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• W2149026054 abstract "To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, non-bilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane. To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, non-bilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane. phosphatidylethanolamine phosphatidylglycerol phosphatidylserine cardiolipin 1,2-dioleoyl-sn-glycero-3-phosphocholine N-methyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine N,N-dimethyl-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine 1,2-dimyristoyl-oleoyl-sn-glycero-3-phosphoethanolamine 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] 1,2-dioleoyl-sn-glycero-3-phosphoglycerol dioleoylglycerol n-octyl β-glucopyranoside dodecyl maltoside inner membrane vesicles polyacrylamide gel electrophoresis Complementary genetic and biochemical approaches have shown that the translocation of proteins across the inner membrane ofEscherichia coli is mediated by the translocase (for reviews see Refs. 1.Wickner W. Leonard M.R. J. Biol. Chem. 1996; 271: 29514-29516Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 2.Danese P.N. Silhavy T.J. Annu. Rev. Genet. 1998; 32: 59-94Crossref PubMed Scopus (187) Google Scholar, 3.Driessen A.J.M. Fekkes P. van der Wolk J.P.W. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (147) Google Scholar). The essential subunits of the translocase are the dissociable peripheral ATPase termed SecA and integral membrane proteins SecY and SecE (4.Akimaru J. Matsuyama S. Tokuda H. Mizushima S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6545-6549Crossref PubMed Scopus (170) Google Scholar, 5.Brundage L. Hendrick J.P. Schiebel E. Driessen A.J.M. Wickner W. Cell. 1990; 62: 649-657Abstract Full Text PDF PubMed Scopus (383) Google Scholar). The SecYE complex forms a preprotein-conducting channel that may associate with SecG or the heterotrimeric SecDFYajC complex (6.Duong F. Wickner W. EMBO J. 1997; 16: 2756-2768Crossref PubMed Scopus (227) Google Scholar). SecA binds with high affinity to the SecYEG complex and functions as a molecular motor that utilizes the binding and hydrolysis of ATP to drive the stepwise translocation of a preprotein across the membrane (7.van der Wolk J.P. de Wit J.G. Driessen A.J.M. EMBO J. 1997; 16: 7297-7304Crossref PubMed Scopus (159) Google Scholar). In addition, the proton motive force accelerates the translocation reaction (8.Driessen A.J.M. EMBO J. 1992; 11: 847-853Crossref PubMed Scopus (94) Google Scholar).Phospholipids have been shown to play an important role in protein translocation. The E. coli inner membrane mostly consists of the zwitterionic phospholipid phosphatidylethanolamine (PE,1 70–75%) and two anionic phospholipids, phosphatidylglycerol (PG, 20–25%) and cardiolipin (CL, 5–10%). The membrane lipid composition is normally tightly regulated, but by the use of strains engineered in the expression of critical phospholipid biosynthetic enzymes, the in vivo manipulation of the bulk phospholipid composition has been achieved (9.Dowhan W. Annu. Rev. Biochem. 1997; 66: 199-232Crossref PubMed Scopus (772) Google Scholar). The major anionic phospholipids PG and CL can be depleted in a strain in which the expression of the pgsA gene encoding the phosphatidylglycerophosphate synthase is controlled. At low PG and CL content (2–3%), protein translocation is severely compromised, whereas lack of only CL did not affect translocation (10.de Vrije T. de Swart R.L. Dowhan W. Tommassen J. de Kruijff B. Nature. 1988; 334: 173-175Crossref PubMed Scopus (204) Google Scholar). Only the negative charge of the polar lipid head group is important as many other anionic phospholipids are able to restore the protein translocation activity of PG and CL-depleted inner membrane vesicles (IMVs) or in reconstituted proteoliposomes (4.Akimaru J. Matsuyama S. Tokuda H. Mizushima S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6545-6549Crossref PubMed Scopus (170) Google Scholar, 11.Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar, 12.Tokuda H. Shiozuka K. Mizushima S. Eur. J. Biochem. 