title: Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation.
abstract: Large nuclear proteins must possess a signal sequence to pass through the nuclear pores. Using an in vitro system, we have been able experimentally to dissect nuclear protein transport into two distinct steps: binding and translocation. In the absence of ATP, we observe a binding of nuclear proteins to the pore that is signal sequence-dependent. Translocation through the pore, on the other hand, strictly requires ATP. These steps, visualized in the fluorescence and electron microscopes, were observed both with a natural nuclear protein, nucleoplasmin, and a synthetic nuclear protein, composed of the signal sequence of SV40 T antigen coupled to HSA. When a mutant signal sequence was coupled to HSA, neither transport nor binding were observed, indicating that both result from the presence of a functional signal sequence. An inhibitor of transport, the lectin WGA, also arrested nuclear proteins in a bound state at the cytoplasmic face of the pore. Therefore, only the translocation step is sensitive to the inhibitor WGA, which is known to bind specifically to proteins of the nuclear pore.
authors.affiliation: Department of Biology, University of California at San Diego, La Jolla 92093.
authors.forename: D D
authors.initials: DD
authors.lastname: Newmeyer
authors.forename: D J
authors.initials: DJ
authors.lastname: Forbes
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D005260:Female; D006046:Gold; D006651:Histocytochemistry; D008099:Liver / Q000648:ultrastructure; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D056489:Nucleoplasmins; D009865:Oocytes / Q000648:ultrastructure; D010750:Phosphoproteins; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012709:Serum Albumin / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D014981:Xenopus
chemical_list: D000952:Antigens, Polyomavirus Transforming; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D021382:Protein Sorting Signals; D012709:Serum Albumin; D014909:Wheat Germ Agglutinins; D006046:Gold; D000255:Adenosine Triphosphate