Exo70M26p is severely impaired in binding to Bem1p. (A) Amino acid sequence of Exo70p domains B and C from 153 to 515. The sequence of domain C is underlined. Surface-exposed charged residues within the C domain are indicated by bold text. They were systematically mutated to alanine either one at a time or in small patches. These mutation sites are numbered from 1 to 15 in order. Two unintentional mutations were found within domain B in our original M13 construct (renamed M28), serine at position 196 was mutated to phenylalanine (S196F), and leucine at position 246 was mutated to serine (L246S). (B) Some of the single mutation sites were combined and given a new name, shown on the left column of the table. (C) GST-tagged WT Exo70p or mutants were immobilized on glutathione beads and incubated with Bem1p-His6. Bound proteins were detected as described in the Materials and methods. (D) The percentage of Bem1p bound to Exo70p WT or mutants was quantified. Error bars represent SD, n = 3. (E) The interactions between Bem1p-His6 and GST-Exo70p WT or three mutants were tested at three different concentrations of Bem1p: 25 nM, 50 nM, and 100 nM. Bound proteins were detected as mentioned in the Materials and methods.
