Diacylglycerol Specifically Blocks Spontaneous Integration of Membrane Proteins and Allows Detection of a Factor-assisted Integration

Abstract

We recently found that the spontaneous integration of M13 procoat is blocked by diacylglycerol (DAG) (Nishiyama, K., Ikegami, A., Moser, M., Schiltz, E., Tokuda, H., and Muller, M. (2006) J. Biol. Chem. 281, 35667–35676). Here, we demonstrate that the spontaneous integration of Pf3 coat, another membrane protein that has been thought to be integrated spontaneously into liposomes, can be blocked by DAG at physiological concentrations. Moreover, the spontaneous integration of the membrane potential-independent version of Pf3 coat (3L-Pf3 coat), which is independent of YidC, was also blocked by DAG. To clarify the mechanism by which DAG blocks spontaneous integration, we examined lipid compounds similar to DAG and DAG derivatives. The blockage of spontaneous integration was specific to DAG, as fatty acids, monoacylglycerol, and phosphatidic acids were not effective for the blockage. When the acyl chains in DAG were shortened even to octanoyl residues, it still blocked spontaneous integration, whereas diheptanoylglycerol did not block it at all. Triacylglycerol was more effective than DAG. However, the lipid A-derivative-dependent integration of M13 procoat could not be reconstituted when triacylglycerol was included in the liposomes. On the other hand, when DAG was included in the liposomes, we found that the integration of 3L-Pf3 coat was strictly dependent on the lipid A-derived integration factor. We propose that the bulky structure of DAG rather than changes in membrane curvature is essential for the blockage of spontaneous integration. We also demonstrated that the blockage of spontaneous integration by DAG is also operative in native membrane vesicles. We recently found that the spontaneous integration of M13 procoat is blocked by diacylglycerol (DAG) (Nishiyama, K., Ikegami, A., Moser, M., Schiltz, E., Tokuda, H., and Muller, M. (2006) J. Biol. Chem. 281, 35667–35676). Here, we demonstrate that the spontaneous integration of Pf3 coat, another membrane protein that has been thought to be integrated spontaneously into liposomes, can be blocked by DAG at physiological concentrations. Moreover, the spontaneous integration of the membrane potential-independent version of Pf3 coat (3L-Pf3 coat), which is independent of YidC, was also blocked by DAG. To clarify the mechanism by which DAG blocks spontaneous integration, we examined lipid compounds similar to DAG and DAG derivatives. The blockage of spontaneous integration was specific to DAG, as fatty acids, monoacylglycerol, and phosphatidic acids were not effective for the blockage. When the acyl chains in DAG were shortened even to octanoyl residues, it still blocked spontaneous integration, whereas diheptanoylglycerol did not block it at all. Triacylglycerol was more effective than DAG. However, the lipid A-derivative-dependent integration of M13 procoat could not be reconstituted when triacylglycerol was included in the liposomes. On the other hand, when DAG was included in the liposomes, we found that the integration of 3L-Pf3 coat was strictly dependent on the lipid A-derived integration factor. We propose that the bulky structure of DAG rather than changes in membrane curvature is essential for the blockage of spontaneous integration. We also demonstrated that the blockage of spontaneous integration by DAG is also operative in native membrane vesicles. Integral membrane proteins in Escherichia coli are integrated into the cytoplasmic membrane through defined steps. Many membrane proteins are cotranslationally targeted to membranes with the help of the signal recognition particle (SRP) 2The abbreviations used are:SRPsignal recognition particleDAGdiacylglycerolINVinverted and inner membrane vesicleDCCDN,N-dicyclohexylcarbodiimidePMFproton motive forceFAfatty acidMAGmonoacylglycerolTAGtriacylglycerolPAphosphatidic acid. 2The abbreviations used are:SRPsignal recognition particleDAGdiacylglycerolINVinverted and inner membrane vesicleDCCDN,N-dicyclohexylcarbodiimidePMFproton motive forceFAfatty acidMAGmonoacylglycerolTAGtriacylglycerolPAphosphatidic acid./SRP receptor system followed by membrane integration at the SecYEG site (for a review, see Ref. 1Luirink J. von Heijne G. Houben E. de Gier J.W. Annu Rev. Microbiol. 2005; 59: 329-355Crossref PubMed Scopus (154) Google Scholar). Several membrane proteins, such as M13 procoat, Pf3 coat, CyoA, and F0 c subunit, are integrated independently of both SRP and SecYEG, as these proteins are integrated into Sec- or SRP-deficient membranes (2Rohrer J. Kuhn A. Science. 