FIG. 3.
Alterations in the first conserved region of YfgL (residues 171 to 181) result in weakened YaeT interactions. (A) Coimmunoprecipitation of YaeT with histidine-tagged YfgL variants. Wild-type (WT) YfgL-His6 (YfgL6His) (lanes 1 and 5) and variants (lanes 2 to 4 and 6 to 8) were produced from pBAD24. All the strains carried a copy of yaeT on the chromosome and on pBAD33. Proteins immunoprecipitated from cell lysates incubated with Penta-His antibody were resolved on an 11% SDS-polyacrylamide gel and silver stained. (B) HisProbe-HRP detection of histidine-tagged YfgL in the same immunoprecipitate samples from A. (C) Silver-stained SDS-polyacrylamide gel of YaeT coimmunoprecipitated with YfgL-His6 variants. The left panel (lanes 1 to 4) is a repeat of the right panel in A (lanes 5 to 8) but without the two 0.5 M NaCl washes. The middle panel (lanes 5 and 6) shows YaeT precipitated by YfgL(R176A)-His6. Prior to immunoprecipitation, cells expressing YfgL(R176A)-His6 were incubated with the DSP cross-linker. The right panel (lanes 7 and 8) shows YaeT coprecipitated with YfgL(S172A)-His6 without cross-linking treatment.