FIGURE 2.

Arl4A binds the ELMO1 RBD through a key evolutionarily conserved RBD residue. A, schematic representation of ELMO1 deletion mutants used in yeast two-hybrid experiments. B, the ELMO1 N terminus is required for Arl4A binding. Yeast strain EGY48 cotransformed with LexA BD fusion construct of ELMO1^WT and deletion mutants, and the B42 fusion constructs of the indicated Arl4As were grown on selective (−histidine, −tryptophan, −leucine) and nonselective (−histidine, −tryptophan) medium for a nutrient selective growth assay. C, the Arl4A-ELMO1 interaction in cellulo requires the ELMO1 RBD. HEK293T cells transfected with the indicated plasmids were cross-linked, lysed, and immunoprecipitated (IP) with an antibody against the Myc epitope (ELMO1). The coprecipitation of the various ELMO1 proteins and Arl4A was analyzed via immunoblotting with anti-Myc (ELMO1) and anti-FLAG-HRP (Arl4A) antibodies, respectively. D, mutation of a key conserved residue in the ELMO RBD (L43A) abolishes Arl4A binding. Yeast strain EGY48 cotransformed with LexA BD fusion construct of ELMO1^WT and ELMO1^L43A and the B42 fusion construct of Arl4A^WT were grown on selective (−histidine, −tryptophan, −leucine) and nonselective (−histidine, −tryptophan) medium for a nutrient selective growth assay.