Figure 4.

MEL-28 interacts with the Nup107–160 complex and is required for Nup107–160 recruitment to chromatin. (A) MEL-28 antibodies co-immunoprecipitate the Nup107–160 complex. The indicated proteins were identified by mass spectrometry after silver staining (left) or by western blotting with respective antisera (right) after immunoprecipitation from cytosol. Two high-molecular-weight bands that stain weakly on the silver gel represent MEL-28 (not shown). The asterisk indicates a background signal for p62. (B) Co-immunopreciptation of Nup107 and MEL-28: immunoprecipitations were performed from cytosol with antibodies against MEL-28, Nup107 or Nup205 as a control. The indicated proteins were identified by western blotting with respective antisera. (C) Depletion of the Nup107–160 complex reduces but does not fully deplete MEL-28: extracts were depleted of the Nup107–160 complex by two passages over an affinity resin. Untreated starting material, mock-depleted extracts and Nup107–160-depleted extracts after each round of depletion were analysed by western blotting with the indicated antisera. (D) Both MEL-28 and the Nup107–160 complex exist as free pools: extracts were separated on a Superose 6 gel filtration column and analysed by western blotting for the indicated proteins. Fraction 1 corresponds to the void; elution of dextran 2000 and ferritin markers for 2,000 and 550 kDa, respectively, are indicated. (E) MEL-28 is recruited to chromatin in the absence of the Nup107–160 complex, but not vice versa: sperm heads were incubated for 20 min in mock-depleted, Nup107–160-depleted, MEL-28-depleted or heat-inactivated extracts, fixed and analysed by immunofluorescence. Chromatin (upper row, blue) is stained with DAPI. MEL-28 or Nup160 (green) is shown in the lower row. Scale bar, 10 μm. 1st, first round; 2nd, second round; DAPI, 4,6-diamidino-2-phenylindole; MEL, maternal effect lethal; Nup, nucleoporin.