Mechanism Underlying the Inner Membrane Retention of Escherichia coli Lipoproteins Caused by Lol Avoidance Signals

Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e. the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane. To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes. Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release. In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue. Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function. Based on these results, the Lol avoidance mechanism is discussed. Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e. the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane. To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes. Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release. In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue. Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function. Based on these results, the Lol avoidance mechanism is discussed. Bacterial lipoproteins are synthesized as precursors in the cytoplasm and then translocated across the inner membrane by the Sec translocation machinery (1Hayashi S. Wu H.C. J. Bioenerg. Biomembr. 1990; 22: 451-471Crossref PubMed Scopus (426) Google Scholar, 2Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar). Subsequent processing to mature lipoproteins occurs on the periplasmic side of the inner membrane. The N-terminal Cys of mature lipoproteins is modified by thioether-linked diacylglycerol and amino-linked acyl chain (3Sankaran K. Wu H.C. J. Biol. Chem. 1994; 269: 19701-19706Abstract Full Text PDF PubMed Google Scholar). In Escherichia coli, lipoproteins are anchored through N-terminal lipids to the periplasmic leaflet of either the inner or outer membrane. It has been proposed that Asp at position 2 makes lipoproteins specific to the inner membrane, whereas other residues direct lipoproteins to the outer membrane (4Yamaguchi K. Fujio Y. Inouye M. Cell. 1988; 53: 423-432Abstract Full Text PDF PubMed Scopus (259) Google Scholar). We recently revealed that the inner membrane retention of lipoproteins is determined by Asp at position 2 but also affected by the residue at position 3 (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Thus, strong inner membrane retention occurs with Asp at position 2 and Asp, Glu, Gln, or Asn at position 3. Other residues at position 3 decrease the inner membrane retention of lipoproteins to various extents depending on the species. Importantly, E. coli native lipoproteins specific to the inner membrane only utilize these strong inner membrane signals (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The Lol system, which is composed of five Lol proteins, is involved in the sorting and outer membrane localization of lipoproteins. The LolCDE complex in the inner membrane belongs to the ATP-binding cassette transporter superfamily and comprises two copies of an ATPase subunit, LolD, and one copy each of the integral membrane subunits LolC and LolE. This complex releases outer membrane-directed lipoproteins from the inner membrane in an ATP-dependent manner (6Yakushi T. Yokota N. Matsuyama S. Tokuda H. J. Biol. Chem. 1998; 273: 32576-32581Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 7Yakushi T. Masuda K. Narita S. Matsuyama S. Tokuda H. Nat. Cell Biol. 2000; 2: 212-218Crossref PubMed Scopus (203) Google Scholar, 8Narita S. Tanaka K. Matsuyama S. Tokuda H. J. Bacteriol. 2002; 184: 1417-1422Crossref PubMed Scopus (63) Google Scholar), leading to the formation of a water-soluble complex comprising one molecule each of lipoprotein and LolA in the periplasm (9Matsuyama S. Tajima T. Tokuda H. EMBO J. 1995; 14: 3365-3372Crossref PubMed Scopus (164) Google Scholar, 10Tajima T. Yokota N. Matsuyama S. Tokuda H. FEBS Lett. 1998; 439: 51-54Crossref PubMed Scopus (78) Google Scholar). The LolA-lipoprotein complex then interacts with outer membrane receptor LolB, which catalyzes the anchoring of lipoproteins to the outer membrane (11Matsuyama S. Yokota N. Tokuda H. EMBO J. 1997; 16: 6947-6955Crossref PubMed Scopus (171) Google Scholar, 12Tanaka K. Matsuyama S. Tokuda H. J. Bacteriol. 2001; 183: 6538-6542Crossref PubMed Scopus (70) Google Scholar). The crystal structures of LolA and LolB are very similar to each other despite the fact that their amino acid sequences are dissimilar (13Takeda K. Miyatake H. Yokota N. Matsuyama S. Tokuda H. Miki K. EMBO J. 2003; 22: 3199-3209Crossref PubMed Scopus (107) Google Scholar). The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing the retention of lipoproteins in the inner membrane (14Masuda K. Matsuyama S. Tokuda H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7390-7395Crossref PubMed Scopus (86) Google Scholar). The Lol avoidance is not caused by a difference in the mode of lipid modification between inner membrane-specific and outer membrane-specific lipoproteins (15Fukuda A. Matsuyama S. Hara T. Nakayama J. Nagasawa H. Tokuda H. J. Biol. Chem. 2002; 277: 43512-43518Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). A LolC mutant, LolC(A40P), that releases lipoproteins possessing the Lol avoidance signal was recently isolated (16Narita S. Kanamaru K. Matsuyama S. Tokuda H. Mol. Microbiol. 