title: Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr.
abstract: A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin alpha and beta in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin alpha, beta, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin beta binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin beta and to importin alpha/beta heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin beta is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant beta or beta45-462 fragment freely exchanges with the endogenous importin beta/Nup153 complex, but cannot displace endogenous importin beta from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153- and Tpr-importin beta complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153- and Tpr-importin beta complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin beta. Models for the roles of these interactions are discussed.
authors.affiliation: Department of Biology, University of California at San Diego, La Jolla, California 92093, USA.
authors.forename: S
authors.initials: S
authors.lastname: Shah
authors.forename: S
authors.initials: S
authors.lastname: Tugendreich
authors.forename: D
authors.initials: D
authors.lastname: Forbes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001755:Blastocyst / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D018076:DNA, Complementary; D019281:Dimerization; D006801:Humans; D028884:Karyopherins; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
chemical_list: D018076:DNA, Complementary; D028884:Karyopherins; D046911:Macromolecular Substances; C079377:NUP153 protein, human; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; C513083:Nup153 protein, rat; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; C073597:TPR protein, human