Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex.
Nup153, one of the best characterized nuclear pore complex proteins (nucleoporins), plays a critical role in the import of proteins into the nucleus as well as in the export of RNAs and proteins from the nucleus. Initially an epitope of Nup153 was found to reside at the distal ring of the NPC, whereas more recently another epitope was localized to the nuclear ring moiety of the NPC. In an effort to more definitively determine the location of Nup153 within the 3-D architecture of the NPC we have generated domain-specific antibodies against distinct domains of Xenopus Nup153. With this approach we have found that the N-terminal domain is exposed at the nuclear ring of the NPC, whereas the zinc-finger domain of Nup153 is exposed at the distal ring of the NPC. In contrast, the C-terminal domain of Nup153 is not restricted to one particular subdomain of the NPC but rather appears to be highly flexible. Exogenous epitope-tagged hNup153 incorporated into Xenopus oocyte NPCs further underscored these findings. Our data illustrate that multiple domain-specific antibodies are essential to understanding the topology of a nucleoporin within the context of the NPC. Moreover, this approach has revealed new clues to the mechanisms by which Nup153 may contribute to nucleocytoplasmic transport.
Fahrenkrog
Birthe
B
M.E. Mueller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstr.70, Basel, Switzerland. birthe.fahrenkrog@unibas.ch
Maco
Bohumil
B
Fager
Ammon M
AM
Köser
Joachim
J
Sauder
Ursula
U
Ullman
Katharine S
KS
Aebi
Ueli
U
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001665:Binding Sites; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008954:Models, Biological; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014981:Xenopus; D016335:Zinc Fingers
D000906:Antibodies; D008164:Luminescent Proteins; C079377:NUP153 protein, human; D028861:Nuclear Pore Complex Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins