The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors.
We have isolated two cytosolic fractions from Xenopus oocytes that contain all of the activity necessary to support both steps of nuclear import in digitonin-permeabilized mammalian cells: binding at the nuclear envelope and translocation through the nuclear pore. The first cytosolic fraction (fraction A) interacts with an import-competent, but not a mutant, nuclear localization sequence-bearing conjugate and stimulates its accumulation at the nuclear envelope in an ATP-independent fashion. The second cytosolic fraction (fraction B) gives no discernible effect when added alone; but when added either together with fraction A, or after fraction A, stimulates the passage of the conjugate from the outer nuclear envelope to the interior of the nucleus in an ATP-dependent fashion.
Moore
M S
MS
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
Blobel
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D003600:Cytosol / Q000502:physiology; D005033:Ethylmaleimide / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000378:metabolism; D014982:Xenopus laevis
D009687:Nuclear Proteins; D010455:Peptides; D014909:Wheat Germ Agglutinins; D005033:Ethylmaleimide