1990; 250: 1418-1421Crossref PubMed Scopus (67) Google Scholar, 3van der Laan M. Urbanus M.L. Ten Hagen-Jongman C.M. Nouwen N. Oudega B. Harms N. Driessen A.J. Luirink J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5801-5806Crossref PubMed Scopus (124) Google Scholar, 4Yi L. Celebi N. Chen M. Dalbey R.E. J. Biol. Chem. 2004; 279: 39260-39267Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 5Wolfe P.B. Rice M. Wickner W. J. Biol. Chem. 1985; 260: 1836-1841Abstract Full Text PDF PubMed Google Scholar, 6Kuhn A. FEMS Microbiol. Rev. 1995; 17: 185-190Crossref PubMed Google Scholar, 7Geller B.L. Wickner W. J. Biol. Chem. 1985; 260: 13281-13285Abstract Full Text PDF PubMed Google Scholar, 8Kiefer D. Kuhn A. EMBO J. 1999; 18: 6299-6306Crossref PubMed Scopus (85) Google Scholar). YidC, a homologue of mitochondrial Oxa1p and Alb3p in chloroplasts, is thought to be involved in the integration of such SecYEG-independent proteins, as YidC depletion caused the accumulation of the precursor form of M13 procoat (9Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (414) Google Scholar). On the other hand, M13 procoat can be efficiently integrated into liposomes composed of polar phospholipids even in the absence of a membrane potential (7Geller B.L. Wickner W. J. Biol. Chem. 1985; 260: 13281-13285Abstract Full Text PDF PubMed Google Scholar), and therefore, these proteins have long been thought to be integrated “spontaneously.” Although the YidC-dependent integration of Pf3 coat (10Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar) and F0 c subunit (11van der Laan M. Bechtluft P. Kol S. Nouwen N. Driessen A.J. J. Cell Biol. 2004; 165: 213-222Crossref PubMed Scopus (173) Google Scholar, 12Kol S. Turrell B.R. de Keyzer J. van der Laan M. Nouwen N. Driessen A.J. J. Biol. Chem. 2006; 281: 29762-29768Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) has been reconstituted in proteoliposomes, the experimental conditions used in those studies allow spontaneous integration. Indeed, the integration of a potential-independent version of Pf3 coat, 3L-Pf3 coat, was reported to occur into liposomes free of YidC, suggesting that the spontaneous mechanism of membrane protein integrations was still operative (10Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar). In in vivo studies, it has been shown that integration of both M13 procoat and Pf3 coat is strongly dependent on YidC (9Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (414) Google Scholar, 13Chen M. Samuelson J.C. Jiang F. Muller M. Kuhn A. Dalbey R.E. J. Biol. Chem. 2002; 277: 7670-7675Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). On the other hand, the generation of a proton motive force is severely impaired by YidC depletion (3van der Laan M. Urbanus M.L. Ten Hagen-Jongman C.M. Nouwen N. Oudega B. Harms N. Driessen A.J. Luirink J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5801-5806Crossref PubMed Scopus (124) Google Scholar), which should inhibit the integration of these potential-dependent proteins. Moreover, M13 procoat is resistant to alkaline even YidC depletion J.C. Jiang F. L. Chen M. de Gier J.W. Kuhn A. Dalbey R.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that even the potential-dependent protein is integrated into membranes in a the YidC for the integration of these membrane proteins is still signal recognition particle diacylglycerol and inner membrane proton motive force fatty triacylglycerol phosphatidic acid. signal recognition particle diacylglycerol and inner membrane proton motive force fatty triacylglycerol phosphatidic acid. We have been the the membrane integration of which S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. B. J. Biol. 2005; PubMed Scopus Google Scholar). integration is dependent on both the SRP system and SecYEG Muller M. J. Cell Biol. 2000; PubMed Scopus Google Scholar, J. B. Muller M. Biol. 1999; PubMed Scopus Google Scholar). On the we found that is spontaneously integrated into liposomes in the absence of SecYEG A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar), to such spontaneous integration of could be in We found that integration into liposomes could be and blocked by diacylglycerol (DAG) in liposomes at a physiological A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar), and to integration in a Moreover, we found a lipid A-derived integration that is involved in the integration of both and M13 procoat A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). In we examined the of on the blockage of spontaneous integration, as we used DAG E. coli of polar We found that the spontaneous integration, that of potential-independent 3L-Pf3 coat, was blocked by DAG at the to the in vivo Moreover, we found that in the of DAG, 3L-Pf3 coat integration was dependent on the lipid A-derived factor. and coli J. Biol. PubMed Scopus Google Scholar) was used as a (9Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (414) Google Scholar) was used to and were by of M. M. M. B.L. Biol. 2006; Scopus Google Scholar). and were by was by the of the W. 1995; PubMed Scopus Google Scholar) and as reported B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). which the of the was as of and was used to the with the as a The of and to both are shown in the and The was into M. Biol. Chem. Scopus Google Scholar) which been with and were E. coli or as M. D. Muller M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). To YidC were in the of and with and in the of The depletion of YidC was by The E. coli of polar and were and were and were and were by with the acyl was with in for at followed by with as A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). and were by Kuhn and used to in M13 procoat A. Wickner W. