Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.
Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.
Paschal
B M
BM
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037, USA.
Gerace
L
L
D000595:Amino Acid Sequence; D001692:Biological Transport; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D039641:Integrin beta Chains; D016023:Integrins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D020931:ran GTP-Binding Protein
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D039641:Integrin beta Chains; D016023:Integrins; D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D011994:Recombinant Proteins; C049312:nuclear pore protein p62; C093515:pat-3 protein, C elegans; D020931:ran GTP-Binding Protein