2003; 49: 167-177Crossref PubMed Scopus (31) Google Scholar). Because both LolA and LolB could interact with inner membrane-specific lipoproteins released by the mutant LolCDE complex, the inner membrane retention signal was found to only function against LolCDE. However, it remains to be clarified why Asp at position 2 has such a specific function. To clarify critical properties required for the Lol avoidance signal, the second residue of lipoproteins was chemically modified, and the release of the modified lipoproteins was examined in proteoliposomes reconstituted with E. coli phospholipids and LolCDE. Here we show that the distance between Cα and the negative charge of the second residue is critical for the Lol avoidance signal. Furthermore, phosphatidylethanolamine was found to be essential for the Lol avoidance mechanism. Materials—Globomycin was a kind gift from Masatoshi Inukai (Sankyo Co.). Sucrose monocaprate and octylglucoside were purchased from Dojindo Laboratories. Avidin-HRP 1The abbreviations used are: avidin-HRP, streptavidin-horseradish peroxidase conjugant; PE, phosphatidylethanolamine; CL, cardiolipin; PC, phosphatidylcholine; IPTG, isopropyl-β-d-thiogalactopyranoside; octylglucoside, n-octyl-β-d-glucopyranoside; sucrose monocaprate, β-d-fructopyranosyl-α-d-glucopyranoside monodecanoate; maleimide biotin, (+)-biotinyl-3-maleimidepropionamidyl-3, 6-dioxaoctanediamine; iodoacetyl biotin, (+)-biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine; biotin PEO-amine, (+)-biotiny-3, 6-dioxaoctanediamine; SNA, sulfo-N-hydroxysuccinimide acetate; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; AMS, 4-acetamido-4′-maleimidylstilbene-2, 2′-disulfonic acid disodium salt; TCEP, tris-(2-carboxyethyl)phosphine hydrochloride; SH, sulfhydryl; MES, 4-morpholineethanesulfonic acid. was from Amersham Biosciences. Maleimide biotin, iodoacetyl biotin, biotin PEO-amine, SNA, and EDC were obtained from Pierce. AMS and TCEP were purchased from Molecular Probes. Synthetic CL and PC were obtained from Avanti Polar Lipids. E. coli phospholipids were also from Avanti Polar Lipids and washed with acetone as reported (17Tokuda H. Shiozuka K. Mizushima S. Eur. J. Biochem. 1990; 192: 583-589Crossref PubMed Scopus (28) Google Scholar). Talon, a metal-chelating resin, was from Clontech. Bacteria and Plasmids—E. coli K12 strain JM83 (F- ara Δ(lacproAB) rpsL ([phis]80 lacZΔM15)) (18Yakushi T. Tajima T. Matsuyama S. Tokuda H. J. Bacteriol. 1997; 179: 2857-2862Crossref PubMed Google Scholar) was grown on L broth at 30 or 37 °C. When required, the medium was supplemented with 50 μg/ml ampicillin or 25 μg/ml chloramphenicol. Plasmids pTPH21 (15Fukuda A. Matsuyama S. Hara T. Nakayama J. Nagasawa H. Tokuda H. J. Biol. Chem. 2002; 277: 43512-43518Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and pKM301 (14Masuda K. Matsuyama S. Tokuda H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7390-7395Crossref PubMed Scopus (86) Google Scholar) carry pal-his and lolE, respectively, under the control of tacPO and lacIq. pKM402 (14Masuda K. Matsuyama S. Tokuda H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7390-7395Crossref PubMed Scopus (86) Google Scholar) carries lolC and lolD-his under the control of PBAD. Construction of Pal Derivatives—To construct plasmids encoding His-tagged Pal(2C), Pal(3C), and Pal(4C) having a single Cys at positions 2, 3, and 4, respectively, under the control of tacPO and lacIq , pTPH21 was mutagenized using a QuikChange site-directed mutagenesis kit (Stratagene) with a specified pair of PCR primers (Table I). To construct plasmids encoding Pal(2D), Pal(2D3C), Pal(2D3D4C), and Pal(2C3D) under the control of tacPO and lacIq , pTPH21 was digested with SalI and HindIII, followed by Klenow enzyme to make the ends blunt, and then subjected to self-ligation. The MfeI-SphI fragment of the the signal of was with the specified in used in in a Construction of Synthetic lipoprotein was as to the signal of Pal at the a and a at the and carboxylic acid in the mature for the The for the lipoprotein was the of under the control of tacPO and lacIq. However, of the lipoprotein was very To the for Asp and were the mature The plasmids were and then the of lipoproteins was A was isolated from the that the of To construct