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The molecular mechanism of translocation through the nuclear pore complex is highly conserved

¹ Department of Anatomy and Cell Biology, University of Florida, College of Medicine, Gainesville, FL 32610, USA
² MRC Laboratory of Molecular Biology, Hills Rd, Cambridge CB2 2QH, UK

View larger version (125K): [in a new window]   Fig. 1. Electron micrographs of amoebae, fixed 30 minutes after microinjection, showing the distribution of colloidal gold particles coated with either wild-type NTF2 (A) or W7A mutant NTF2 (B). There was significantly less binding of the mutant NTF2-gold to the nuclear pores (arrows). Since the mutation inhibits binding to FxFG repeats, the results suggest that these moieties are present in the pore complexes. The structures (nl) seen extending from the inner surface of the envelope are components of the nuclear lamina. In A. proteus, the lamina forms an elaborate honeycomb-like supporting structure. c, cytoplasm; n, nucleus. Bar, 100 nm.

View larger version (114K): [in a new window]   Fig. 2. The distribution of MAb414-coated gold particles in an amoebae fixed for electron microscopy 30 minutes after injection. Since particles coated with antibody are unable to penetrate the pore complexes (arrows), the gold localized to the nucleoporins along the cytoplasmic faces of the pores. c, cytoplasm; n, nucleus. Bar, 100 nm.

View larger version (38K): [in a new window]   Fig. 3. Western blot analysis for Ran. Blots were probed with monoclonal antibody to human Ran, and similar bands were detected in both the amoebae extract and the A431 cell lysate, which served as a positive control for Ran.

View larger version (54K): [in a new window]   Fig. 4. (A) Fluorescent micrographs illustrating the effect of transportin on the nuclear uptake of GST/GFP/M9 (wt or mt) fusion proteins. The micrographs were taken 60 minutes after the injection of GST/GFP/M9wt alone, GST/GFP/M9mt plus transportin or GST/GFP/M9wt plus transportin. N, nucleus; CV, contractile vacuole. (B) The nuclear uptake kinetics, expressed as N/C fluorescent ratios, are shown for amoebae injected with GST/GFP/M9wt alone (——), GST/GFP/M9mt plus transportin (——), GST/GFP/M9wt plus transportin (——), or fluorescein-labeled BSA (...[UNK]...), which served as a background control. Respectively, 13, 6, 17 and 7 cells were analyzed.

View larger version (108K): [in a new window]   Fig. 5. The distribution of transportin-coated gold in amoebae fixed 30 minutes after injection. Particles bound to the pore complexes (arrows) and were also present within the nucleoplasm. c, cytoplasm; n, nucleus. Bar, 100 nm.

View larger version (150K): [in a new window]   Fig. 6. The nucleocytoplasmic distribution of gold particles coated with BSA-bipartite NLS conjugates, which were injected either alone (A) or along with importin /ß (B). In the presence of the receptor, particles were associated with the pores (arrows) and also accumulated in the nucleus. c, cytoplasm; n, nucleus. Bar, 100 nm.

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