Data on dimer formation between importin α subtypes

This article describes data related to the research article titled “Functional characterization of importin α8 as a classical nuclear localization signal receptor” [1]. A GST pull-down assay showed that both importin α1 and α8, which are classical nuclear localization signal (cNLS) receptors, can form a dimer with importin α6, α7, or α8. Importin α8 has higher dimer-forming ability than importin α1. In addition, our data show that either importin α1 or importin α8 can form a heterodimer with importin α3, which exists in a preformed complex with cNLS substrates such as the conventional SV40TNLS or the p53 protein, resulting in the release of the cNLS substrates from importin α3.

These data are valuable to researchers interested in the molecular mechanisms by which the importin α-cNLS substrate complex dissociates in the nucleus. These data show that importin α1 and α8 have substantial differences in dimer-forming ability, despite both proteins belonging to the same subfamily.
These data provide a new insight into the function of nuclear-localized importin αs.

Data
To examine whether importin αs can directly bind with other importin α subtypes, FLAG-tagged importin α6, α7, or α8 recombinant proteins were incubated with either GST-importin α8, or GSTimportin α1, and analyzed by western blotting (Fig. 1A). To investigate the effect of heterodimerization of importin αs on its substrate binding, either an importin α3-SV40TNLS complex, or an importin α3-p53 complex was incubated with increasing amounts of FLAG-importin α8, or α1 recombinant proteins, and analyzed by western blotting (Figs. 1B and 2).
The human cDNA encoding the tumor protein p53 (NM_000546) was amplified from MCF7 cells by PCR performed using the following primers: p53 Forward: 5 0 -CACGGATCCATGGAGGAGCCG-CAGTCAGATC-3 0 and p53 Reverse: 5 0 -GGACTCGAGTCAGTCTGAGTCAGGCCCTTCTG-3 0 . The PCR program was as follows: one cycle of 2 min at 94°C; 40 cycles of 15 s at 94°C, 30 sec at 64°C, and 1 min 20 s at 68°C; and one cycle of 10 min at 68°C. The PCR product was subcloned into the BamHI and XhoI sites of pGEX6P1, and then verified by sequencing.

Recombinant protein purification
Recombinant proteins fused to GST were purified as follows: The expression vectors were transformed into Escherichia coli Rosetta, and then the cells were grown at 37°C in LB medium containing  Fig. 1A: Bacterially produced FLAG-h-importin α6, α7, and α8 recombinant proteins (100 pmol each) were incubated with GST, GST-h-importin α8 (KPNA7), or GST-m-importin α1 (KPNA2) immobilized on GSH beads in 200 μL of transport buffer (TB; 20 mM HEPES at pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, 1 mM DTT, 500 μM PMSF, and 1 μg/mL each of aprotinin, pepstatin, and leupeptin) with 0.1% Triton X-100 at 4°C for 1 h. After washing five times with TB containing 0.1% Triton X-100, the beads were suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (50 mM Tris-HCl at pH 6.8, 34.7 mM SDS, 50% glycerol, 25% β-mercaptoethanol, and bromophenol blue). Bound proteins were analyzed by western blotting with specific antibodies described.   1% Triton X-100 at 4°C for 1 h. After washing the beads, either 50 pmol or 500 pmol of FLAG-h-importin α8 or FLAG-h-importin α1 was added to the importin α3-p53 complex at 4°C for 1 h. The beads were then washed five times with TB containing 0.1% Triton X-100, and bound proteins were analyzed by western blotting with the specific antibodies described below.

Western blotting
Samples were loaded on a 10% SDS-PAGE gel, and the separated proteins in the gel were transferred onto an Immobilon-P membrane (PVDF membrane; Merck Millipore, Darmstadt, Germany) using a semi-dry transfer blotting system (Trans-Blot Turbo Transfer System, BioRad Laboratories, Inc., Hercules, CA, USA). The membrane was blocked with blocking buffer consisting of 3% skim milk in Tris-buffered saline (TBS; TAKARA BIO, Shiga, Japan) with 0.05% Tween (TBS-T) for 1 h. The membrane was probed with primary antibodies diluted in Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO, Osaka, Japan) at 4°C overnight, and then incubated with the HRP-conjugated secondary antibody diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO) at room temperature (RT) for 1 h. After the membrane was washed with TBS-T, it was developed with Chemi-Lumi One L or Super (Nacalai Tesque). The intensity of each western blot signal was quantified by Image J software (http://rsbweb.nih.gov/ij/).