In Vitro Synthesis of Lactose Permease to Probe the Mechanism of Membrane Insertion and Folding
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m300332200
ISSN1083-351X
AutoresShushi Nagamori, J. Vázquez, Adam B. Weinglass, H. Ronald Kaback,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoInsertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein fromEscherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N6) and probe changes in conformation during insertion into the membrane. Specifically, when N6 remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N6 is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N6 is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N6 is degraded by FtsH protease in vivo, and N6 with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N6 remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N6 and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains. Insertion and folding of polytopic membrane proteins is an important unsolved biological problem. To study this issue, lactose permease, a membrane transport protein fromEscherichia coli, is transcribed, translated, and inserted into inside-out membrane vesicles in vitro. The protein is in a native conformation as judged by sensitivity to protease, binding of a monoclonal antibody directed against a conformational epitope, and importantly, by functional assays. By exploiting this system it is possible to express the N-terminal six helices of the permease (N6) and probe changes in conformation during insertion into the membrane. Specifically, when N6 remains attached to the ribosome it is readily extracted from the membrane with urea, whereas after release from the ribosome or translation of additional helices, those polypeptides are not urea extractable. Furthermore, the accessibility of an engineered Factor Xa site to Xa protease is reduced significantly when N6 is released from the ribosome or more helices are translated. Finally, spontaneous disulfide formation between Cys residues at positions 126 (Helix IV) and 144 (Helix V) is observed when N6 is released from the ribosome and inserted into the membrane. Moreover, in contrast to full-length permease, N6 is degraded by FtsH protease in vivo, and N6 with a single Cys residue at position 148 does not react with N-ethylmaleimide. Taken together, the findings indicate that N6 remains in a hydrophilic environment until it is released from the ribosome or additional helices are translated and continues to fold into a quasi-native conformation after insertion into the bilayer. Furthermore, there is synergism between N6 and the C-terminal half of permease during assembly, as opposed to assembly of the two halves as independent domains. signal recognition particle N-terminal six helices of lactose permease C-terminal six helices of lactose permease lactose permease phosphatidylethanolamine N-ethylmaleimide monoclonal antibody 6′-(N-dansyl)-1-thio-β-d-galactopyranoside inside out right-side out potassium phosphate dodecyl-β-d-maltopyranoside biotin acceptor domain Most inner membrane proteins in Escherichia coli are targeted to the membrane by the signal recognition particle (SRP)1 pathway and insert into the membrane via the Sec machinery (1Herskovits A.A. Bochkareva E.S. Bibi E. Mol. Microbiol. 2000; 38: 927-939Crossref PubMed Scopus (97) Google Scholar, 2Dalbey R.E. Chen M.Y. Jiang F.L. Samuelson J.C. Curr. Opin. Cell Biol. 2000; 12: 435-442Crossref PubMed Scopus (17) Google Scholar, 3de Gier J.W. Luirink J. Mol. Microbiol. 2001; 40: 314-322Crossref PubMed Scopus (82) Google Scholar, 4Bernstein H.D. Curr. Opin. Microbiol. 