Characterization of lipoprotein-sorting signal in Pseudomonas aeruginosa

　　Bacterial lipoprotein is a subset of membrane proteins, which are modified at their amino terminus with lipid. In Gram-negative bacteria, lipoproteins are anchored with their lipid moiety to outer leaflet of cytoplasmic membrane, inner or outer leaflet of outer membrane. Localization of lipoproteins to the inner leaflet of outer membrane requires a specific targeting machinery, the Lol proteins, which enable hydrophobic lipoproteins to detach from cytoplasmic membrane and to move across the hydrophilic periplasmic space. In Escherichia coli, cytoplasmic-membrane specific lipoproteins have aspartate at the next residue (+2 residue) to the amino terminal cystein, whereas outer-membrane specific lipoproteins have other amino acids at this site. Therefore, the +2 residue has been described as the lipoprotein-sorting signal, and it has recently shown that aspartate interacts with phosphatidylethanolamine of the cytoplasmic membrane, thus acting as the Lol-avoidance signal. In Gram-negative bacteria other than E. coli, on the other hand, cytoplasmic-membrane specific lipoproteins without aspartate at +2 residue can be found. For example, MexA, a membrane fusion protein of RND-type multidrug efflux system in Pseudomonas aeruginosa, is known to be localized to the cytoplasmic membrane in spite of glycine at +2 residue, suggesting that alternative sorting signal may be effective in this bacterium. To address mechanism underlying Lol avoidance of cytoplasmic-membrane specific lipoproteins in P. aeruginosa, and to understand universal rule for lipoprotein localization in Gram-negative bacteria, we characterized the sorting signal of MexA. First, we substituted the +2 residue of MexA and determined their membrane localization by membrane fractionation and Western blotting. We further cloned the genes encoding Lol proteins of P. aeruginosa, and expressed them in E. coli. P. aeruginosa Lol-proteins dependent release of E. coli lipoproteins from cytoplasmic membrane was assessed to characterize lipoprotein-sorting signal that is recognized by P. aeruginosa Lol proteins. By integrating these results, we will summarize lipoprotein-sorting signal in P. aeruginosa.