FIGURE 1.
Identification of a widely accepted transmembrane substrate for rhomboid proteases. A, comparison of cleavage efficiency of model substrates LacYTM2, Gurken, TatA, and Spitz by bacterial rhomboid proteases GlpG (E. coli), AarA (P. stuartii), YqgP (B. subtilis), and BtioR3 (B. thetaiotaomicron) in vitro. Equal concentrations of purified recombinant substrates were exposed to purified recombinant rhomboid proteases. Cleavage products were separated by SDS-PAGE, stained, and quantified densitometrically to determine initial reaction rates, which were converted to molar catalytic activities to allow comparisons. Displayed values are representative of two independent experiments. B, cleavage of synthetic LacYTM2 transmembrane peptide KSp31 by GlpG. Purified synthetic peptide KSp31 was incubated with purified recombinant GlpG or its inactive mutant S201T in the presence of 0.05% (w/v) DDM, and the reaction mixtures were analyzed by MALDI mass spectrometry. The theoretical molecular masses of the expected cleavage products at the native cleavage site are denoted below the peptide sequence, and unambiguously match those experimentally determined and displayed in the mass spectra. The star-marked peak with molecular mass of 1893.3 is an unidentified minor contaminant in the preparation of KSp31. C, monitoring of cleavage of peptide substrate KSp31 by rhomboid protease GlpG using CE. The N-terminal cleavage product (P) of KSp31 was separated by free-flow CE in the background electrolyte composed of 100 mm H3PO4 and 69 mm Tris, pH 2.5, in bare fused silica capillary at separation voltage +25 kV. Samples for CE were prepared by mixing 20 μl of reaction mixture at selected reaction times (0–90 min) with 2 μl of 2.2 mm tyramine (T) as an internal standard. Samples were injected into the capillary by 20 mbar pressure for 10 s. Quantitative analysis was based on the ratio of corrected (migration time normalized) peak areas of peptides of interest and the internal standard. Analyses were performed in triplicate. P, cleaved N-terminal peptide; X, system peak. D, the importance of the transmembrane domain of the substrate for its recognition and cleavage by rhomboid. A series of synthetic peptides covering LacYTM2 with progressive truncations of its transmembrane domain from the C terminus was exposed to GlpG and initial rates of cleavage were quantified by capillary electrophoresis as denoted in panel C.