Self-catalyzed Cleavage of the Yeast Nucleoporin Nup145p Precursor

Abstract

Nup145p is a component of the nuclear pore complex of Saccharomyces cerevisiae and is essential for mRNA export. Nup145p and its apparent vertebrate homologue are the only known nucleoporins to be composed of two functionally independent peptide moieties resulting from the post-translational cleavage of a large precursor molecule. In this study, the proteolytic cleavage site of Nup145p has been mapped upstream of an evolutionary conserved serine residue. Cleavage occurs at the same site when a precursor is artificially expressed in Escherichia coli. A hydroxyl-containing residue is critical for the reaction, although a thiol-containing residue offers an acceptable replacement. In vitro kinetics experiments using a purified precursor molecule demonstrate that the cleavage is self-catalyzed and that the catalytic domain lies within the N-terminal moiety. Taken altogether, our data are consistent with a proteolytic mechanism involving an N>O acyl rearrangement and a subsequent ester intermediate uncovered in other selfprocessing proteins.

The Fission Yeast Nup107-120 Complex Functionally Interacts with the Small GTPase Ran/Spi1 and Is Required for mRNA Export, Nuclear Pore Distribution, and Proper Cell Division

“…The ϳ110-kDa polypeptide, reproducibly detected in both the GFP-Nup85 and GFP-Seh1 fractions, was identified as the C-terminal domain of an S. pombe nucleoporin similar to S. cerevisiae Nup145 and mammalian Nup98/96 and designated Nup189 in fission yeast (61). Nup189 was previously shown to be cleaved in vivo, most likely by autoproteolysis, as demonstrated in S. cerevisiae and mammals (19,64), leading to a 95-kDa N-terminal product that interacted with Ned1 in a two-hybrid screen (61). In contrast, the protein identified in our complex corresponds to the S. pombe orthologue of ScNup145-C.…”

Reconstitution of Nup157 and Nup145N into the Nup84 Complex*[boxs]

“…Notably, the conserved Nup145 is made as a precursor that is posttranslationally cleaved into two functionally separated domains, Nup145N and Nup145C in yeast (38,39) or Nup98 and Nup96 in mammals (34,40). The autoproteolytic cleavage of Nup145 is not essential in yeast but becomes essential in cells lacking Nup188 (38,39). Moreover, self-cleavage of the Nup98-Nup96 precursor in mammals is crucial for NPC assembly (34,40).…”

Multiple Conformations in the Ligand-binding Site of the Yeast Nuclear Pore-targeting Domain of Nup116p

“…The Nup116p homolog Nup145p possesses autoproteolytic activity that is unique among yeast nucleoporins (14). This protein is expressed as a single polypeptide chain, cleaving itself post-translationally near the * This work was supported in part by National Institutes of Health Grant GM36373 (to P. A. S.) and Grants GM47467 and CA68262 (to G. W.).…”