Figure 5.

Mex67 relies on multiple FG repeats for binding the NPC and fulfills its essential function only at the NPC. (A) Example nuclei are shown for different strains expressing Nups that have deleted FG/GFLG/FxFG regions as indicated. Images are single slices of z-stacks acquired on a spinning disk microscope. Slices were selected for being equatorial planes using an ER marker channel. Scale bar, 2 µm. (B) Intensity of Mex67 at the nuclear envelope using NuRIM in different strains with deletions of various FG repeats. Shown is the mean of three biological replicates normalized to wild-type cells. Error bars represent the SEM. Stars indicate significant P values of a paired t test compared with wild type (**, P < 0.01; ***, P < 0.001). Schematic indicates approximate positions of the NPC components in the NPC structure. Note that this applies to the body of the protein and not necessarily to its FG repeat region. (C) N-terminal fusion of Mex67 to Nup116 rescues deletion of MEX67 locus. Strains in which a deletion of the MEX67 locus was covered with Mex67 expressed from a plasmid containing the URA3 selection marker gene were engineered to express fusions of Mex67 to different nucleoporins. Streaks of these strains on 5-FOA–containing plate indicate rescue only by the N-terminal Nup116 fusion. Asterisk indicates position of Mex67 fusion.