Defining the essential functional regions of the nucleoporin Nup145p

Abstract

Studies of the essential nucleoporin Nup145p have shown that its depletion is coincident with a block in RNA export and that deletion of its amino-terminal domain results in clustering of nuclear pore complexes. To further define the functional domains of Nup145p, we have characterized a panel of nup145 mutants. Deletions from both the amino terminus and the carboxy terminus resulted in temperature sensitive mutants that accumulated polyadenylated RNA in the nucleus at the non-permissive temperature. In addition, these mutants also displayed constitutive clustering of nuclear pore complexes in localized patches of the nuclear envelope. These results suggested that an internal region of Nup145p consisting of amino acids 593–893 is essential for function. Accordingly, when this region was deleted, growth was not supported at any temperature, whereas the region alone was able to complement a null mutation when expressed on a high copy plasmid. Previous studies have suggested that Nup145p is cleaved into two polypeptides of approximately 65 and 80 kDa. Interestingly, our experiments suggest that cleavage occurs in vivo. However, a small internal deletion of 17 amino acid residues that abolished cleavage had no effect on cell growth. Therefore, cleavage is not necessary for Nup145p function. When a sequence harboring the Nup145p cleavage site required for Nup145p cleavage was inserted in a chimeric protein, it was not sufficient for mediating cleavage. Cleavage likely requires a second region from amino acid residues 247–524 in addition to the cleavage site.

Dissecting the Structural Dynamics of the Nuclear Pore Complex

“…These two Nups are normally generated by autocatalytic cleavage of a single precursor Nup145 protein; upon cleavage and release, Nup145N can re-bind Nup145C, largely maintaining its prior form. This cleavage is neither essential for growth nor necessary for localization of these Nups to the NPC (Emtage et al, 1997;Teixeira et al, 1997Teixeira et al, , 1999. Thus, elimination of the cleavage site effectively fuses the two proteins (Nup145NC) with no gross phenotypic effects.…”