A conserved biogenesis pathway for nucleoporins: Proteolytic processing ofa 186-kilodalton precursor generates Nup98 and the novel nucleoporin, Nup96

The mammalian nuclear pore complex (NPC) is comprised of similar to 50 uniq ue proteins, collectively known as nucleoporins. Through fractionation of r at liver nuclei, we have isolated >30 potentially novel nucleoporins and ha ve begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel nucleoporin with a predicted molecul ar mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two nucleoporins: Nup98, a previously characterize d GLFG-repeat containing nucleoporin, and Nup96, Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively splic ed mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for N up98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces c erevisiae homologues, N-Nup145p and C-Nup145p, are also produced through pr oteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the n ucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the n ucleoplasmic side of the NPC was found to be dependent on proteolytic cleav age, suggesting that the cleavage process may regulate NPC assembly. Finall y, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13-related proteins was identified, revealing that a major su b-complex of the NPC is conserved between yeast and mammals.