Figure 11.

The WA Domain of DIS3/SCAR2 Is Required for Direct Binding to ARPC3.

(A) DIS3/SCAR2 and human WAVE1 interact with ARPC3 in a yeast two-hybrid assay. The bait and prey plasmids that were cotransformed into yeast are defined to the left of the corresponding yeast patches and β-galactosidase assay results to the right. DIS3-WA contains amino acids 1032 to 1399; DIS3-ΔWA contains amino acids 1032 to 1238; ARPC3, DIS2 (ARPC2), and HSWAVE1 are full-length proteins.

(B) The DIS3-ΔWA and DIS3-WA two-hybrid constructs are expressed at comparable levels in yeast. Top, lanes 1 to 9, duplicate yeast protein soluble fractions probed with anti-HA antibody. Bottom, same blot probed with control anti-tubulin antibody. Lanes 1-4, duplicate protein samples extracted from two individual colonies (cly-1 and cly-2) of the yeast strain expressing HA-tagged DIS3-WA; lanes 5 to 8, same as 1 to 4, but expressing HA-tagged DIS3-ΔWA; lane 9, yeast extracts from the strain harboring the empty vector pACT2. All the strains used also contained the ARPC3 bait construct.

(C) Direct interaction of DIS3/SCAR2 and ARPC3 in a GST pull-down assay. Top, lanes 1 to 6, fractions probed with an anti-ARPC3 antibody; bottom, fractions probed with an anti-ROP2 antibody. Lane 1, 5% of total binding reaction; lane 2, unbound; lane 3, GST-DIS3-WA–associated fraction; lane 4, 5% of total binding reaction with GST alone control beads; lane 5, unbound; lane 6, GST bead-associated fraction.