Fig. 4.

Characterization of FG repeat domain polymers formed by Nup54 and Nup98. (A) Transmission electron micrographs of polymeric fibers of the FG domains of Nup54 and Nup98 negatively stained with uranyl acetate. (Scale bar: 200 nm.) (B) X-ray diffraction patterns of lyophilized Nup54 and Nup98 FG domain polymers. (C) SDD-AGE of amyloid polymers formed by yeast Sup35NM, Nup54 FG domain, or Nup98 FG domain. Incubation with increasing amounts of SDS did not substantially affect ySup35NM aggregates. Both Nup54 and Nup98 fibers were fully depolymerized under similar conditions. (D) Chemical footprinting of Nup54 fibers using the acetylation reagent NAI, as analyzed by SILAC mass spectrometry. The reactivity of specific residues in Nup54 to NAI was compared in the fully polymerized state and the denatured state. The degree of protection from acetylation on polymerization is reflected by the ratio of acetylation in denatured/native conditions (y axis). (E) WT or phenylalanine-to-proline double-mutant Nup54 monomers were incubated with WT Nup54 polymer seeds. An increase in the fluorescence of thioflavin-T (y axis) as a function of time (x axis) reflects seeded polymerization of Nup54 monomers.