MAJOR BINDING-SITES FOR THE NUCLEAR IMPORT RECEPTOR ARE THE INTERNAL NUCLEOPORIN NUP153 AND THE ADJACENT NUCLEAR FILAMENT PROTEIN TPR

A major question in nuclear import concerns the identity of the nucleo porin(s) that interact with the nuclear localization sequences (NLS) r eceptor and its cargo as they traverse the nuclear pore, Ligand blotti ng and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies h ave 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 subcomp lexes in an assembly competent state. We have then assessed our conclu sions in the context of assembled nuclear pores themselves. We have us ed immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrat ed that it is possible to obtain immunofluorescence localization of nu cleoporins to subregions of the nuclear pore and its associated struct ures. By immunoprecipitation, we find the nucleoporin Nup153 and the p ore-associated filament protein Tpr, previously shown to reside at dis tinct sites on the intranuclear side of assembled pores. are each in s table subcomplexes with importin alpha and beta in Xenopus egg extract s, Importin subunits are not in stable complexes with nucleoporins Nup 62, 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 impo rtin alpha, beta, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin be ta binds directly to Nup153 and in vitro can do so at multiple sites i n 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 wit h importin beta is fundamentally different from that of Nup153 is addi tionally demonstrated by the finding that recombinant beta or beta(45- 162) fragment freely exchanges with the endogenous importin beta/Nup15 3 complex, but cannot displace endogenous importin beta from a Tpr com plex. However, the GTP analogue GMP-PNP is able to disassemble both Nu p153- and Tpr-importin beta complexes. Importantly, analysis of extrac ts of isolated nuclei indicates that Nup153- and Tpr-importin beta com plexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are majo r physiological binding sites for importin beta, Models for the roles of these interactions are discussed.