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.
Roy
Hervé
H
Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA. roy.79@osu.edu
Ibba
Michael
M
D019881:Aminoacyltransferases; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D008239:Lysine / Q000096:biosynthesis*; D010715:Phosphatidylglycerols / Q000096:biosynthesis*; D012357:RNA, Transfer, Lys / Q000378:metabolism*
D001426:Bacterial Proteins; D003902:Detergents; D010715:Phosphatidylglycerols; D012357:RNA, Transfer, Lys; C002285:lysylphosphatidylglycerol; D019881:Aminoacyltransferases; C429146:mprF protein, Staphylococcus aureus; D008239:Lysine