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Vol. 11, Issue 10, 3381-3396, October 2000
and
*Department of Biology, University of California at San Diego, La
Jolla, California 92093; The study of the nuclear pore in vertebrates would benefit from a
strategy to directly identify new nucleoporins and interactions between
those nucleoporins. We have developed a novel two-step "organelle
trap" assay involving affinity selection and in vitro pore assembly.
In the first step, soluble proteins derived from Xenopus
egg extracts are applied to a column containing a ligand of interest.
The bound proteins are then tagged by biotinylation and eluted. In the
second step, potential nucleoporins are selected for by virtue of their
ability to assemble into annulate lamellae, a cytoplasmic mimic of
nuclear pores. The incorporated proteins are then recognized by their
biotin tag. Here we use the lectin wheat germ agglutinin (WGA) as
ligand; WGA inhibits nuclear transport and has been shown to directly
bind three known nucleoporins from Xenopus extract,
Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues. Under reduced-stringency
conditions, three additional proteins bind to WGA-Sepharose and are
revealed by the organelle trap assay. We identified all three as
partner nucleoporins. Two were discovered to be Xenopus
Nup93 and Nup205. The third is a novel vertebrate nucleoporin, Nup188.
This new vertebrate protein, Xenopus Nup188, exists in a
complex with xNup93 and xNup205. The Nup93-Nup188-Nup205 complex does
not bind directly to WGA but binds indirectly via the
N-acetylglucosamine-modified nucleoporins. A gene
encoding human Nup188 was also identified. The discovery of vertebrate
Nup188, related to a yeast nucleoporin, and its novel protein-protein
interactions illustrates the power of the two-step organelle trap assay
and identifies new building blocks for constructing the nuclear pore.
Department of Cell Biology,
Emory University School of Medicine, Atlanta, Georgia 30322; and
Clayton Laboratory for Peptide Biology, The Salk
Institute for Biological Studies, La Jolla, California 92037
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