Cell, Vol. 69, 939-950,
June
12, 1992, Copyright
0 1992 by Cell Press
The Two Steps of Nuclear Import, Targeting to the Nuclear Envelope and Translocation through the Nuclear Pore, Require Different Cytosolic Factors Mary Shannon Moore and Giinter Laboratory of Cell Biology Howard Hughes Medical Institute The Rockefeller University New York, New York 10021
Blobel
Summary We have isolated two cytosolic fractions from Xenopus oocytes that contain all of the activity necessary to support both steps of nuclear import in digitoninpermeabilized 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. Introduction The passage of macromolecules between the nucleus and the cytoplasm occurs through openings in the nuclear envelope called nuclear pores (for review, see Dingwall and Laskey, 1986). These passageways through the double membrane of the nuclear envelope are formed by nuclear pore complexes, large (1.24 x 1O8 daltons) and geometrically complex structures that extend into both the cytoplasm and the nucleoplasm (Reichelt et al., 1990; Akey 1989). The nuclear pore acts as an aqueous channel allowing free diffusion of small macromolecules at a rate inversely proportional to their mass. Dextrans with a molecular weight of less than 20 kd diffuse very rapidly through nuclear pores, those of 40 kd more slowly, and those of 70 kd not at all (Paine et al., 1975). Both smaller (
June
12, 1992, Copyright
0 1992 by Cell Press
The Two Steps of Nuclear Import, Targeting to the Nuclear Envelope and Translocation through the Nuclear Pore, Require Different Cytosolic Factors Mary Shannon Moore and Giinter Laboratory of Cell Biology Howard Hughes Medical Institute The Rockefeller University New York, New York 10021
Blobel
Summary We have isolated two cytosolic fractions from Xenopus oocytes that contain all of the activity necessary to support both steps of nuclear import in digitoninpermeabilized 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. Introduction The passage of macromolecules between the nucleus and the cytoplasm occurs through openings in the nuclear envelope called nuclear pores (for review, see Dingwall and Laskey, 1986). These passageways through the double membrane of the nuclear envelope are formed by nuclear pore complexes, large (1.24 x 1O8 daltons) and geometrically complex structures that extend into both the cytoplasm and the nucleoplasm (Reichelt et al., 1990; Akey 1989). The nuclear pore acts as an aqueous channel allowing free diffusion of small macromolecules at a rate inversely proportional to their mass. Dextrans with a molecular weight of less than 20 kd diffuse very rapidly through nuclear pores, those of 40 kd more slowly, and those of 70 kd not at all (Paine et al., 1975). Both smaller (