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar), Pf3 coat (2Rohrer J. Kuhn A. Science. 1990; 250: 1418-1421Crossref PubMed Scopus (67) Google Scholar), and 3L-Pf3 coat D. Kuhn A. EMBO J. 1999; 18: 6299-6306Crossref PubMed Scopus (85) Google Scholar), 3L-Pf3 coat into the of the membrane of Pf3 coat, integration independent of the membrane potential D. Kuhn A. EMBO J. 1999; 18: 6299-6306Crossref PubMed Scopus (85) Google Scholar, J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar). of DAG or other were as phospholipids and DAG or other were in and a of and To the lipid was to a of of and liposomes were by of a To liposomes, the lipid in as was and and through the by of to the of the into the were reconstituted as A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). the was with polar phospholipids and of or in of were reconstituted by the A. The was with of and by for at in of were and J. S. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). was to the integration of membrane proteins and integration were as A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). The in protein composed of acids other than and and and M. G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), B. Muller M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), J. B. Muller M. Biol. 1999; PubMed Scopus Google Scholar), and were with and of or of in and The were at for and in The was with of and for at followed by The other was with were on M. S. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). were and by of a of the DAG in the was in the followed by at for by for were in of the with were by was to the at and on for to membranes followed by for at of the was to as J. Biol. Chem. Full Text PDF PubMed Google Scholar). of 3L-Pf3 of the and YidC the of DAG on the blockage of spontaneous integration, we of membrane proteins that are to be integrated into liposomes are M13 procoat A. Wickner W. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar), which we used in the A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar), Pf3 coat, and 3L-Pf3 coat, a potential-independent version of Pf3 coat D. Kuhn A. EMBO J. 1999; 18: 6299-6306Crossref PubMed Scopus (85) Google Scholar). M13 procoat is integrated to M13 procoat, that the signal is not M. M. Kuhn A. PubMed Scopus Google Scholar). reported D. Kuhn A. EMBO J. 1999; 18: 6299-6306Crossref PubMed Scopus (85) Google Scholar, A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar), of these proteins were integrated into the efficiently In the of M13 procoat and Pf3 coat, integration was by on the of to the proton motive force that the integration of these proteins is by the membrane a of and On the other hand, 3L-Pf3 coat integration was not on the of as reported D. Kuhn A. EMBO J. 1999; 18: 6299-6306Crossref PubMed Scopus (85) Google Scholar). When were 3L-Pf3 coat integration was rather with that into Moreover, 3L-Pf3 coat integration into was not of the or absence of In the integration of M13 procoat and Pf3 coat into was with (10Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar, 13Chen M. Samuelson J.C. Jiang F. Muller M. Kuhn A. Dalbey R.E. J. Biol. Chem. 2002; 277: 7670-7675Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). the integration of 3L-Pf3 coat was independent of both and YidC, whereas M13 procoat and Pf3 coat integration was in the absence of YidC or these and the that the integration of 3L-Pf3 coat is both Sec- and (10Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar) that 3L-Pf3 coat to be integrated into membranes spontaneously help by integration We examined the integration liposomes When liposomes composed of polar phospholipids were the membrane proteins used were integrated more efficiently than into with 3L-Pf3 coat integration into liposomes was more than that of the other proteins, was not in liposomes. In when liposomes DAG were integration for of these proteins was these proteins are not spontaneously integrated into liposomes DAG is as for integration A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). DAG examined the blockage of spontaneous integration is specific to DAG. In to DAG, fatty acids or triacylglycerol was into liposomes followed by of the integration the integration for Pf3 coat was the as that for M13 procoat and not M13 procoat was used as a potential-dependent The integration for both M13 procoat and 3L-Pf3 coat with liposomes or were the as those with liposomes composed of polar phospholipids On the other hand, liposomes with integration as did with DAG both DAG and were effective for the blockage of spontaneous integration, liposomes with of DAG or were followed by of integration the DAG or in liposomes, the integration for M13 procoat and 3L-Pf3 coat the integration of both membrane proteins was blocked when or more DAG was In the of the blockage was even with for both membrane proteins. When the integration was as a of the DAG or and it was found that blockage by is more effective than that by DAG. Although spontaneous integration is blocked by liposomes with of the structure these it was that the blockage of spontaneous integration is specific to of the of DAG or in liposomes on blockage of spontaneous