Monoclonal antibodies to NTF2 inhibit nuclear protein import by preventing nuclear translocation of the GTPase Ran.
Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.
Steggerda
S M
SM
Center for Cell Signaling, Department of Biochemistry and Molecular Genetics, and Cell and Molecular Biology Program, University of Virginia Health Sciences Center, Charlottesville, Virginia, 22908, USA.
Black
B E
BE
Paschal
B M
BM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000276:immunology* / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D019921:Biotinylation; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin / Q000008:administration & dosage / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; C062521:RCC1 protein, human; D011993:Recombinant Fusion Proteins; D019809:Streptavidin; D020931:ran GTP-Binding Protein