O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import.
Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component.
Sterne-Marr
R
R
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Blevitt
J M
JM
Gerace
L
L
D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats
D006023:Glycoproteins; D009687:Nuclear Proteins; D005033:Ethylmaleimide