2000; 3: 203-209Crossref PubMed Scopus (40) Google Scholar). It has been suggested that bacterial SRP (Ffh protein and 4.5 S RNA) binds to the hydrophobic region of nascent membrane proteins, and subsequently, the ribosome-nascent chain complex and SRP interact with the SRP receptor (FtsY) at the membrane surface. Recently, it has been proposed that FtsY is a primary membrane-docking site for the ribosome, and SRP binds to nascent protein after FtsY-ribosome binding (5Herskovits A.A. Shimoni E. Minsky A. Bibi E. J. Cell Biol. 2002; 159: 403-410Crossref PubMed Scopus (57) Google Scholar). The Sec machinery is comprised of SecY, SecE, and SecG proteins in the cytoplasmic membrane (6Driessen A.J.M. Manting E.H. van der Does C. Nat. Struct. Biol. 2001; 8: 492-498Crossref PubMed Scopus (177) Google Scholar, 7Mori H. Ito K. Trends Microbiol. 2001; 9: 494-500Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 8Müller M. Koch H.G. Beck K. Schafer U. Prog. Nucleic Acid Res. Mol. Biol. 2001; 66: 107-157Crossref PubMed Google Scholar, 9Van Voorst F. De Kruijff B. Biochem. J. 2000; 347: 601-612Crossref PubMed Scopus (63) Google Scholar) and contributes to the topology of some membrane proteins (10Prinz W.A. Boyd D.H. Ehrmann M. Beckwith J. J. Biol. Chem. 1998; 273: 8419-8424Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). It has been shown (11Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (298) Google Scholar, 12Samuelson 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 (417) Google Scholar) that the Sec machinery and YidC exist as a complex, and several inner membrane proteins interact with YidC during insertion. Thus, YidC appears to play a key role in inner membrane protein insertion (12Samuelson 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 (417) Google Scholar). SecD, SecF, and YajC form another heterotrimeric complex that binds to the SecYEG complex (13Duong F. Wickner W. EMBO J. 1997; 16: 4871-4879Crossref PubMed Scopus (165) Google Scholar). In addition, it has been reported (14Nouwen N. Driessen A.J.M. Mol. Microbiol. 2002; 44: 1397-1405Crossref PubMed Scopus (134) Google Scholar) that YidC interacts directly with SecDF rather than SecYEG, which forms an even larger complex with SecYEG. Furthermore, SecA, a cytosolic protein with ATPase activity, is also important for insertion of inner membrane proteins with large periplasmic loops (15Andersson H. von Heijne G. EMBO J. 1993; 12: 683-691Crossref PubMed Scopus (125) Google Scholar, 16Koch H.G. Hengelage T. Neumann-Haefelin C. MacFarlane J. Hoffschulte H.K. Schimz K.L. Mechler B. Müller M. Mol. Biol. Cell. 1999; 10: 2163-2173Crossref PubMed Scopus (128) Google Scholar). Although some details regarding the mechanism of integral membrane protein insertion have been elucidated, there are still many issues that require resolution, particularly those related to insertion of polytopic membrane proteins. Examples include folding of intermediates, interaction between helices during translation/insertion, the order and timing of helices exiting from the translocon into the lipid bilayer, and topological determinants.In an effort to study some of these issues we have chosen to use the lactose permease of E. coli (LacY) as a model system. LacY is a member of the Major Facilitator Superfamily (17Saier Jr., M.H. Mol. Microbiol. 2000; 35: 699-710Crossref PubMed Scopus (231) Google Scholar) and catalyzes the coupled stoichiometric translocation of galactosides and H+(symport) (18Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics: Transport Processes in Eukaryotic and Prokaryotic Organisms. II. Elsevier Science Publishers B. V., Amsterdam1996Google Scholar). The protein has been solubilized from the membrane and purified to homogeneity in a completely functional state (19Viitanen P. Garcia M.L. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1629-1633Crossref PubMed Scopus (74) Google Scholar, 20Viitanen P. Newman M.J. Foster D.L. Wilson T.H. Kaback H.R. Methods Enzymol. 1986; 125: 429-452Crossref PubMed Scopus (180) Google Scholar) and is a 12-helix bundle with the N and C termini on the cytoplasmic face of the membrane (21Foster D.L. Boublik M. Kaback H.R. J. Biol. Chem. 1983; 258: 31-34Abstract Full Text PDF PubMed Google Scholar, 22Calamia J. Manoil C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4937-4941Crossref PubMed Scopus (231) Google Scholar, 23Kaback H.R. Wu J. Q. Rev. Biophys. 