Figure 6.

Numerous binding sites for Ran on the cytoplasmic fibrils of the NPC, partially competed away by RanBP1. (A) Vertical sections. RanGDP binds to the cytoplasmic fibrils of the NPC in large numbers. RanGDP was conjugated to 5-nm gold beads and incubated with digitonin-permeabilized HeLa cells as described in Materials and Methods. Vertical ultra-thin sections through several NPCs were photographed at an original magnification of ×26,000 (Left). (Bars = 100 μm.) (B) Distance of RanGDP-gold particles from the midplane of the NPC. Photographs of vertical sections through 25 NPCs obtained as in A were analyzed by using Adobe photoshop and nih image software to determine the distance of RanGDP 5-nm gold particles from the midplane of the NPC. (C) Surface views. RanBP1 competes away a substantial portion of RanGDP, leaving a peripheral ring of RanGDP bound to the NPC. RanGDP was conjugated to 5-nm gold beads and incubated with digitonin-permeabilized HeLa cells as in A in the presence (Right) or absence (Left) of 4 μM RanBP1. Grazing sections parallel to the surface of the nucleus were photographed in semithin sections at ×33,000 original magnification. RanGDP-gold particles were counted in 23 and 27 NPCs in the absence and presence of RanBP1, respectively. (Bar = 100 μm.)