integration. M13 procoat was in in the of liposomes DAG or at the The integration was as in the to the integration in was the of DAG or The integration of liposomes with than of DAG or is also shown in the 3L-Pf3 coat was in in the of the liposomes as in A. the of integration in was as in coat integration is dependent on the is in liposomes. The was reconstituted liposomes (DAG) or M13 procoat and 3L-Pf3 coat were in in the of liposomes or with the integration as The integration was as shown in the to as the of by clarify of DAG is for the blockage of spontaneous integration, we examined of DAG or DAG have the to block spontaneous integration. In the of the of acyl chains in DAG was The of DAG used are shown in DAG with or acyl chains the integration as DAG for both M13 procoat and 3L-Pf3 coat In DAG with or acyl chains blocked spontaneous integration and When the integration were as a of the of acyl chains it that the acyl of is a for of spontaneous integration. is that DAG in vivo is of Microbiol. Rev. PubMed Google Scholar). another form of DAG, was We found that form of DAG was as effective for the blockage of spontaneous integration as physiological DAG and that the acyl to can be at the or In the in we used DAG with acyl we examined DAG with acyl Although the of for and is and than that for DAG was to block the spontaneous integration of both M13 procoat and 3L-Pf3 coat and suggesting that the is not for the blockage of spontaneous integration. a DAG with a was also was found that still spontaneous integration of both M13 procoat and 3L-Pf3 coat when with liposomes DAG and phosphatidic is not a lipid for of spontaneous integration. In these we used liposomes by the of which is F. D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). To the of the structure the of spontaneous integration, we liposomes by of and the spontaneous integration of 3L-Pf3 coat with those in the integration of 3L-Pf3 coat was the the and liposomes in the and absence of DAG, strongly suggesting that the blockage of spontaneous integration by DAG was not caused by changes in the membrane curvature or the In to block spontaneous integration, the of DAG is as the to block spontaneous integration. However, the bulky structure of DAG is rather than the as for The in the of membrane curvature not to the blockage of spontaneous integration by DAG. of DAG in E. DAG of E. coli has been reported to be that of phospholipids J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). We the of the DAG in a lipid to However, it to even suggesting that both DAG and phospholipids could not be these we by with We used to as DAG is by the PubMed Scopus Google Scholar). The DAG of was to be which when in liposomes a on the blockage of spontaneous integration of the DAG in DAG of E. coli was and as a to which were the of independent are also were in the of The DAG of E. coli was and as a to which were the of independent are also The were in the of in a Several are to DAG. The are C.M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), J. PubMed Google Scholar, W. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar), and J. PubMed Google Scholar, J. PubMed Google Scholar, L. W. J. PubMed Google Scholar). To the of DAG in we these in However, the and DAG at a similar as the DAG at a similar The a DAG which at the of DAG. In the the DAG therefore, was as reported J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the DAG of the that the When was in or in the the DAG was not at that the DAG is strictly in the the of DAG in of DAG and the of 3L-Pf3 that the blockage of spontaneous integration by DAG in studies is operative in native we reconstituted of DAG by the were with and to the that is in E. coli A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). The were with followed by with was that DAG be to the membrane proteins were reconstituted When or were used as the spontaneous integration of 3L-Pf3 coat into the blocked of the that these also YidC YidC is not by the A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). In when were with 3L-Pf3 coat integration Although the integration was still was dependent both on and on of that DAG depletion in spontaneous integration. The is to the that by has a to block spontaneous integration on the in 3L-Pf3 coat was found not to be spontaneously integrated into both liposomes and native proteoliposomes, we examined 3L-Pf3 coat integration on the lipid A-derived as M13 procoat integration A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). DAG and the integration M13 procoat as reported A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). were also as to 3L-Pf3 coat integration that the potential-independent membrane protein is also integrated in integration it is that 3L-Pf3 coat integration not occur spontaneously on the integration factor. was found to be a more effective lipid for the blockage of spontaneous integration than DAG and we examined the of on integration integration of membrane proteins were not integrated into even the integration been The of the in both of were the not strongly that liposomes membrane that not blocks spontaneous integration also the integration integration, has a to block spontaneous integration. integration into liposomes has long been thought to occur to be of the protein integration into of M13 procoat and Pf3 coat (7Geller B.L. Wickner W. J. Biol. Chem. 1985; 260: 13281-13285Abstract Full Text PDF PubMed Google Scholar, 8Kiefer D. Kuhn A. EMBO J. 1999; 18: 6299-6306Crossref PubMed Scopus (85) Google Scholar). the of YidC, M13 procoat was not to be spontaneously as YidC depletion caused a in M13 procoat integration (9Samuelson J.C. Chen M. Jiang F. Moller I. Wiedmann M. Kuhn A. Phillips G.J. Dalbey R.E. Nature. 