was as to Asp and at the N-terminal second and positions of the mature The of the at the of was found to be in which the and are and the for the second and residues of are with the of in The amino acid sequences of and by and respectively, are in of and LolCDE complex was in JM83 pKM301 and pKM402 as reported (14Masuda K. Matsuyama S. Tokuda H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7390-7395Crossref PubMed Scopus (86) Google Scholar). Pal and were in JM83 the His-tagged were with a metal-chelating as reported (15Fukuda A. Matsuyama S. Hara T. Nakayama J. Nagasawa H. Tokuda H. J. Biol. Chem. 2002; 277: 43512-43518Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). LolCDE was at in 50 2 and sucrose Pal were at in 50 sucrose monocaprate and TCEP at 2 and were in LolA was as reported (9Matsuyama S. Tajima T. Tokuda H. EMBO J. 1995; 14: 3365-3372Crossref PubMed Scopus (164) Google Scholar). S. Tanaka K. Matsuyama S. Tokuda H. J. Bacteriol. 2002; 184: 1417-1422Crossref PubMed Scopus (63) Google Scholar), K. Tokuda H. Mizushima S. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar), and K. Mizushima S. Tokuda H. EMBO J. 1993; PubMed Scopus Google Scholar) were in as were obtained from of from reported (14Masuda K. Matsuyama S. Tokuda H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7390-7395Crossref PubMed Scopus (86) Google Scholar) was E. coli phospholipids and LolCDE were on for in of 50 2 and sucrose The was with 50 and and then against of the The reconstituted were by at for 2 and in of 50 2 and the was with of a the lipoprotein followed by on for 30 to anchoring of lipoproteins to were and in of 50 2 and Because of in a and were reconstituted proteoliposomes with LolCDE. The release of lipoproteins from reconstituted proteoliposomes was examined as reported T. Masuda K. Narita S. Matsuyama S. Tokuda H. Nat. Cell Biol. 2000; 2: 212-218Crossref PubMed Scopus (203) Google Scholar). The obtained was with of 50 2 TCEP, and of followed by at 30 for proteoliposomes and the the lipoproteins in each were determined by and with specified or of the was to the of and modification of lipoproteins was with maleimide biotin, iodoacetyl biotin, or AMS at 30 for 2 The reaction was by the of and the modified lipoproteins were with followed by in the of biotin to the carboxylic acid of lipoproteins by of a EDC, lipoproteins were at 30 for 2 in octylglucoside, 50 EDC, and biotin The reaction was against of octylglucoside for 2 and then of octylglucoside for of Cys was to the of Scopus Google Scholar). acid was by of acid with 50 of for 2 at were with in a of acid and 2 of and then at for 30 acid was to the lipoprotein followed by at for the reaction with the lipoproteins were with acetone and then in sucrose E. coli phospholipids in 50 octylglucoside were with 25 for at and then against 50 to the Other for Pal and and was to PubMed Scopus Google Scholar) and M. S. Mizushima S. J. Biol. Chem. Full Text PDF PubMed Google Scholar), H. Matsuyama S. Tokuda H. Mizushima S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and (11Matsuyama S. Yokota N. Tokuda H. EMBO J. 1997; 16: 6947-6955Crossref PubMed Scopus (171) Google Scholar) were as at 2 the and LolCDE-dependent of membrane lipoprotein Pal has Cys residue for the N-terminal Pal(2C), which Cys at position 2 (Table and a at the was and in E. It was that the not lipoprotein sorting (15Fukuda A. Matsuyama S. Hara T. Nakayama J. Nagasawa H. Tokuda H. J. Biol. Chem. 2002; 277: 43512-43518Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). localization of was then examined in with in (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), Cys at position 2 did not function as the Lol avoidance signal, and was localized in the outer membrane not was and with was anchored to proteoliposomes reconstituted with LolCDE and then subjected to the release reaction in the and of LolA introduction of molecules such as iodoacetyl biotin or AMS did not inhibit the release. released the with the of biotin to the released the are not the modified in the as a complex with LolA and was outer in a these that introduction of nonprotein molecules to the second residue of outer membrane-specific lipoproteins has on their with Lol was modified with acid to a negative charge to the second However, also did not inhibit the which was on both LolCDE and LolA whereas possessing Asp at position 2 was not released from proteoliposomes. It has been reported that at position 2 not the Lol avoidance function (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). that the introduction of a negative charge is not for of an outer membrane lipoprotein an inner membrane We examined through a the release of was either with TCEP or with and then reconstituted