Figure 5.

The C-terminal Nup53 membrane binding site is essential for interphasic nuclear pore complex (NPC) formation. (A) Nuclei were preassembled in mock or Nup53-depleted extracts supplemented with wild-type full-length protein (1–320), constructs with a mutated N-terminal membrane binding site (1–320 R105E/K106E, 1–320 S94E/T100E), or constructs lacking the C-terminal membrane interaction site (1–319, 1–312), respectively. After 90 min, the samples were supplemented with cytosol depleted of Nup53 and FG-containing nucleoporins. After 60 min, FITC-labelled 70-kDa dextran and Hoechst was added. Bar: 10 μm. The right panel shows the quantitation of three independent experiments with >300 randomly chosen chromatin substrates per sample. Error bars represent the range. (B) Nuclei were assembled in Nup53-depleted extracts supplemented with wild-type full-length protein (1–320), a construct with a mutated N-terminal membrane binding site (1–320 R105E/K106E), or a construct lacking the last C-terminal amino acid (1–319), respectively, for 120 min. Where indicated, de novo NPC assembly was blocked by the addition of 2 μM importin β after 50 min and nuclei were further incubated for 70 min. For each construct, total NPC numbers per nucleus identified by mAB414 immunofluorescence were quantified from 20 nuclei in 2 independent experiments and normalized to the wild-type full-length protein. Error bars represent the s.e. of the mean.