Ben-Efraim I , Frosst PD , Gerace L .

GM41955 NIGMS NIH HHS , R01 GM041955-16 NIGMS NIH HHS , R01 GM041955-17 NIGMS NIH HHS , R01 GM041955 NIGMS NIH HHS

	Figure 1. Purification of recombinant Tpr from SF9 cells and C-Tpr (residues 1626-2348) from bacteria. Panel A: Coomassie stained SDS-PAGE (8%) of a representative 6×His-Tpr purification. Shown are the cell lysate following centrifugation, washes (800 mM NaCl, 20 mM HEPES pH7.5, 10% glycerol, 15 mM imidazole and protease inhibitors) to remove material non-specifically bound to the resin and seven elution fractions (100 mM NaCl, 20 mM HEPES, 150 mM imidazole and 5 mM beta-mercaptoethanol with protease inhibitors). Panel B: Western analysis of the samples in Fig.1 Panel A, probed with a combination of antibodies to the N-terminus and the C-terminus of Tpr. Panel C: Coomassie stained SDS-PAGE (8%) of a representative purified CTpr(1626-2348).
	Figure 2. Characterization of the binding of CRM1 or importin β to Tpr or C-Tpr. Panel A: Binding was analyzed in the absence of RanGTP and NES (empty circles) or presence of RanGTP and NES (filled triangles) or absence of RanGTP and presence of NES (filled circles). Increasing eqimolar concentrations of CRM1, RanGTP and NES were incubated with Tpr and bound CRM1 was quantified as described in materials and methods. Panel B: Binding to Tpr was analyzed with increasing concentrations of importin β (filled circles) or importin β I178D (inverted triangles). Binding to C-Tpr was analyzed with increasing concentrations of importin β. The results are from duplicates of a single typical experiment. The standard deviation was <5% of the mean. The data were fitted to the equation B(Y) = Bmax × Y/(Kd + Y) (using Kaleida Graph software) where Y is the concentration of CRM1 (Panel A) or importin β/importin β I178D (Panel B) and B is the amount of CRM1(Panel A) or importin β/importin β I178D (Panel B) specifically bound. The correlation coefficients of the data to the fitted curves were >0.99.
	Figure 3. The binding of importin β to Tpr in the solid phase assay is sensitive to RanGTP. The binding of 100 nM importin β to Tpr adsorbed to microtiter wells was analyzed in the absence, or in the presence of 0.1, 0.2, 0.5 and 1 μM RanGMP-PNP or RanGDPβS, as indicated. The bound importin β was quantified as described in materials and methods.
	Figure 4. Characterization of the binding of ΔIBB-importin α to Tpr and demonstration of different binding sites for ΔIBB-importin α and importin β on Tpr. Panel A: Increasing concentrations of ΔIBB-importin α were incubated with immobilized Tpr or C-Tpr and the bound ΔIBB-importin α was quantified as described in materials and methods. The data were analyzed as described in Fig. 1. Panel B: The binding of 60 nM importin β to Tpr pre-adsorbed to microtiter wells was analyzed in the absence of ΔIBB-importin α or in the presence of 60, 120, 300 and 600 nM ΔIBB-importin α, as indicated. Bound importin β and ΔIBB-importin α were quantified as described in Materials and Methods.
	Figure 5. C-Tpr import into the nucleus is cytosol, temperature and energy dependent and is blocked by WGA. Digitonin-permeabilized NRK cells were incubated with 8 pmoles of Cy5-C-Tpr for 20 min at 30°C (panel A) or 4°C (panel B) in the presence of cytosol and an energy-regenerating system. Panel C: 8 μg WGA was added. Panel D: the ATP regenerating system was replaced by an ATP-depleting system, 0.8885 U hexokinase + glucose. Samples were visualized by confocal microscopy.
	Figure 6. Role of importin α/β nuclear import pathway in the nuclear distribution of C-Tpr. Digitonin-permeabilized NRK cells were incubated with 8 pmoles Cy5-C-Tpr (Panels A, C, E, G) or 8 pmoles Cy3-BSA-NLS (coupled with a synthetic peptide comprising the NLS of SV40 T-antigen; Panels B, D, F, H) and nuclear import was conducted with recombinant factors as described in Materials and Methods. Panels A, B: nuclear import of Cy5-C-Tpr and Cy5-BSA-NLS performed at 30°C; panels C, D: importin α was omitted; panels E, F: importin β was omitted; panels G, H: Cy5-C-Tpr and Cy3-BSA-NLS competed with 10-fold molar excess of unlabeled cargo. Samples were visualized by confocal fluorescent microscopy.

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