Monitoring Lys-tRNA(Lys) phosphatidylglycerol transferase activity.

In some bacteria Lys-tRNA(Lys) is used both in translation and for the specific addition of Lys to phosphatidylglycerol in the cytoplasmic membrane. This reaction is catalyzed by the membrane protein MprF, and the lysyl-phosphatidylglycerol formed contributes to the resistance of these bacteria to various cationic antibacterial molecules. Obtaining proteins and reconstituting an in vitro system mimicking membrane conditions is a major challenge to studying the function of membrane proteins, especially when labile substrates such as Lys-tRNA(Lys) are required. Here we report methods to obtain a stable enriched membrane fraction containing MprF, and the techniques necessary to quantitatively monitor its activity in vitro and in vivo.