Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process.
To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures.
Bataillé
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Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Helser
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Fried
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D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D002501:Centrifugation, Zonal; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008845:Microinjections; D009685:Nuclear Envelope / Q000502:physiology; D009865:Oocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D013696:Temperature; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D014982:Xenopus laevis
D046911:Macromolecular Substances; D014909:Wheat Germ Agglutinins; D000255:Adenosine Triphosphate