Giesecke A , Stewart M .

Wellcome Trust , MC_U105178939 Medical Research Council , MRC_MC_U105178939 Medical Research Council

	FIGURE 1. Schematic illustration of how TPX2 is activated by RanGTP in the vicinity of the chromosomes. In the bulk of the cytoplasm, where the RanGTP concentration is low, importin-α binds to importin-β via its IBB domain freeing it to bind to TPX2 thereby inactivating its role in mitotic spindle assembly (1–3). However near the chromosomes, where the RanGTP concentration is elevated (11), RanGTP binds to importin-β leading to the release of TPX2 from importin-α, after which TPX2 is active in mitotic spindle assembly.
	FIGURE 2. The TPX2270–289 NLS binds to a range of Xenopus and mouse importin-α constructs. A, lanes 1 and 4 are crude bacterial lysate expressing the importin-α isoform; lanes 2 and 5 are the pull-down using GST- TPX2270–289; and lanes 3 and 6 are the pull-down using GST alone. GST-TPX2270–289 was able to bind mouse ΔIBB-importin-α2 and Xenopus ΔIBB-importin-α1a specifically from crude bacterial lysates, whereas virtually no binding was seen to GST alone. B, GST-TPX2270–289 binds to a range of ΔIBB importin-α isoforms derived from either mouse or Xenopus.
	FIGURE 3. Crystal structure of the TPX2:ΔIBB-importin-α complex. A, electron density (blue) for the TPX2 NLS peptide (red) superimposed on the importin-α structure (yellow); B, interaction interface between TPX2 residues 283–288 and the minor NLS-binding site on importin-α; C, interaction interface between TPX2 residues 322–331 and the major NLS-binding site on importin-α; D, schematic illustration of the interactions of importin-α with nucleoplasmin and TPX2, respectively.
	FIGURE 4. Nonclassical TPX2 NLS binds to the minor binding site of ΔIBB importin-α. Pull-down assays with ΔIBB importin-α variants from mouse (A) and Xenopus (B). Clarified bacterial cell lysates expressing the indicated GST fusion proteins were immobilized on glutathione-Sepharose and incubated with cell lysates containing the respective importin-α variants. Bound proteins were eluted with 2× SDS sample buffer and analyzed by SDS-PAGE. The ΔIBB importin-αD and ΔIBB importin-αE are variants in which binding to the major and minor binding sites, respectively, is inhibited, whereas the ΔIBB importin-αED variant harbors mutations in both sites.
	FIGURE 5. TPX2284–287 is the stronger binding site for ΔIBB importin-α. Pull-down assays were performed with crude bacterial cell lysates expressing the indicated GST-TPX2 NLS fragments to assess their ability to co-precipitate ΔIBB importin-α. Whereas TPX2 fragments containing residues 284–287 (GST-TPX2270–350, lane 1, and GST-TPX2270–289, lane 2) were able to pull-down ΔIBB-importin-α, the fragment containing only residues 322–331 (GST-TPX2290–350, lane 3) was not, consistent with residues 284–287 representing the major importin-α-binding site on TPX2.
	FIGURE 6. Pull-down competition assay. Beads containing 4.5 μg of GST-SV40 NLS (lanes 1–3) or 4.5 μg of GST-nucleoplasmin NLS (lanes 4–6) were incubated with 10 μg of ΔIBB importin-α ± 30 μm of the indicated TPX2 NLS-GFP fragments. Following incubation, the beads were separated and washed by centrifugation. The bound lane shows the material remaining on the beads, whereas the unbound is the material remaining in solution. Although it has been proteolyzed to a considerable extent TPX2276–351-GFP can compete with both the SV40, and the nucleoplasmin NLS for binding to ΔIBB-importin-α, as evidenced by the displacement of ΔIBB-importin-α from the GST-NLS into the unbound fraction, whereas TPX2276–291-GFP competes less effectively, so that more ΔIBB-importin-α remains bound. Asterisks represent impurities or proteolytic fragments.

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