Escherichia coli lipoproteins are anchored to either the inner or outer membrane through fatty acyl chains covalently attached to an N-terminal cysteine. Aspartate at position 2 functions to retain lipoproteins in the inner membrane, although the retention is perturbed depending on the residue at position 3. We previously revealed that LolCDE and LolA play critical roles in this lipoprotein sorting. To clarify the sorting signals, the LolA-dependent release of lipoprotein derivatives having various residues at positions 2 and 3 was examined in spheroplasts. When the residue at position 3 was serine, only aspartate at position 2 caused the retention of lipoproteins in spheroplasts. We then examined the release of derivatives having aspartate at position 2 and various residues at position 3. Strong inner membrane retention occurred with a limited number of species of residues at position 3. These residues were present at position 3 of native lipoproteins having aspartate at position 2, whereas residues that inhibited the retention were not. It was also found that a strong inner membrane retention signal having residues other than aspartate at position 2 could be formed through the combination of the residues at positions 2 and 3. These results indicate that the inner membrane localization of native lipoproteins is ensured by the use of a limited number of strong inner membrane retention signals.Various bacteria possess lipoproteins, which have a lipidmodified cysteine at the N terminus. In Escherichia coli, lipoproteins are anchored to the periplasmic side of either the inner or outer membrane through N-terminal fatty acyl chains (1, 2). Lipoproteins are synthesized as precursors in the cytoplasm and then are translocated across the inner membrane, followed by sequential modification reactions leading to the formation of mature lipoproteins on the periplasmic side of the inner membrane (1, 2). Five Lol proteins are involved in the sorting and outer membrane localization of lipoproteins (3-5). LolCDE in the inner membrane releases outer membrane-directed lipoproteins from the inner membrane in an ATP-dependent manner (5, 6), leading to the formation of a complex between the lipoprotein and LolA (3), a periplasmic chaperone. The LolA-lipoprotein complex crosses the periplasm and then interacts with outer membrane receptor LolB, which mediates the anchoring of lipoproteins to the outer membrane (4). The lipoprotein-sorting signal is recognized at the release step, and the inner membrane-specific lipoproteins are not released (3-5).Inouye and collaborators (7) first revealed the importance of aspartate at position 2 for the inner membrane localization of E. coli lipoproteins. They showed that replacement of serine at position 2 of an outer membrane-specific lipoprotein by aspartate caused the protein to remain in the inner membrane. Furthermore, replacement of aspartate at position 2 of an inner membrane-specific lipoprotein by another residue caused outer membrane localization of the protein. Taken together, these re...

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