2000; 406: 637-641Crossref PubMed Scopus (414) Google Scholar). Moreover, it has been reported that the integration of Pf3 coat into liposomes is by YidC (10Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar). However, potential-independent 3L-Pf3 coat, has been shown to be still integrated into liposomes, suggesting a spontaneous of integration (10Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar). We demonstrated that even the spontaneous integration of 3L-Pf3 coat is blocked by a physiological DAG. To clarify the mechanism the blockage of spontaneous integration, we examined the of on the integration The of acyl chains was and should be or for the blockage. Moreover, the of a to DAG spontaneous integration to the the of DAG to be for the blockage. However, which more than was to block spontaneous integration, as were and that a is not for the blockage. acyl chains of a should be to the it is that the bulky structure of DAG is essential for the blockage. On the of liposomes, the of phospholipids are to be to allow the of the The structure with these be therefore, a spontaneous integration of membrane proteins. The bulky structure of DAG be to such in the lipid of liposomes to the compounds such as and DAG with acyl chains not form a bulky structure to the is with the that was more than DAG for the blockage of the spontaneous integration. However, liposomes could not the lipid A-derived suggesting that the membrane was when is in the liposomes. also E. coli DAG not J. Biol. Chem. Full Text PDF PubMed Google Scholar). is that DAG the of the membrane R. PubMed Scopus Google Scholar). However, liposomes the of DAG on the of spontaneous integration as the and it is not a in membrane curvature therefore, in the which the blockage of spontaneous integration by DAG. In the of DAG, when spontaneous integration the integration of the 3L-Pf3 coat, was found to on the lipid A-derived factor. Moreover, reconstituted native which YidC not the integration even for 3L-Pf3 YidC was for integration. has been reported that YidC depletion a of (3van der Laan M. Urbanus M.L. Ten Hagen-Jongman C.M. Nouwen N. Oudega B. Harms N. Driessen A.J. Luirink J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5801-5806Crossref PubMed Scopus (124) Google Scholar). Indeed, were in the generation of not which should the of integration of potential-dependent On the other hand, it has been reported that the integration of potential-independent version of M13 procoat and Pf3 coat is also by YidC depletion M. Samuelson J.C. Jiang F. Muller M. Kuhn A. Dalbey R.E. J. Biol. Chem. 2002; 277: 7670-7675Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, J.C. Jiang F. L. Chen M. de Gier J.W. Kuhn A. Dalbey R.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). integration on the of the of integration are to be those of integration, such as the of integration, not be M13 procoat is resistant to alkaline even YidC depletion J.C. Jiang F. L. Chen M. de Gier J.W. Kuhn A. Dalbey R.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that the of integration not the of in the of integration, YidC was not involved in the integration, as by the of a A. M. E. Muller M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar), the integration of chains of can be efficiently with YidC G. D. Dalbey R.E. J. Muller M. EMBO PubMed Scopus Google Scholar). has been that YidC as a membrane for proteins as Pf3 coat integration was found to be by However, those experimental conditions spontaneous integration into liposomes of YidC could still have (10Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar) to the of DAG. Moreover, 3L-Pf3 coat was efficiently integrated independently of YidC (10Serek J. Bauer-Manz G. Struhalla G. van den Berg L. Kiefer D. Dalbey R. Kuhn A. EMBO J. 2004; 23: 294-301Crossref PubMed Scopus (166) Google Scholar). we that 3L-Pf3 coat is not integrated into liposomes when spontaneous integration is blocked by DAG and that of membrane integration the of a lipid A-derived integration factor. that is as a membrane protein The DAG in vivo to be strictly as the of of and and did not in DAG it has been that DAG by the which is involved in of the is a in E. coli J. Biol. Chem. Full Text PDF PubMed Google Scholar). Moreover, the DAG which blockage of spontaneous integration, was similar to that of Indeed, in native proteoliposomes, in which DAG was to by with and spontaneous integration was that the blockage of spontaneous integration by DAG also in native that such as also have to spontaneous integration that is it is that DAG other than the precursor of We are to Kuhn of for the of the We of for