Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro.

Abstract:

:Nuclear import of plasmid DNA mediated by a nuclear localization signal (NLS) derived from SV40 T antigen was investigated in a cell-free extract. In vitro assembled sea urchin male pronuclei were incubated in a 100,000g supernatant of a zebrafish fertilized egg lysate, together with fluorescently labeled plasmid DNA bound to NLS or nuclear import deficient reverse NLS (revNLS) peptides. After 3 hr, DNA-NLS, but not DNA-revNLS, complexes were bound around the nuclear periphery. We demonstrate that nuclear import of DNA-NLS complexes is a two-step process involving binding to, and translocation across, the nuclear envelope. Binding is ATP-independent, occurs at 0 degree C and is Ca(2+)-independent. By contrast, translocation requires ATP hydrolysis, Ca2+, is temperature dependent and is blocked by the lectin wheat germ agglutinin. Both binding and translocation are competitively inhibited by albumin-NLS conjugates, require heat-labile cytosolic factors, and are inhibited by N-ethylmaleimide treatment of the cytosol. Binding and translocation are differentially affected by cytosol dilutions, suggesting that at least two distinct soluble fractions are required for nuclear import. The requirements for NLS-mediated nuclear import of plasmid DNA are similar to those for nuclear import of protein-NLS conjugates in permeabilized cells.

journal_name

Mol Reprod Dev

authors

Collas P,Aleström P

doi

10.1002/(SICI)1098-2795(199612)45:4<431::AID-MRD4>

subject

Has Abstract

pub_date

1996-12-01 00:00:00

pages

431-8

issue

4

eissn

1040-452X

issn

1098-2795

pii

10.1002/(SICI)1098-2795(199612)45:4<431::AID-MRD4>

journal_volume

45

pub_type

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