Autoproteolysis in nucleoporin biogenesis

We have molecularly characterized a proteolytic cleavage in conserved nucle ar pore complex proteins. This cleavage, previously demonstrated to be esse ntial for the biogenesis of two nuclear pore complex proteins in mammals (N up98 and Nup96) and yeast (Nup145-N and Nup145-C), occurs between Phe and S er residues within a highly conserved domain in a polyprotein precursor. He re, we show that a protease is not involved in the cleavage event. By using a combination of domain mapping and site-directed mutagenesis, we demonstr ate that the human nuclear pore complex protein Nup98 specifically cleaves itself between F863 and S864. A region of Nup98, amino acids 715-920, is ab le to cleave, whereas a smaller region, amino acids 772-920, does not cleav e. In addition, we have generated a Nup98 mutant that cleaves under defined conditions in vitro. Further, the two cleaved fragments of Nup98 form a co mplex, providing a possible mechanism whereby specific, yet low-affinity, b inding between Nup98 and Nup96 is responsible for the nuclear targeting of Nup96. Although apparently unrelated evolutionarily, Nup98 has converged on an autoproteolytic biogenesis mechanism similar to that of hedgehog protei ns, the inteins, and the N-terminal nucleophile proteins.