1990; 192: 583-589Crossref PubMed Scopus (28) Google Scholar). The efficiency of protein translocation is directly proportional to the amount of anionic phospholipids (13.Kusters R. Dowhan W. de Kruijff B. J. Biol. Chem. 1991; 266: 8659-8662Abstract Full Text PDF PubMed Google Scholar). Anionic phospholipids influence various steps in the preprotein translocation cascade as follows. (i) They promote the interaction of SecA with the membrane surface (14.Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 15.Ulbrandt N.D. London E. Oliver D.B. J. Biol. Chem. 1992; 267: 15184-15192Abstract Full Text PDF PubMed Google Scholar, 16.Breukink E. Demel R.A. de Korte-Kool G. de Kruijff B. Biochemistry. 1992; 31: 1119-1124Crossref PubMed Scopus (156) Google Scholar) and SecYEG (11.Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar) and are needed for the SecA translocation ATPase activity,i.e. the preprotein-stimulated ATPase activity of the SecYEG-bound SecA (12.Tokuda H. Shiozuka K. Mizushima S. Eur. J. Biochem. 1990; 192: 583-589Crossref PubMed Scopus (28) Google Scholar, 14.Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar). At low levels of anionic phospholipids, excess SecA can compensate for the reduced translocation activity (17.Kusters R. Huijbregts R. de Kruijff B. FEBS Lett. 1992; 308: 97-100Crossref PubMed Scopus (16) Google Scholar), suggesting a role of these lipids in the targeting of preproteins and SecA to the membrane. In addition, the endogenous SecA ATPase activity at low Mg2+ concentration is stimulated by the presence of anionic phospholipids, an activity termed SecA lipid ATPase (14.Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar). (ii) Membrane insertion of the positively charged signal sequence of a preprotein is dependent on anionic phospholipids (18.Keller R.C. Killian J.A. de Kruijff B. Biochemistry. 1992; 31: 1672-1677Crossref PubMed Scopus (85) Google Scholar, 19.Keller R.C. ten Berge D. Nouwen N. Snel M.M. Tommassen J. Marsh D. de Kruijff B. Biochemistry. 1996; 35: 3063-3071Crossref PubMed Scopus (39) Google Scholar, 20.Leenhouts J.M. van den Wijngaard P.W. de Kroon A.I. de Kruijff B. FEBS Lett. 1995; 370: 189-192Crossref PubMed Scopus (29) Google Scholar, 21.van Raalte A.L. Demel R.A. Verberkmoes G. Breukink E. Keller R.C. de Kruijff B. Eur. J. Biochem. 1996; 235: 207-214Crossref PubMed Scopus (8) Google Scholar, 22.Phoenix D.A. Kusters R. Hikita C. Mizushima S. de Kruijff B. J. Biol. Chem. 1993; 268: 17069-17073Abstract Full Text PDF PubMed Google Scholar). (iii) Anionic phospholipids stabilize the SecYEG complex during octyl glucoside solubilization (12.Tokuda H. Shiozuka K. Mizushima S. Eur. J. Biochem. 1990; 192: 583-589Crossref PubMed Scopus (28) Google Scholar, 23.Driessen A.J.M. Wickner W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3107-3111Crossref PubMed Scopus (34) Google Scholar) and (iv) influence the acquisition of the correct topology of membrane proteins (24.van Klompenburg W. Nilsson I. von Heijne G. de Kruijff B. EMBO J. 1997; 16: 4261-4266Crossref PubMed Scopus (172) Google Scholar).The cold-sensitive growth defect of a secG null strain (25.Kontinen V.P. Tokuda H. FEBS Lett. 1995; 364: 157-160Crossref PubMed Scopus (25) Google Scholar, 26.Shimizu H. Nishiyama K. Tokuda H. Mol. Microbiol. 1997; 26: 1013-1021Crossref PubMed Scopus (44) Google Scholar, 27.Kontinen V.P. Helander I.M. Tokuda H. FEBS Lett. 1996; 389: 281-284Crossref PubMed Scopus (10) Google Scholar, 28.Suzuki H. Nishiyama K. Tokuda H. J. Biol. Chem. 1999; 274: 31020-31024Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) can be suppressed by various gene products involved in phospholipid biosynthesis along with the pgsA gene. Likewise, the cold-sensitive growth defect of the secAcsR11mutant strain is suppressed by overproduction of the PgsA protein (28.Suzuki H. Nishiyama K. Tokuda H. J. Biol. Chem. 1999; 274: 31020-31024Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). These effects have been attributed to an increase in the anionic phospholipid content that restores the secretion defect of these strains by facilitating the SecA catalytic cycle at low temperature (28.Suzuki H. Nishiyama K. Tokuda H. J. Biol. Chem. 1999; 274: 31020-31024Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The exact mechanism of this activation, however, remains obscure as studies with photoreactive phospholipid analogues suggest that the SecYEG-bound, membrane-inserted form of SecA is not in contact with phospholipids (29.van Voorst F. van der Does C. Brunner J. Driessen A.J.M. de Kruijff B. Biochemistry. 