Figure 7.

Partial rescue of nuclear assembly and function in RCC1 or RanBP1 deficient extracts by Ran mutants. (A) Nuclear protein import can be rescued by Ran mutants. XB buffer (second row from top), RanBP1 (50 μg/ml; third row), or RCC1 (10 μg/ml; bottom row) was added to codepleted extracts in the absence (two left columns) or presence of RanG19V (middle two columns; 40 μg/ml) or RanT24N (right two columns; 40 μg/ml) proteins. Similar samples were prepared with mock-depleted extracts (top row). The extracts were incubated on ice for 15 min, and standard nuclear assembly assays were performed. Rhodamine-labeled protein import substrate were added after 60 min. The samples were examined 120 min after the start of the assembly reaction. The parts with uppercase locants show typical nuclei from each reaction obtained with Hoechst 33258 DNA dye and with the lowercase locants show the accumulation of nuclear transport substrate in the same nuclei. Bar, 3.0 × 10⁻⁶ m. (B) DNA replication was partially restored by Ran mutants. In the same nuclear assembly assay as in A, samples were taken after 120 min for DNA replication assays and results were quantified on a Phosphorimager.