1997; 30: 333-364Crossref PubMed Scopus (121) Google Scholar). In addition, LacY is physiologically (24Sahin-Tóth M. Lawrence M.C. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5421-5425Crossref PubMed Scopus (137) Google Scholar) and structurally a monomer in the membrane (25Costello M.J. Escaig J. Matsushita K. Viitanen P.V. Menick D.R. Kaback H.R. J. Biol. Chem. 1987; 262: 17072-17082Abstract Full Text PDF PubMed Google Scholar, 26Sun J. Kaback H.R. Biochemistry. 1997; 36: 11959-11965Crossref PubMed Scopus (54) Google Scholar, 27Guan L. Murphy F.D. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3475-3480Crossref PubMed Scopus (47) Google Scholar). Analysis of an extensive library of mutants, particularly Cys replacement mutants (28Frillingos S. Sahin-Tóth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (318) Google Scholar), in conjunction with a battery of site-directed biochemical and biophysical techniques has led to the formulation of a tertiary-structure model (29Sorgen P.L. Hu Y. Guan L. Kaback H.R. Girvin M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14037-14040Crossref PubMed Scopus (82) Google Scholar) as well as a hypothesis for the mechanism of lactose/H+ symport (30Kaback H.R. Sahin-Tóth M. Weinglass A.B. Nat. Rev. Mol. Cell Biol. 2001; 2: 610-622Crossref PubMed Scopus (249) Google Scholar).Like most inner membrane proteins, LacY is inserted into the membrane co-translationally (31Stochaj U. Ehring R. Eur. J. Biochem. 1987; 163: 653-658Crossref PubMed Scopus (11) Google Scholar, 32Ahrem B. Hoffschulte H.K. Müller M. J. Cell Biol. 1989; 108: 1637-1646Crossref PubMed Scopus (31) Google Scholar), and insertion involves SRP and FtsY (33MacFarlane J. Müller M. Eur. J. Biochem. 1995; 233: 766-771Crossref PubMed Scopus (99) Google Scholar,34Seluanov A. Bibi E. J. Biol. Chem. 1997; 272: 2053-2055Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) as well as SecY (35Ito K. Akiyama Y. Mol. Microbiol. 1991; 5: 2243-2253Crossref PubMed Scopus (77) Google Scholar). Phosphatidylethanolamine (PE) is also important for the late maturation of LacY and required for proper assembly and function (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). PE can even reverse misfolding of LacY topology after insertion into membranes devoid of PE (37Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar). However, the mechanism by which LacY is inserted into the membrane and folds into its final tertiary conformation is far from clear.Remarkably, co-expression of LacY in two contiguous, non-overlapping fragments with a covalent discontinuity in either cytoplasmic or periplasmic loops leads to complementation resulting in resistance of the fragments to proteolysis and functional LacY (38Bibi E. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4325-4329Crossref PubMed Scopus (166) Google Scholar, 39Wrubel W. Stochaj U. Sonnewald U. Theres C. Ehring R. J. Bacteriol. 1990; 172: 5374-5381Crossref PubMed Google Scholar, 40Zen K.H. McKenna E. Bibi E. Hardy D. Kaback H.R. Biochemistry. 1994; 33: 8198-8206Crossref PubMed Scopus (80) Google Scholar, 41Sahin-Tóth M. Kaback H.R. Friedlander M. Biochemistry. 1996; 35: 2016-2021Crossref PubMed Scopus (15) Google Scholar). Furthermore, a salt bridge between Asp-237 (Helix VII) and Lys-358 (Helix XI) plays an important role in membrane insertion (42Dunten R.L. Sahin-Tóth M. Kaback H.R. Biochemistry. 1993; 32: 3139-3145Crossref PubMed Scopus (114) Google Scholar, 43Frillingos S. Sahin-Tóth M. Lengeler J.W. Kaback H.R. Biochemistry. 1995; 34: 9368-9373Crossref PubMed Scopus (25) Google Scholar), indicating that Helix VII must interact with Helix XI before insertion of LacY into the bilayer. In addition, the relatively long middle cytoplasmic loop is important for functional expression, possibly acting as a time delay to allow the N-terminal six helices (N6) to clear the translocon before the last six helices are inserted (44Weinglass A.B. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8938-8943Crossref PubMed Scopus (38) Google Scholar). To analyze these phenomena more completely, as well as the insertion mechanism of LacY, an in vitro transcription/translation/insertion system is described in which LacY appears to be inserted in a native, functional conformation. In this study, we focus on insertion of the N-terminal half of LacY (N6).DISCUSSIONAlthough previous studies (32Ahrem B. Hoffschulte H.K. Müller M. J. Cell Biol. 1989; 108: 1637-1646Crossref PubMed Scopus (31) Google Scholar, 64Bochkareva E. Seluanov A. Bibi E. Girshovich A. J. Biol. Chem. 1996; 271: 22256-22261Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) indicate that LacY transcribed and translated in vitro can be inserted into ISO membrane vesicles with the correct topology (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar), questions remain as to whether or not the inserted protein is in a native conformation. Because LacY, like many other membrane proteins, is resistant to traditional means of structural analysis, alternative approaches have been developed to study topology and discern the overall three-dimensional fold (see Refs. 29Sorgen P.L. Hu Y. Guan L. Kaback H.R. Girvin M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14037-14040Crossref PubMed Scopus (82) Google Scholar and 30Kaback H.R. Sahin-Tóth M. Weinglass A.B. Nat. Rev. Mol. Cell Biol. 2001; 2: 610-622Crossref PubMed Scopus (249) Google Scholar). The studies presented here, which utilize some of these approaches, provide convincing evidence that LacY synthesized and inserted into the membrane in vitro is in a native conformation. (a) By using engineered Factor Xa protease sites in cytoplasmic or periplasmic loops, the topology of the polypeptide with respect to the membrane appears to be correct. (b) A mAb that binds to a discontinuous epitope comprised of residues in cytoplasmic loops VIII/IX and X/XI (61Sun J. Li J. Carrasco N. Kaback H.R. Biochemistry. 1997; 36: 274-280Crossref PubMed Scopus (27) Google Scholar) binds to LacY synthesized and inserted in vitro as well as it binds when LacY is synthesized and inserted in vivo. (c) Cys residues at positions 126 and 144 in the in vitro system undergo spontaneous cross-linking as observed in vivo (53Wolin C.D. Kaback H.R. Biochemistry. 2000; 39: 6130-6135Crossref PubMed Scopus (43) Google Scholar). (d) LacY synthesized and inserted in vitroexhibits lactose/Dns6-Gal counter-flow and significant ligand protection against alkylation of Cys-148. Therefore, transcription, translation, and insertion of LacY into ISO vesiclesin vitro represents a system in which the mechanism of insertion of a polytopic membrane protein and its folding into a tertiary conformation can be studied reliably.Despite conjecture regarding co-translational insertion of polytopic membrane proteins, it is unclear how many transmembrane helices are accommodated by the translocon before insertion into the bilayer. Furthermore, little information is available regarding whether folding into a tertiary conformation begins in the translocon or only after migration into the bilayer with the assistance of chaperones such as PE (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar, 37Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar, 70Bogdanov M. Dowhan W. J. Biol. Chem. 1999; 274: 36827-36830Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The observation that the first six helices of LacY are extracted with urea when the ribosome remains attached, whereas the first eight helices are not, suggests that the translocon can accommodate at least six transmembrane helices, a conclusion similar to that drawn from findings obtained with P-glycoprotein (71Borel A.C. Simon S.M. Biochemistry. 