Identification of a soluble precursor complex essential for nuclear pore assembly in vitro.
We analysed the soluble form in which the nuclear pore complex protein p68 is stored in Xenopus laevis eggs and its involvement in pore complex assembly processes. We have shown previously that p68, which is the major wheat germ agglutinin (WGA)-binding glycoprotein of nuclear pore complexes from Xenopus oocytes, is located in the pore channel and participates in mediated transport of karyophilic proteins. Using a monoclonal antibody directed against p68 (PI1) we removed this protein from Xenopus egg extract by immunoadsorption. On addition of lambda DNA the immuno-depleted extract supported reconstitution of nuclei which were surrounded by a continuous double-membrane envelope but lacked pore complexes and were unable to import karyophilic proteins such as nucleoplasmin or lamin LIII. Essentially identical results were obtained with extract depleted of WGA-binding proteins. Our finding that both the anti-p68 antibody and WGA efficiently removed components from the extract necessary for pore complex assembly but did not interfere with nuclear membrane formation demonstrates that these processes are independent of each other. Analysis of the immunoprecipitate on silver-stained SDS-polyacrylamide gels indicated that the antibody adsorbed other proteins besides p68, notably two high molecular weight components. By sucrose gradient centrifugation and gel filtration we showed that p68 together with associated protein(s) forms a stable, approximately globular complex with an Mr of 254,000, a Stokes radius of 5.2 nm and a sedimentation coefficient of 11.3 S. Our finding that p68 occurs in the form of larger macromolecular assemblies offers an explanation for the distinctly punctate immunofluorescence pattern observed in the cytoplasm of mitotic cells after staining with antibodies to p68.
Dabauvalle
M C
MC
Institute of Zoology I, University of Würzburg, Federal Republic of Germany.
Loos
K
K
Scheer
U
U
D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D053487:DEAD-box RNA Helicases; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008938:Mitosis; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism / Q000648:ultrastructure; D011494:Protein Kinases; D020365:RNA Helicases; D014909:Wheat Germ Agglutinins / Q000378:metabolism; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D014909:Wheat Germ Agglutinins; D011494:Protein Kinases; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases