The location of the transport gate in the nuclear pore complex

Signal-mediated nuclear transport is a gated process that occurs through a central transporter element located within the pore complex, The purpose of this investigation was to identify the region of the transporter that functions as the gate; i.e. the region that restricts passive diffusion of macromolecules through the pores. To accomplish this: small gold particles coated with polyethylene glycol (PEG; total particle diameter 40-70 Angstrom) or large PEG-particles (total diameter 110-270 Angstrom) were microinjected into the cytoplasm or nucleoplasm of Xenopus oocytes. Since PEG does not contain either nuclear import or export signals, it is assumed that the particles distribute by simple diffusion. The cells were fixed after 5 or 30 minutes and subsequently examined using TEM. The distribution of the particles located adjacent to and within the pore complexes was then mapped. The results obtained at both 5 and 30 minutes after cytoplasmic injections of small gold were basically the same. The particles readily entered the transporter but, on the average, were approximately 11 times more concentrated in the cytoplasmic half of this structure, The opposite distribution was observed following nuclear injections, i.e. the particles that were located in the transporter were approximately 7 times more numerous in the nuclear half, Our data indicate that there is a single transport gate located in the central domain of the transporter that restricts passive diffusion, The large particles that were injected into the cytoplasm migrated to the surface of the pore complex, but entered the transporter less frequently than small gold, Interestingly, the diffusion of large PEG-particles to the surface of the pores following nuclear injection was greatly restricted; however, this was not the case for similar size particles that were coated with protein containing nuclear export signals (NES). The latter results suggest that the NES is not only required for translocation, but also for migration within the nucleoplasm.