Transportin mediates nuclear entry of DNA in vertebrate systems.
Delivery of DNA to the cell nucleus is an essential step in many types of viral infection, transfection, gene transfer by the plant pathogen Agrobacterium tumefaciens and in strategies for gene therapy. Thus, the mechanism by which DNA crosses the nuclear pore complex (NPC) is of great interest. Using nuclei reconstituted in vitro in Xenopus egg extracts, we previously studied DNA passage through the nuclear pores using a single-molecule approach based on optical tweezers. Fluorescently labeled DNA molecules were also seen to accumulate within nuclei. Here we find that this import of DNA relies on a soluble protein receptor of the importin family. To identify this receptor, we used different pathway-specific cargoes in competition studies as well as pathway-specific dominant negative inhibitors derived from the nucleoporin Nup153. We found that inhibition of the receptor transportin suppresses DNA import. In contrast, inhibition of importin beta has little effect on the nuclear accumulation of DNA. The dependence on transportin was fully confirmed in assays using permeabilized HeLa cells and a mammalian cell extract. We conclude that the nuclear import of DNA observed in these different vertebrate systems is largely mediated by the receptor transportin. We further report that histones, a known cargo of transportin, can act as an adaptor for the binding of transportin to DNA.
Lachish-Zalait
Aurelie
A
Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel.
Lau
Corine K
CK
Fichtman
Boris
B
Zimmerman
Ella
E
Harel
Amnon
A
Gaylord
Michelle R
MR
Forbes
Douglass J
DJ
Elbaum
Michael
M
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000378:metabolism / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D022022:Nuclear Pore / Q000378:metabolism; D010063:Ovum / Q000166:cytology; D029867:Xenopus Proteins / Q000378:metabolism / Q000502:physiology; D014982:Xenopus laevis
D028884:Karyopherins; D029867:Xenopus Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA