (D) Bacterial lysates containing recombinant near-full length Nup98 (13) (lane 1), its C terminus (lane 2), or its repeat-containing N terminus (lane 3) were electrophoresed and transferred to nitrocellulose. The blot was stained with amido black (Left) and subjected to overlay assay with karyopherin β3 as in A (Right). Small arrows indicate the positions of the Nup98 bands in the amido black-stained gel.

Karyopherin β3 binds to ribosomal proteins in an overlay assay. Ribosomal proteins were separated by reverse-phase HPLC, electrophoresed, transferred to nitrocellulose membrane, stained with amido black (Left), and subjected to overlay assay with recombinant karyopherin β3 (Right). Fractions 6–16 are shown; the remaining fractions were negative by overlay assay (not shown). Small arrows indicate the positive bands on the amido black-stained gel.

Human karyopherin β3 is homologous to yeast Kap121p and human karyopherin β1. (A) The amino acid sequence of human karyopherin β3 (hβ3) was aligned with that of Kap121p (yβ3) by the Clustal method using the Multiple Sequence Alignment module of the Lasergene software (dnastar). The aligned sequences were imported into SeqVu 1.1 (Garvan Institute) to highlight homologies (boxed sequences) and identities (shaded sequences); the setting used for homologies was GES scale at 85%. (B) The amino acid sequences of human karyopherin β1 and β3 were aligned as in A. (C) Comparison of Ran binding regions in RanBP1 (32), human karyopherin β1 (21) and β3; the numbers indicate amino acid positions.

Immunofluorescence staining for karyopherin β3. HeLa cells treated with or without digitonin as indicated were incubated with anti-karyopherin β3 primary antibody and Cy3-conjugated secondary antibody. (Bar = 10 μm.)

Karyopherin β3 binds to Ran-GTP. GST-karyopherin β3 fusion protein was immobilized on beads as in Fig. 4C and either Ran-GDP or Ran-GTP was added. The bound and half of the unbound fractions were electrophoresed and subjected to immunoblotting with anti-Ran antibody.