FIG 6.

Identification of amino acids critical for PI3P binding by RavB. (A) Multiple sequence alignment of ten effectors or predicted effectors harboring LED035. The conserved amino acid tyrosine and the adjacent arginine-histidine or histidine-serine that were mutated are marked with red arrows if the mutation affected the PI3P binding or with black arrows if the mutation did not affect PI3P binding (see panel B). (B) Binding of affinity-purified GST fusion proteins of the wild-type effector RavB or the three mutants with mutations in the single tyrosine, the adjacent arginine-histidine, and the adjacent histidine-serine (Y185A, RH213-214AA, and HS257-258AA) of the same effector (300 pmol) to 2-fold serial dilutions of PIs (100 to 1.56 pmol) immobilized on nitrocellulose membranes was analyzed by a protein-lipid overlay assay using an anti-GST antibody.