Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor.
We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin-permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro nuclear import. Transport inhibition by GTP gamma S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography. Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient nuclear import. Biochemical complementation with bacterially expressed protein demonstrated that this essential GTP gamma S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate nuclear protein import with these other nuclear activities.
Melchior
F
F
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Paschal
B
B
Evans
J
J
Gerace
L
L
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D020931:ran GTP-Binding Protein
D009687:Nuclear Proteins; D009842:Oligopeptides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein