Molecular determinants of binding between Gly-Leu-Phe-Gly nucleoporins and the nuclear pore complex.
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.
Ratner
Gary A
GA
Department of Cell Biology and Biochemistry, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA.
Hodel
Alec E
AE
Powers
Maureen A
MA
D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D006801:Humans; D022022:Nuclear Pore / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D020815:Polyproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D028861:Nuclear Pore Complex Proteins; D009842:Oligopeptides; D020815:Polyproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins