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.

Adams, PHENIX: building new software for automated crystallographic structure determination. 2002, Pubmed

Adams, PHENIX: building new software for automated crystallographic structure determination. 2002, Pubmed
Bastiaens, Gradients in the self-organization of the mitotic spindle. 2006, Pubmed , Xenbase
Bibby, A cancer-associated aurora A mutant is mislocalized and misregulated due to loss of interaction with TPX2. 2009, Pubmed
Brunet, Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts. 2004, Pubmed , Xenbase
Catimel, Biophysical characterization of interactions involving importin-alpha during nuclear import. 2001, Pubmed
Clarke, Spatial and temporal coordination of mitosis by Ran GTPase. 2008, Pubmed
Colledge, Extensive mutagenesis of the nuclear location signal of simian virus 40 large-T antigen. 1986, Pubmed
Conti, Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha. 2000, Pubmed , Xenbase
Conti, Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha. 1998, Pubmed
Cook, Structural biology of nucleocytoplasmic transport. 2007, Pubmed
Davis, MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. 2007, Pubmed
Dias, The molecular basis for the regulation of the cap-binding complex by the importins. 2009, Pubmed
Dougherty, Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. 1996, Pubmed
Eckerdt, Spindle pole regulation by a discrete Eg5-interacting domain in TPX2. 2008, Pubmed , Xenbase
Ems-McClung, Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal. 2004, Pubmed , Xenbase
Emsley, Coot: model-building tools for molecular graphics. 2004, Pubmed
Fontes, Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-alpha. 2000, Pubmed
Fontes, Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha. 2003, Pubmed , Xenbase
Groen, Functional overlap of microtubule assembly factors in chromatin-promoted spindle assembly. 2009, Pubmed , Xenbase
Gruss, Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity. 2001, Pubmed , Xenbase
Gruss, Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells. 2002, Pubmed , Xenbase
Gruss, The mechanism of spindle assembly: functions of Ran and its target TPX2. 2004, Pubmed
Harreman, Regulation of nuclear import by phosphorylation adjacent to nuclear localization signals. 2004, Pubmed
Hodel, Dissection of a nuclear localization signal. 2001, Pubmed
Hodel, Nuclear localization signal receptor affinity correlates with in vivo localization in Saccharomyces cerevisiae. 2006, Pubmed
Kalab, The RanGTP gradient - a GPS for the mitotic spindle. 2008, Pubmed
Kapust, Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. 2001, Pubmed
Kobe, Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha. 1999, Pubmed
Kosugi, Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. 2009, Pubmed
Kosugi, Six classes of nuclear localization signals specific to different binding grooves of importin alpha. 2009, Pubmed
Lange, Expanding the definition of the classical bipartite nuclear localization signal. 2010, Pubmed
Lange, Classical nuclear localization signals: definition, function, and interaction with importin alpha. 2007, Pubmed
Leung, Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding. 2003, Pubmed
Matsuura, Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import. 2003, Pubmed
Matsuura, Structural basis for the assembly of a nuclear export complex. 2004, Pubmed
Matsuura, Nup50/Npap60 function in nuclear protein import complex disassembly and importin recycling. 2005, Pubmed
Schatz, Importin alpha-regulated nucleation of microtubules by TPX2. 2003, Pubmed , Xenbase
Stewart, Molecular mechanism of the nuclear protein import cycle. 2007, Pubmed
Tsai, A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly. 2003, Pubmed , Xenbase
Tulu, Molecular requirements for kinetochore-associated microtubule formation in mammalian cells. 2006, Pubmed
Walczak, Mechanisms of mitotic spindle assembly and function. 2008, Pubmed , Xenbase