proteoliposomes. The was only with The of and were released on the of whereas LolA did not the release of Pal(2C), indicating that only the lipoprotein as a for the release of Cys at 2 the of at position 2 not function as the Lol avoidance signal (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), it that the distance between the negative charge and Cα of the chain is critical for the Lol avoidance signal. To Cys of was with acid to cysteic of which the charge distance is similar to that of Asp in 2, the release of was not affected by with acid. in proteoliposomes the with acid. the release of was by We also and which Cys at positions 3 and 4, of the release reaction by acid was on the position of of the release was with and with are with the that Asp at position 2 functions as the Lol avoidance signal, whereas that at position not Inouye M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the acid of the Pal possessing Cys a with indicating that was to of Cys to cysteic acid inhibits the release of lipoproteins depending on the position of Pal were with acid as under and then anchored to proteoliposomes reconstituted with LolCDE. The release of Pal was examined as in and by and with of the was to the of Asp at 2 Lol the of the Asp negative charge for the Lol avoidance and were The has negative charge for that of the carboxylic whereas the has Asp at position 2 followed by Gln, thereby having a strong Lol avoidance signal, (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). lipoproteins and at the for their and In processing of the to the mature was by an of signal was localized in the outer membrane, whereas was found in the inner membrane indicating that membrane sorting of these lipoproteins to the (4Yamaguchi K. Fujio Y. Inouye M. Cell. 1988; 53: 423-432Abstract Full Text PDF PubMed Scopus (259) Google Scholar). and were and with EDC in the of biotin both lipoproteins with avidin-HRP, indicating that the was modified with biotin not and with or were reconstituted proteoliposomes and then subjected to the release reaction or not with was released from proteoliposomes on the of indicating that the modification with biotin not inhibit the release LolA did not the release of that not been with the other with a of was released on the of indicating that the carboxylic acid of Asp at position 2 was with biotin, and the negative charge required for the Lol avoidance function was Cys at 2 of to from the of Cys at position 2, 3, or of lipoproteins was examined in proteoliposomes. Pal possessing Cys (Table were modified with maleimide biotin in the of a sucrose monocaprate, and with When modification with maleimide biotin was examined Pal having Cys at position 2 were not modified, whereas having Cys at position 3 or were Asp residue to position 2 or 3 or both did not the of that lipoproteins are anchored to Cys at position 2 is not to the for the Lol of Asp at that the residue at position 2 of lipoproteins was in a lipid the of the negative charge of Asp, it to be that modification of the Lol avoidance function of Asp at position 2. To E. coli phospholipids PE, and CL as the were with revealed that was modified, i.e. the of with the of a not were reconstituted with or E. coli The and LolCDE-dependent release of was not affected by the was not released from proteoliposomes reconstituted from phospholipids as the other was released from proteoliposomes in and LolCDE-dependent phospholipids were To the of for the Lol avoidance proteoliposomes were reconstituted with CL was released from as as This release was by an of LolCDE T. Masuda K. Narita S. Matsuyama S. Tokuda H. Nat. Cell Biol. 2000; 2: 212-218Crossref PubMed Scopus (203) Google Scholar). the other the Lol avoidance signal in proteoliposomes reconstituted with PC these that the charge of phospholipids is for the Lol avoidance function of Asp at position 2. The N-terminal second residue of lipoproteins has been to a role in the of membrane (4Yamaguchi K. Fujio Y. Inouye M. Cell. 1988; 53: 423-432Abstract Full Text PDF PubMed Scopus (259) Google Scholar, M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, A. A.P. Mol. Microbiol. PubMed Scopus Google Scholar). However, introduction of nonprotein molecules to the second residue did not inhibit the release of outer membrane-specific lipoproteins. that LolCDE releases outer membrane-specific lipoproteins the second residue. It was recently found that an the N-terminal acyl chain was not by LolCDE (15Fukuda A. Matsuyama S. Hara T. Nakayama J. Nagasawa H. Tokuda H. J. Biol. Chem. 2002; 277: 43512-43518Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). LolCDE to only the N-terminal Cys possessing acyl the of lipoproteins. lipoproteins Asp at position It that Asp at position 2 in combination with certain residues at position 3 makes the N-terminal of lipoproteins thereby the recognition of the Cys by LolCDE. Cys (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) at position 2 as the Lol avoidance signal, whereas oxidation of Cys at position 2 to cysteic acid resulted in generation of the Lol avoidance signal. modification of the carboxylic acid of Asp at position 2 abolished Lol avoidance function. results, that the Lol avoidance signal a negative charge that is a certain distance from Cα of the second residue which is not from the modification of and of with CL abolished the Lol avoidance function of Asp at position 2. In contrast, the Lol avoidance signal in proteoliposomes reconstituted with results, that the and between Asp at position 2 and phospholipids having a charge is for the Lol avoidance mechanism. It that a charge of phospholipids is required for the of LolCDE, be at When Glu, Asp, Gln, or Asn is at position 3, Asp at position 2 a very strong Lol avoidance signal (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In contrast, or at position 3 the Lol avoidance function of Asp at position 2. Other residues also the of the Lol avoidance signal. that the Lol avoidance function of Asp are or their that not with Asp at position 2. It that the residue not the and between Asp at position 2 and for the Lol avoidance mechanism. between Asp at position 2 and is to be by between Cys at position and the molecule with Asp at position 2 the other possessing a side chain is at position 2, the molecules involved in the with and the formation with Cys at position be the is not The residues that the Lol avoidance signal are also to a or with the molecule with Asp at position 2. Based on these we that the Lol avoidance signal the formation of a complex This complex has five acyl and be in LolCDE. The release of the and to that the of acyl is critical for the recognition by LolCDE. The complex is to be to such a complex, thereby causing the release of lipoproteins having the Lol avoidance signal. at position 3 the Lol avoidance function of Asp at position 2, whereas at position 3 did not (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). of the by the charge at position 3 to be on the side chain of the residue. A. A.P. Mol. Microbiol. PubMed Scopus Google Scholar) reported that not only Asp but also residues such as and at position 2 followed by Asn at position 3 the inner membrane retention of E. coli native lipoproteins not and at position 2. We reported that the residue at position 3 is these residues at position 2 not the inner membrane retention of lipoproteins (5Terada M. Kuroda T. Matsuyama S. Tokuda H. J. Biol. Chem. 2001; 276: 47690-47694Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). It that the mechanism of inner membrane retention caused by these residues at position 2 is from the mechanism by which native lipoproteins in the inner membrane. It also be that Asp at position 2 not be the Lol avoidance signal in other Lol are in various T. Masuda K. Narita S. Matsuyama S. Tokuda H. Nat. Cell Biol. 2000; 2: 212-218Crossref PubMed Scopus (203) Google Scholar). in is an inner membrane lipoprotein possessing and at positions 2 and 3, In the between and the second residue is not to the Lol avoidance mechanism. Because membrane localization and sorting signals been determined for only a lipoprotein in other it remains to be determined the Lol avoidance mechanism is to the inner membrane-specific lipoproteins in However, the Lol avoidance mechanism is to be required for the localization of of on the outer of the outer membrane. and A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar) examined the membrane localization of in E. coli with or a of comprising the having Asp at position 2 was localized on the outer of the outer membrane with the In contrast, was localized on the periplasmic of the inner membrane in the of the indicating that Asp at position 2 functions as the Lol avoidance signal in the of the of Asp with residue resulted in the localization of on both the periplasmic and the outer of the outer membrane the was However, was localized on the periplasmic of the outer membrane in the of the and that in E. coli K. a Lol avoidance signal to be translocated to the outer of the outer membrane through the the Lol the localization of to the periplasmic of the outer membrane. The critical for the Lol avoidance function were revealed in Because the of LolCDE also the Lol avoidance function (16Narita S. Kanamaru K. Matsuyama S. Tokuda H. Mol. Microbiol. 2003; 49: 167-177Crossref PubMed Scopus (31) Google Scholar), it is of to the localized of LolCDE that the Lol We for Masatoshi Inukai (Sankyo for the and for