1998; 37: 12261-12268Crossref PubMed Scopus (35) Google Scholar, 30.Eichler J. Brunner J. Wickner W. EMBO J. 1997; 16: 2188-2196Crossref PubMed Scopus (57) Google Scholar).The other main phospholipid of the E. coli inner membrane is the type II lipid PE. PE adopts a non-bilayer hexagonal II phase conformation (31.Ellens H. Bentz J. Szoka F.C. Biochemistry. 1986; 25: 4141-4147Crossref PubMed Scopus (145) Google Scholar). Deletion of the pssA gene (32.DeChavigny A. Heacock P.N. Dowhan W. J. Biol. Chem. 1991; 266: 5323-5332Abstract Full Text PDF PubMed Google Scholar), which encodes the phosphatidylserine (PS) synthase, renders cells devoid of the amino-based phospholipids PS and PE. This results in severe pleiotropic effects on membrane protein function and the inactivation of protein translocation (33.Rietveld A.G. Killian J.A. Dowhan W. de Kruijff B. J. Biol. Chem. 1993; 268: 12427-12433Abstract Full Text PDF PubMed Google Scholar, 34.Rietveld A.G. Chupin V.V. Koorengevel M.C. Wienk H.L. Dowhan W. de Kruijff B. J. Biol. Chem. 1994; 269: 28670-28675Abstract Full Text PDF PubMed Google Scholar, 35.Rietveld A.G. Koorengevel M.C. de Kruijff B. EMBO J. 1995; 14: 5506-5513Crossref PubMed Scopus (140) Google Scholar). For growth, this strain is dependent on the presence of a high concentration of divalent cations (Ca2+, Mg2+, or Sr2+) (32.DeChavigny A. Heacock P.N. Dowhan W. J. Biol. Chem. 1991; 266: 5323-5332Abstract Full Text PDF PubMed Google Scholar, 35.Rietveld A.G. Koorengevel M.C. de Kruijff B. EMBO J. 1995; 14: 5506-5513Crossref PubMed Scopus (140) Google Scholar). These cations stimulate the bilayer to non-bilayer transition of CL, which comprises 44% of the total phospholipid in this strain (36.Rand R.P. Sengupta S. Biochim. Biophys. Acta. 1972; 255: 484-492Crossref PubMed Scopus (264) Google Scholar). It therefore appears that the polymorphic behavior of these lipids is essential for growth, a role normally fulfilled by PE. The requirement for non-bilayer lipids for protein translocation appears less strict than for anionic lipids (35.Rietveld A.G. Koorengevel M.C. de Kruijff B. EMBO J. 1995; 14: 5506-5513Crossref PubMed Scopus (140) Google Scholar), but the mechanism by which these lipids act on protein translocation is unknown. Non-bilayer lipids only marginally affect the SecA lipid ATPase activity (37.Ahn T. Kim H. J. Biol. Chem. 1998; 273: 21692-21698Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and appear not essential for the functional reconstitution of protein translocation using octyl glucoside (OG)-purified SecYEG complex (11.Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar).The reconstitution of preprotein translocation with only a limited set of purified Sec proteins provides a unique opportunity to assess systematically the lipid requirement for preprotein translocation. We now report on a method that allows purification of the SecYEG complex in a delipidated state. The activity of the delipidated SecYEG can be restored after reconstitution into liposomes with a defined phospholipid composition. The data with the purified E. coliSecYEG complex not only confirm the hypothesis that anionic lipids are essential for activity but also show that non-bilayer lipids stimulate the activity of the reconstituted translocase. Strikingly, with theBacillus subtilis SecYEG both lipid classes are essential for activity. Overall, optimal activity is observed when anionic and non-bilayer lipids are present at a concentration that matches that of the natural membrane.DISCUSSIONThe E. coli preprotein translocase depends for its activity on specific classes of phospholipids. Previous in vivo and in vitro studies have shown that anionic phospholipids are essential for activity, whereas non-bilayer lipids are stimulatory (for review see Ref. 51.Brundage L. Fimmel C.J. Mizushima S. Wickner W. J. Biol. Chem. 1992; 267: 4166-4170Abstract Full Text PDF PubMed Google Scholar). A systematic study of the phospholipid requirement of the purified translocase has not been reported despite the fact that the lipid composition of the