FIG. 3.

Abi-1 interacts with Wave-1 through a conserved amino-terminal WAB domain. (A) Endogenous Abi-1 interacts with Wave-1. Endogenous Abi-1 was immunoprecipitated from brain extracts by using anti-Abi-1 antibody (6987), and the immunopurified complex was immunoblotted (IB) with polyclonal Wave-1 antibody. Preimmune serum was used as a negative control. (B) The region comprising amino acids 1 to 111 of Abi-1 is necessary and sufficient to bind Wave-1. Different Flag-tagged Abi-1 forms and GFP-Wave-1 were coexpressed in 293T cells, and the Flag-Abi-1 forms were immunoprecipitated (IP) by using anti-Flag antibody. In lane 2, GFP was coexpressed with Flag-Abi-1 as a negative control. The coprecipitating GFP-Wave-1 was detected with anti-GFP antibody. Due to low expression levels of Flag-Abi-1SNARE, the ratio of Flag-tagged constructs to GFP-Wave-1 vector was 7:1 in all lanes. (C) EYFP-Abi-1 or EYFP-Abi-1Δ100-163 was cotransfected into 293T cells with Myc-Wave-1, and the Abi-1 forms were immunoprecipitated with anti-GFP antibody. Purified immunocomplexes were immunoblotted with Myc antibody to detect Wave-1 (upper panel). Whole-cell lysates were immunoblotted for Myc and GFP. (D) Abi-1 amino acids 18 to 79 are required for interaction with Wave-1. The indicated Flag-Abi-1 deletion mutants were coexpressed with GFP-Wave-1 and immunoprecipitated with anti-Flag. The coprecipitated GFP-Wave-1 protein was detected with anti-GFP antibodies (upper panel). GFP-Wave-1 protein levels in total cell lysates (lower panel) and the immunoprecipitated Flag-Abi-1 forms (middle panel) were detected with the indicated antibodies. (E) Abi-1 and Wave-1 interact directly. Abi-1 and Abi-1ΔSNARE were synthesized in vitro by using wheat germ extracts and incubated with GST-Wave-1NT or GST alone; ∼5% of the input was loaded in the first two lanes. (F) Schematic representation of the WAB domain in Abi-1. The Abi-1 nomenclature is given in the legend to Fig. 2G.