Abstract
The vertebrate nucleoporin Nup98 can be expressed in two distinct forms from differentially spliced mRNAs, either as a 98-kDa protein or as the 195-kDa Nup98/Nup96 polyprotein. Both forms undergo autoproteolytic processing to generate the 90-kDa Nup98 and either an 8-kDa tail or the nucleoporin Nup96. An equivalent cleavage event occurs in one yeast ortholog, Nup145, to produce Nup145N and Nup145C. We previously proposed that Nup145N, and possibly the other orthologs Nup116 and Nup100, might bind to Nup145C as demonstrated for Nup98 and Nup96. Here we have further investigated the interaction of both yeast and vertebrate Gly-Leu-Phe-Gly nucleoporins with the nuclear pore. We find that dynamic Nup98 binding can be recapitulated in vitro and that simultaneous translation and folding as a polyprotein are not required to allow subsequent binding between Nup98 and Nup96. We show that Nup145N and Nup145C do indeed bind to each other, and we have determined the dissociation constants for these interactions in vitro. Additionally, we characterize two sites of molecular interaction for each binding pair. Of the yeast orthologs, Nup116 binds far less robustly to Nup145C than does Nup145N, and Nup100 binding is barely detectable. Thus, we conclude that Nup116 and Nup100 likely use means of incorporation into the nuclear pore complex that are distinct from those used by Nup145N.
Highlights
Structural studies, most recently by cryoelectron microscopy, have revealed with considerable detail an nuclear pore complexes (NPCs) with 8-fold symmetry about an axis perpendicular to the plane of the nuclear envelope [1,2,3]
Nups of the core region are distributed symmetrically with respect to the plane of the envelope, whereas the cytoplasmic and nuclear fibrils are each composed of a distinct set of nucleoporins
The Nup98 C-terminal domain undergoes an autocatalytic cleavage that releases either an 8-kDa protein tail or the nucleoporin Nup96 depending upon whether Nup98 was expressed as a single protein or as the polyprotein precursor, Nup98/Nup96 (Fig. 1 and Fontoura et al [15])
Summary
Production of Recombinant Proteins—For expression of GST fusions, fragments of Nup, Nup145, Nup100, or Nup116 genes were cloned into pGEX-4T vectors and expressed in BL21(DE3) cells induced at either 37 °C for 3 h or 17 °C overnight. 1.5 mg of purified tail-GFP-His-tagged protein in 500 l of BB1 was added to the washed beads and incubated for 3 h at 4 °C to allow binding. 37.5 l of washed beads were added to 500 l of clarified lysate containing GST fusion protein, corresponding to 80 ml of expression culture. These samples were incubated to allow binding and washed in the same manner as the trans binding assays. 1.5 mg of purified GFP-Histagged protein in 1 ml of BB1 was added to the washed beads, and the samples were incubated, washed, and processed in the same manner as trans binding assays. Where is the fraction of tail protein bound given as the fraction of the polarization maximum observed at saturating concentrations of head protein, HT is the total concentration of head protein, bound and unbound, in each reaction, and TT equals 10.0 nM, i.e. the total concentration of tail peptide, both bound and unbound, in each reaction
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