The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPγS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPγS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.