An In Vitro Assay For LpxC Degradation By The Membrane-Bound Protease FtsH And Adaptor LapB From Escherichia coli

This protocol presents a sensitive, quantitative in vitro degradation assay of LpxC using reconstituted AAA+ (ATPase associated with diverse cellular activities) protease FtsH and adaptor LapB in proteoliposomes. AAA+ proteases are ATP-dependent molecular machines that maintain protein homeostasis and regulate diverse cellular processes in the cytoplasm or at cellular membranes. Compared with their cytoplasmic counterparts, membrane-bound AAA+ proteases remain poorly characterized due to technical challenges in reconstituting and measuring their activity in vitro. Here, we use the FtsH–LapB–LpxC system as a model to develop a fluorescence-based degradation assay in proteoliposomes. FtsH and LapB are reconstituted into proteoliposomes to mimic the native membrane environment, and LpxC is covalently labeled with the fluorescent dye Atto488. Degradation is initiated by combining proteoliposomes with labeled LpxC and ATP and terminated by trichloroacetic acid (TCA). A successful assay is defined by a time-dependent increase in fluorescence in the soluble supernatant fraction after TCA precipitation, corresponding to the release of fluorescent peptide fragments generated by LpxC proteolysis. This signal enables direct quantification of initial degradation rates under defined conditions. Compared with traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-based assays, this method provides substantially improved sensitivity and enables quantitative kinetic analysis. Moreover, the proteoliposome system allows systematic investigation of how protein components, such as adaptors and anti-adaptors, as well as lipid composition, influence LpxC degradation. Although developed for the FtsH–LapB–LpxC system, this assay is readily adaptable to other membrane-bound AAA+ proteases and substrates, providing a general platform for studying membrane-associated proteolysis in vitro.