Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors.
BACKGROUND
Proteins generally enter or exit the nucleus as cargo of one of a small family of import and export receptors. These receptors bear distant homology to importin beta, a subunit of the receptor for proteins with classical nuclear localisation sequences (NLSs). To understand the mechanism of nuclear transport, the next question involves identifying the nuclear pore proteins that interact with the different transport receptors as they dock at the pore and translocate through it.


RESULTS
Two pathways of nuclear import were found to intersect at a single nucleoporin, Nup153, localized on the intranuclear side of the nuclear pore. Nup153 contains separate binding sites for importin alpha/beta, which mediates classical NLS import, and for transportin, which mediates import of different nuclear proteins. Strikingly, a Nup153 fragment containing the importin beta binding site acted as a dominant-negative inhibitor of NLS import, with no effect on transportin-mediated import. Conversely, a Nup153 fragment containing the transportin binding site acted as a strong dominant-negative inhibitor of transportin import, with no effect on classical NLS import. The interaction of transportin with Nup153 could be disrupted by a non-hydrolyzable form of GTP or by a GTPase-deficient mutant of Ran, and was not observed if transportin carried cargo. Neither Nup153 fragment affected binding of the export receptor Crm1 at the nuclear rim.


CONCLUSIONS
Two nuclear import pathways, mediated by importin beta and transportin, converge on a single nucleoporin, Nup153. Dominant-negative fragments of Nup153 can now be used to distinguish different nuclear import pathways and, potentially, to dissect nuclear export.
Shah
S
S
Department of Biology 0347, University of California at San Diego, La Jolla, California 92093-0347, USA.
Forbes
D J
DJ
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins; D009685:Nuclear Envelope / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000378:metabolism* / Q000494:pharmacology; D010063:Ovum / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014981:Xenopus; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein
D002352:Carrier Proteins; D006165:Guanylyl Imidodiphosphate; D028884:Karyopherins; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein; C079377:NUP153 protein, human