Identification of a new vertebrate nucleoporin, Nup188, with the use of a novel organelle trap assay

The study of the nuclear pore in vertebrates would benefit from a strategy to directly identify new nucleoporins and interactions between those nucleo porins. 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 containi ng a ligand of interest. The bound proteins are then tagged by biotinylatio n and eluted. In the second step, potential nucleoporins are selected for b y virtue of their ability to assemble into annulate lamellae, a cytoplasmic mimic of nuclear pores. The incorporated proteins are then recognized by t heir biotin tag. Here we use the lectin wheat germ agglutinin (WGA) as liga nd; WGA inhibits nuclear transport and has been shown to directly bind thre e known nucleoporins from Xenopus extract, Nup62, Nup98, and Nup214, all of which contain N-acetylglucosamine residues. Under reduced-stringency condi tions, 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 ve rtebrate nucleoporin, Nup188. This new vertebrate protein, Xenopus Nup188, exists in a complex with xNup93 and xNup205. The Nup93-Nup188-Nup205 comple x does not bind directly to WGA but binds indirectly via the N-acetylglucos amine-modified nucleoporins. A gene encoding human Nup188 was also identifi ed. The discovery of vertebrate Nup188, related to a yeast nucleoporin, and its novel protein-protein interactions illustrates the power of the two-st ep organelle trap assay and identifies new building blocks for constructing the nuclear pore.