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. 2007 Oct;18(10):4155–4167. doi: 10.1091/mbc.E07-02-0094

Figure 3.

Figure 3.

Identification of a DAD-like region in for3p necessary for interaction with the N terminus. (A) Mapping of the DAD domain by two-hybrid assay. Interaction of C-terminal for3p fragments cloned in the pGBD vector with pGAD-for3N(1-702) was assayed by growth on −His plates. (B) Sequence alignment of for3p DAD with defined DAD domains from S. cerevisiae, Drosophila, and mouse formins. Stretches of basic residues are shown in blue. Mutation of the residues indicated in red to alanines abolished interaction with for3p N terminus. (C) Two-hybrid assay on SC-His plate of pGAD-for3N(1-702), pGAD-bud6C(581-1385) and empty pGAD with wild-type, LLT-AAA, and RKK-AAA pGBD-for3C(1261-1461). Numbers refer to constructs indicated in A. Mutations in the DAD region specifically abolish interaction with for3N but not with bud6C. (D) 6His-for3C binds directly and specifically to MBP-for3N. Binding of bacterially expressed proteins was assayed by affinity column. 6His-for3C (aa 630-1461) binds to MBP-for3N(1-702), but not MBP alone. This interaction is compromised by the LLT-AAA mutation (DAD*). 6His-tagged protein fragments were detected by Western blotting with an anti-6His antibody. Coomassie staining of MBP-fusions indicates equal loading.