THE LOCATION OF THE TRANSPORT GATE IN THE NUCLEAR-PORE COMPLEX

Signal-mediated nuclear transport is a gated process that occurs throu gh a central transporter element located within the pore complex, The purpose of this investigation was to identify the region of the transp orter that functions as the gate; i.e. the region that restricts passi ve diffusion of macromolecules through the pores. To accomplish this: small gold particles coated with polyethylene glycol (PEG; total parti cle diameter 40-70 Angstrom) or large PEG-particles (total diameter 11 0-270 Angstrom) were microinjected into the cytoplasm or nucleoplasm o f 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 subsequentl y examined using TEM. The distribution of the particles located adjace nt to and within the pore complexes was then mapped. The results obtai ned at both 5 and 30 minutes after cytoplasmic injections of small gol d were basically the same. The particles readily entered the transport er but, on the average, were approximately 11 times more concentrated in the cytoplasmic half of this structure, The opposite distribution w as observed following nuclear injections, i.e. the particles that were located in the transporter were approximately 7 times more numerous i n 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 cyt oplasm migrated to the surface of the pore complex, but entered the tr ansporter less frequently than small gold, Interestingly, the diffusio n 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 nuclea r export signals (NES). The latter results suggest that the NES is not only required for translocation, but also for migration within the nu cleoplasm.