Figure 2.

YmcA, YlbF and YaaT form a ternary complex. (A) Co-purification scheme of the proteins from E. coli. First, GST-YmcA was expressed, purified, and the GST tag cleaved. Separate cultures of cells expressing GST-YlbF and His₆-SUMO-YaaT were mixed and lysed together, and the lysates were incubated with the pure YmcA to allow for complex formation. This mixture was passed over nickel resin to isolate His₆-SUMO-YaaT and associated proteins, and after cleavage of the SUMO tag the preparation was passed over glutathione (GSH) resin to isolate the protein complexes containing GST-YlbF. After cleavage of the GST-tag from YlbF, all three untagged proteins were present in the eluate. (B) Co-purified proteins were run on a Superose 12 size exclusion column, as described in Experimental procedures. Depicted are the sample absorbance at 215 nm (A₂₁₅, solid black line) and absorbance at 280 nm (A₂₈₀, dashed black line), which has a low intensity due to the presence of a single tryptophan residue in the complex (YaaT). Included for comparison is a mixture of molecular weight sizing standards (A₂₈₀, dotted grey line). From left to right the standard peaks are: thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa). Peaks A and B correspond to contaminating species that are roughly 20 and 45 kDa in size, respectively. Peak C represented the intact YmcA-YlbF-YaaT complex, and corresponds to ~80 kDa in size. (C) Fractions taken from peak C (labeled 1, 2, and 3) were TCA precipitated, resolved by SDS-PAGE on a 12.5% Tris-tricine gel and bands visualized by staining with Coomassie blue. The input lane contained 5 µg of partially purified protein complex. Bands labeled X₁-X₃ represented major contaminating proteins in the preparation. All samples shown were run on the same gel with irrelevant lanes removed.