Fig 6.

Composition and assembly of YknWXYZ complex. (A) Combinations of YknW^His, YknX^His, and YknZ^His proteins were expressed in B. subtilis HB6127 cells. Control cells carried an empty vector. Miniscale purifications using 6His tags of proteins were carried out from DSP-treated and untreated cells. Purified proteins (0.1 to 0.3 μg) were loaded onto 12% SDS-PAGE gels. Heat denaturation and the reducing agent in the sample buffer were omitted. After separation, proteins were detected using Coomassie brilliant blue (top panel) or transferred onto a PVDF membrane for immunoblotting with anti-YknX (middle panel) or anti-YknW (bottom panel). Oligomers are indicated by stars. A 35-kDa protein present in YknX^His purified from cells overproducing YknW is indicated by an arrow. (B) Total membranes isolated from cells producing YknX^His, YknYZ^His, and YknXYZ^His were mixed with membranes of cells containing either an empty vector (V) or YknW (W) before solubilization in 5% TX-100. Purification of His-tagged proteins was carried out as described for panel A. Purified proteins (0.4 μg) were analyzed by 12% SDS-PAGE followed by Coomassie staining (top panel) or by immunoblotting with anti-YknW antibodies (bottom panel). For immunoblotting analysis, protein samples were denatured by boiling in SDS sample buffer. (C) Schematic representation of YknWXYZ complex. YknW possesses partial activity and modulates the assembly of the complex. PG, peptidoglycan; CM, cytoplasmic membrane; S, substrate.