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J. Cell Biol.,
Volume 141, Number 1, April 6, 1998 31-49
Department of Biology, University of California at San Diego, La Jolla, California 92093
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 
and 
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 , , and
an NLS substrate, consistent with an involvement of
this nucleoporin in a terminal step of nuclear import.
Importin 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 and to importin / heterodimers, but only to those
that do not carry an NLS substrate. That the complex of
Tpr with importin is fundamentally different from
that of Nup153 is additionally demonstrated by the
finding that recombinant or 45-462 fragment freely exchanges with the endogenous importin /Nup153
complex, but cannot displace endogenous importin from a Tpr complex. However, the GTP analogue
GMP-PNP is able to disassemble both Nup153- and
Tpr-importin complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153- and
Tpr-importin complexes exist in assembled nuclear
pores. Thus, Nup153 and Tpr are major physiological
binding sites for importin . Models for the roles of
these interactions are discussed.
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