reconstituted proteoliposomes can be manipulated in a convenient and systematic manner. We now show that functional reconstitution of the purified, delipidated E. coli SecYEG requires anionic phospholipids, whereas non-bilayer lipids stimulate translocation. These results confirm the studies performed in the crude membrane system but, in addition, extend these observations to the B. subtilis SecYEG. Remarkably, the activity of the B. subtilis SecYEG strictly depends on the presence of non-bilayer lipids. For optimal activity, anionic and non-bilayer lipids need to present at amounts corresponding to the phospholipid composition of the native inner membrane.In order to analyze the phospholipid requirement of the purified translocase, it is desirable to first delipidate the enzyme and subsequently restore its activity by reconstitution in liposomes with a defined phospholipid composition. The SecYEG complex has been purified from IMVs after solubilization with OG. However, to obtain a functional SecYEG complex it is necessary to include phospholipids in the buffers used during the purification (Refs. 5.Brundage L. Hendrick J.P. Schiebel E. Driessen A.J.M. Wickner W. Cell. 1990; 62: 649-657Abstract Full Text PDF PubMed Scopus (383) Google Scholar, 12.Tokuda H. Shiozuka K. Mizushima S. Eur. J. Biochem. 1990; 192: 583-589Crossref PubMed Scopus (28) Google Scholar, and 23.Driessen A.J.M. Wickner W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3107-3111Crossref PubMed Scopus (34) Google Scholar and this study). Delipidation of OG-purified SecYEG leads to the irreversible inactivation of the enzyme (Table I). We now show that SecYEG can be purified in a delipidated and functional form when DDM is used as a detergent instead of OG. OG is a detergent with a rather short acyl chain of only eight carbon moieties. The acyl chain of DDM is longer and thus may more closely resemble the interaction of the phospholipid acyl chain with the SecYEG complex present in detergent micelles. In this respect, it was previously noted that OG-solubilized SecYEG is thermolabile (39.van der Does C. Manting E.H. Kaufmann A. Lutz M. Driessen A.J.M. Biochemistry. 1998; 37: 201-210Crossref PubMed Scopus (94) Google Scholar, 51.Brundage L. Fimmel C.J. Mizushima S. Wickner W. J. Biol. Chem. 1992; 267: 4166-4170Abstract Full Text PDF PubMed Google Scholar, 52.Duong F. Wickner W. EMBO J. 1999; 18: 3263-3270Crossref PubMed Scopus (63) Google Scholar). DDM-purified SecYEG complex is less susceptible to such thermal inactivation. 2A. Veenendaal, manuscript in preparation. The DDM-purified SecYEG complex, like the OG-purified, lipid-supplemented SecYEG complex (39.van der Does C. Manting E.H. Kaufmann A. Lutz M. Driessen A.J.M. Biochemistry. 1998; 37: 201-210Crossref PubMed Scopus (94) Google Scholar), supports the high affinity binding of SecA, the nucleotide-induced conformational states of SecA, and the endogenous SecA ATPase activity. 3C. van der Does, J. Swaving, W. van Klompenburg, and A. J. M. Driessen, unpublished results. However, it has not been possible to detect SecA translocation ATPase activity with the detergent-solubilized translocase even though the enzyme is stable at the temperatures that support this activity in reconstituted liposomes. Electron microscopic studies on the B. subtilis SecYE complex indicate the presence of oligomeric forms that may constitute the preprotein conducting channel (53.Meyer T.H. Ménétret J.F. Breitling R. Miller K.R. Akey C.W. Rapoport T.A. J. Mol. Biol. 1999; 285: 1789-1800Crossref PubMed Scopus (125) Google Scholar). This oligomeric form may be a stable state of the SecYE complex, but alternatively, the SecYE oligomer may be a dynamic entity, disassembling and assembling in response to the demand for translocation. When reconstituted into a lipid membrane, the kinetics of such an assembly event will largely be dictated by the lateral protein diffusion rate. In detergent micelles, however, assembly will be limited by the rate of collision within the three-dimensional space and/or the ability of the micelles to fuse. These events may be effective only when the enzyme is present at a very high concentration. Strikingly, preprotein translocation in detergent solution has been reported for the Sec61p isolated from the endoplasmic membrane of yeast (54.Matlack K.E. Plath K. Misselwitz B. Rapoport T.A. Science. 1997; 277: 938-941Crossref PubMed Scopus (63) Google Scholar) but is observed only at a very high Sec61p concentration.Although the purification method in the absence of phospholipids was developed to be able to examine the effects of small quantities of phospholipids on the activity of the translocase, the data show that high concentrations of anionic and non-bilayer lipids are needed to saturate the translocation activity. It thus appears that phospholipids act on protein translocation in a more global sense. The E. coli SecYEG complex is maximally active in a mixture of 30% DOPG and 70% DOPE. As far as the ratio between anionic and non-bilayer lipids concerns, this mixture “more or less” corresponds to the lipid composition of the E. coli inner membrane. The activity in the optimal synthetic mixture is about 75% of that found with natural E. coli phospholipids, showing that substantial activity can be recovered with the defined system. ReconstitutedB. subtilis SecYEG complex is maximally active in 70% DOPG and 30% DOPE, i.e. at a ratio that closely matches that of anionic to non-bilayer lipids in the native B. subtilisinner membrane. It is remarkable that both systems differ in their quantitative lipid requirement and are most active at their physiological lipid conditions. In this respect, the lipid requirement of the E. coli SecYEG complex did not change significantly when pre-PhoB translocation was assayed3 instead of pro-OmpA.Anionic phospholipids have been shown to fulfill at least a dual role,i.e. they promote SecA membrane binding and insertion and stimulate the interaction of the signal sequence of preproteins and the membrane (50.van Klompenburg W. de Kruijff B. J. Membr. Biol. 1998; 162: 1-7Crossref PubMed Scopus (43) Google Scholar). These lipids may indirectly stimulate targeting of SecA to the SecYEG complex, for instance by promoting the low affinity membrane binding of SecA. This may result in a membrane-bound pool of SecA protein that could have a kinetic advantage relative to the cytosolic pool to associate with SecYEG complexes that have completed a translocation reaction. Experimental evidence for such mechanism is, however, difficult to obtain. Another possible role for anionic phospholipids may be found in the putative assembly of the SecYEG complex into larger functional oligomers. The collective cold-sensitive secretion defect of the secG null and secAcsR11strain (28.Suzuki H. Nishiyama K. Tokuda H. J. Biol. Chem. 1999; 274: 31020-31024Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and many other Sec mutants may be found in a compromised channel assembly activity.The reconstitution studies with the purified SecYEG complex provide compelling evidence that non-bilayer lipids stimulate translocation. The activating effect of DOPE can be mimicked with dioleoylglycerol, a lipid that like DOPE adopts a non-bilayer conformation, whereas bilayer-forming PE derivatives fail to stimulate translocation. These data provide strong evidence that the lipid shape is the major factor for activation rather than the amino group of PE. In contrast to theE. coli SecYEG, the activity of the B. subtilisSecYEG strictly required the presence of non-bilayer lipids. In contrast, the amino group of PE is needed for the functional reconstitution of the lactose permease of E. coli (55.Chen C.C. Wilson T.H. J. Biol. Chem. 1984; 259: 10150-10158Abstract Full Text PDF PubMed Google Scholar, 56.Seto-Young D. Chen C.C. Wilson T.H. J. Membr. Biol. 1985; 84: 259-267Crossref PubMed Scopus (32) Google Scholar) and leucine permease of Lactococcus lactis (57.Driessen A.J.M. Zheng T. In't Veld G. Op den Kamp J.A.F. Konings W.N. Biochemistry. 1988; 27: 865-872Crossref PubMed Scopus (47) Google Scholar). PE stimulates the folding of the lactose permease into its active conformation (58.Bogdanov M. Umeda M. Dowhan W. J. Biol. Chem. 1999; 274: 12339-12345Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 59.Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Likewise, non-bilayer lipids may be needed for folding and/or assembly of the SecYEG complex.Our data suggest that the absolute amounts of bilayer (or anionic) and non-bilayer lipids are essential for optimal activity. Since these lipids are needed at high concentration, it seems that the protein translocation activity is determined by the collective, physical properties of the membrane. The requirement for non-bilayer lipids could relate to their effect on the lateral membrane pressure and/or optimal matching of the protein-lipid interface and thus affect the conformation of the active translocase. Complementary genetic and biochemical approaches have shown that the translocation of proteins across the inner membrane ofEscherichia coli is mediated by the translocase (for reviews see Refs. 1.Wickner W. Leonard M.R. J. Biol. Chem. 1996; 271: 29514-29516Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 2.Danese P.N. Silhavy T.J. Annu. Rev. Genet. 1998; 32: 59-94Crossref PubMed Scopus (187) Google Scholar, 3.Driessen A.J.M. Fekkes P. van der Wolk J.P.W. Curr. Opin. Microbiol. 1998; 1: 216-222Crossref PubMed Scopus (147) Google Scholar). The essential subunits of the translocase are the dissociable peripheral ATPase termed SecA and integral membrane proteins SecY and SecE (4.Akimaru J. Matsuyama S. Tokuda H. Mizushima S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6545-6549Crossref PubMed Scopus (170) Google Scholar, 5.Brundage L. Hendrick J.P. Schiebel E. Driessen A.J.M. Wickner W. Cell. 1990; 62: 649-657Abstract Full Text PDF PubMed Scopus (383) Google Scholar). The SecYE complex forms a preprotein-conducting channel that may associate with SecG or the heterotrimeric SecDFYajC complex (6.Duong F. Wickner W. EMBO J. 1997; 16: 2756-2768Crossref PubMed Scopus (227) Google Scholar). SecA binds with high affinity to the SecYEG complex and functions as a molecular motor that utilizes the binding and hydrolysis of ATP to drive the stepwise translocation of a preprotein across the membrane (7.van der Wolk J.P. de Wit J.G. Driessen A.J.M. EMBO J. 1997; 16: 7297-7304Crossref PubMed Scopus (159) Google Scholar). In addition, the proton motive force accelerates the translocation reaction (8.Driessen A.J.M. EMBO J. 1992; 11: 847-853Crossref PubMed Scopus (94) Google Scholar). Phospholipids have been shown to play an important role in protein translocation. The E. coli inner membrane mostly consists of the zwitterionic phospholipid phosphatidylethanolamine (PE,1 70–75%) and two anionic phospholipids, phosphatidylglycerol (PG, 20–25%) and cardiolipin (CL, 5–10%). The membrane lipid composition is normally tightly regulated, but by the use of strains engineered in the expression of critical phospholipid biosynthetic enzymes, the in vivo manipulation of the bulk phospholipid composition has been achieved (9.Dowhan W. Annu. Rev. Biochem. 1997; 66: 199-232Crossref PubMed Scopus (772) Google Scholar). The major anionic phospholipids PG and CL can be depleted in a strain in which the expression of the pgsA gene encoding the phosphatidylglycerophosphate synthase is controlled. At low PG and CL content (2–3%), protein translocation is severely compromised, whereas lack of only CL did not affect translocation (10.de Vrije T. de Swart R.L. Dowhan W. Tommassen J. de Kruijff B. Nature. 1988; 334: 173-175Crossref PubMed Scopus (204) Google Scholar). Only the negative charge of the polar lipid head group is important as many other anionic phospholipids are able to restore the protein translocation activity of PG and CL-depleted inner membrane vesicles (IMVs) or in reconstituted proteoliposomes (4.Akimaru J. Matsuyama S. Tokuda H. Mizushima S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6545-6549Crossref PubMed Scopus (170) Google Scholar, 11.Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar, 12.Tokuda H. Shiozuka K. Mizushima S. Eur. J. Biochem. 1990; 192: 583-589Crossref PubMed Scopus (28) Google Scholar). The efficiency of protein translocation is directly proportional to the amount of anionic phospholipids (13.Kusters R. Dowhan W. de Kruijff B. J. Biol. Chem. 1991; 266: 8659-8662Abstract Full Text PDF PubMed Google Scholar). Anionic phospholipids influence various steps in the preprotein translocation cascade as follows. (i) They promote the interaction of SecA with the membrane surface (14.Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 15.Ulbrandt N.D. London E. Oliver D.B. J. Biol. Chem. 1992; 267: 15184-15192Abstract Full Text PDF PubMed Google Scholar, 16.Breukink E. Demel R.A. de Korte-Kool G. de Kruijff B. Biochemistry. 1992; 31: 1119-1124Crossref PubMed Scopus (156) Google Scholar) and SecYEG (11.Hendrick J.P. Wickner W. J. Biol. Chem. 1991; 266: 24596-24600Abstract Full Text PDF PubMed Google Scholar) and are needed for the SecA translocation ATPase activity,i.e. the preprotein-stimulated ATPase activity of the SecYEG-bound SecA (12.Tokuda H. Shiozuka K. Mizushima S. Eur. J. Biochem. 1990; 192: 583-589Crossref PubMed Scopus (28) Google Scholar, 14.Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar). At low levels of anionic phospholipids, excess SecA can compensate for the reduced translocation activity (17.Kusters R. Huijbregts R. de Kruijff B. FEBS Lett. 1992; 308: 97-100Crossref PubMed Scopus (16) Google Scholar), suggesting a role of these lipids in the targeting of preproteins and SecA to the membrane. In addition, the endogenous SecA ATPase activity at low Mg2+ concentration is stimulated by the presence of anionic phospholipids, an activity termed SecA lipid ATPase (14.Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (461) Google Scholar). (ii) Membrane insertion of the positively charged signal sequence of a preprotein is dependent on anionic phospholipids (18.Keller R.C. Killian J.A. de Kruijff B. Biochemistry. 1992; 31: 1672-1677Crossref PubMed Scopus (85) Google Scholar, 19.Keller R.C. ten Berge D. Nouwen N. Snel M.M. Tommassen J. Marsh D. de Kruijff B. Biochemistry. 1996; 35: 3063-3071Crossref PubMed Scopus (39) Google Scholar, 20.Leenhouts J.M. van den Wijngaard P.W. de Kroon" @default.
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• W2149026054 cites W1480251791 @default.
• W2149026054 cites W1495024113 @default.
• W2149026054 cites W1536139652 @default.
• W2149026054 cites W1548309530 @default.
• W2149026054 cites W1555008776 @default.
• W2149026054 cites W1567962789 @default.
• W2149026054 cites W1572059244 @default.
• W2149026054 cites W1590719173 @default.
• W2149026054 cites W1602075505 @default.
• W2149026054 cites W1602373369 @default.
• W2149026054 cites W1707474365 @default.
• W2149026054 cites W180358250 @default.
• W2149026054 cites W1946720111 @default.
• W2149026054 cites W1965731203 @default.
• W2149026054 cites W1965918112 @default.
• W2149026054 cites W1968143836 @default.
• W2149026054 cites W1969414258 @default.
• W2149026054 cites W1977262269 @default.
• W2149026054 cites W1978467616 @default.
• W2149026054 cites W1986756715 @default.
• W2149026054 cites W2002707099 @default.
• W2149026054 cites W2003709444 @default.
• W2149026054 cites W2006921115 @default.
• W2149026054 cites W2008223866 @default.
• W2149026054 cites W2008415333 @default.
• W2149026054 cites W2009507746 @default.
• W2149026054 cites W2016606940 @default.
• W2149026054 cites W2021911762 @default.
• W2149026054 cites W2026811970 @default.
• W2149026054 cites W2027161586 @default.
• W2149026054 cites W2035172570 @default.
• W2149026054 cites W2035595494 @default.
• W2149026054 cites W2041651125 @default.
• W2149026054 cites W2043542175 @default.
• W2149026054 cites W2043841819 @default.
• W2149026054 cites W2045250199 @default.
• W2149026054 cites W2048052250 @default.
• W2149026054 cites W2048737734 @default.
• W2149026054 cites W2049701913 @default.
• W2149026054 cites W2056891601 @default.
• W2149026054 cites W2063360444 @default.
• W2149026054 cites W2067410749 @default.
• W2149026054 cites W2070336844 @default.
• W2149026054 cites W2077723148 @default.
• W2149026054 cites W2080304004 @default.
• W2149026054 cites W2083553357 @default.
• W2149026054 cites W2091721805 @default.
• W2149026054 cites W2095056890 @default.
• W2149026054 cites W2095407363 @default.
• W2149026054 cites W2097534440 @default.
• W2149026054 cites W2110945620 @default.
• W2149026054 cites W2110965426 @default.
• W2149026054 cites W2120754295 @default.
• W2149026054 cites W2140636157 @default.
• W2149026054 cites W2152397724 @default.
• W2149026054 cites W2163258998 @default.
• W2149026054 cites W2163862717 @default.
• W2149026054 cites W2169769186 @default.
• W2149026054 cites W2281077886 @default.
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