Figure 5.

Two Point Mutations in the LRR Domain of RanGAP1 Abolish the GAP Activity of the Protein but Not Its Localization and Protein-Protein Interactions.

(A) Schematic representation of mutations and deletions of RanGAP1. The wild-type protein contains the N-terminal WPP domain, the middle LRR domain, and the C-terminal acidic tail. The variant that loses nuclear envelope and mitotic targeting but retains the GAP activity has two point mutations in the WPP domain (WPP/AAP). Two point mutations in the LRR domain (N219A and D330A) block the GAP activity. RanGAP1 variants containing both the mutations in the targeting domain and one of the LRR domain mutations were generated as well.

(B) Schematic 3D representation of RanGAP1. The mutations abolishing the GAP activity are marked and positioned at the top loops of the LRR domain crescent, where Ran protein might interact with RanGAP1 (left, side view; right, top view).

(C) Yeast complementation. The RanGAP1 variants shown in (A) were transformed into the S. cerevisiae rna1-1 temperature-sensitive mutant, and the yeast was grown on selective media at the permissive (30°C) and restrictive (37°C) temperatures. RanGAP1 and RanGAP1^AAP complement the temperature sensitivity. However, RanGAP1 variants containing the N219A or D330A mutations (RanGAP1^N219A, RanGAP1^D330A, RanGAP1^AAP+N219A, and RanGAP1^AAP+D330A), as well as the empty vector control, were unable to rescue the phenotype.

(D) Point mutations affecting RanGAP1 GAP activity and/or subcellular localization do not interfere with proper protein folding. RanGAP1-GFP (a to f) and RanGAP1^N219A-GFP (h) localize in the same manner, while RanGAP1^AAP-GFP is exclusively cytoplasmic (g). Triangles indicate nuclear envelope localization (a and h). Arrowheads point to mitotic locations of the fusion proteins. Bars = 20 μm.

(E) Yeast two-hybrid assay. The RanGAP1 variants shown in (A) were transformed into S. cerevisiae and tested for interaction with a GTP-locked form of Ran (Ran1Q72L) or with WIP1. RanGAP1, RanGAP1AAP, RanGAP1N219A, and RanGAP1AAP+N219A all interacted with Ran1Q72L, while those variants containing the D330A mutation did not. RanGAP1 variants with an intact WPP domain interacted with WIP1, while those carrying the AAP mutation did not. Original colony streaks for this table are shown in Supplemental Figure 7. Positive interactions are marked with a “+,” negative interactions are marked with a “−,” and a blank rectangle indicates the combination was not tested.