1996; 35: 10587-10594Crossref PubMed Scopus (30) Google Scholar). Similarly, an in vitro study of the polytopic membrane protein MtlA suggests more than one pair of transmembrane domains are assembled before the exit from translocon (72Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (136) Google Scholar). On the other hand, the N6 portion of LacY may not be fully accommodated by the translocon, and part of N6 may be in contact with lipid, which would explain why the polypeptide is not completely extracted with urea (Fig. 3).Photo cross-linking studies on certain membrane proteins indicate that the transmembrane domains may indeed interact with lipid during insertion (72Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (136) Google Scholar, 73Urbanus M.L. Scotti P.A. Froderberg L. Saaf A. de Gier J.W. Brunner J. Samuelson J.C. Dalbey R.E. Oudega B. Luirink J. EMBO Rep. 2001; 2: 524-529Crossref PubMed Scopus (153) Google Scholar). With LacY, the first or second transmembrane helix might interact with phospholipid, whereas helices III to VI are still in the translocon. However, if this is the case, it is unlikely that the first two helices migrate very far from the translocon. Indeed, it has been suggested that in some endoplasmic reticulum membrane proteins, a transmembrane helix can re-enter the translocon after its initial release into the lipid phase if it has affinity for other helices still within the translocon (74Mothes W. Heinrich S.U. Graf R. Nilsson I. von Heijne G. Brunner J. Rapoport T.A. Cell. 1997; 89: 523-533Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Taken as a whole, the data suggest that the N6 half of LacY must remain near the translocon after exit into the bilayer as the C6 half is translated. Once the C6 half exits the translocon, folding into a final tertiary structure likely occurs within the bilayer, resulting in a protease-resistant, functional molecule.The differential sensitivity of engineered Factor Xa protease sites in cytoplasmic loop IV/V to cleavage in N6 with attached ribosomes versus fully translated N6 suggests that the conformation of the two polypeptides differs. The observation that fully translated N6 is not extracted with urea whereas a significant amount of the translocation intermediate is urea-soluble (Fig. 4) implies that a folding event occurs after N6 exits the translocon. Cross-linking data with Cys residues at positions 126 (Helix IV) and 144 (Helix V) also support the interpretation that N6 folds after insertion into the bilayer (Fig. 5). However, it seems unlikely that N6 synthesized and insertedin vitro has the same tertiary structure as it does in native, full-length LacY since N6 is clearly sensitive to proteolysis by FtsH protease in the absence of C6 but stabilized in its presence (Fig. 7A). Furthermore, N6 with a single Cys residue at position 148 does not react with NEM (Fig. 7B).Unlike the two-dimensional projection map of OxlT (75Heymann J.A. Sarker R. Hirai T. Shi D. Milne J.L. Maloney P.C. Subramaniam S. EMBO J. 2001; 20: 4408-4413Crossref PubMed Scopus (55) Google Scholar), which indicates that each helix in the two halves of the protein occupy symmetry-related positions, neither the Na+/H+antiporter NhaA (76Williams R.W. Methods Enzymol. 1986; 130: 311-331Crossref PubMed Scopus (156) Google Scholar, 77Williams K.A. Geldmacher-Kaufer U. Padan E. Schuldiner S. Kuhlbrandt W. EMBO J. 1999; 18: 3558-3563Crossref PubMed Scopus (110) Google Scholar), the Na+/sugar symporter MelB (78Hacksell I. Rigaud J.L. Purhonen P. Pourcher T. Hebert H. Leblanc G. EMBO J. 2002; 21: 3569-3574Crossref PubMed Scopus (41) Google Scholar), nor LacY (29Sorgen P.L. Hu Y. Guan L. Kaback H.R. Girvin M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14037-14040Crossref PubMed Scopus (82) Google Scholar) exhibit such symmetry. Rather, in these transport proteins, helices from the N- and C-terminal halves of the polypeptides interdigitate. Thus, it is not surprising that N6 in LacY probably does not insert into the bilayer in a native conformation, although some of the structural features of the native, full-length protein are observed. This being the case, it seems clear that folding of LacY into a native, functional conformation must occur in the bilayer after both halves of the protein have exited the translocon, a conclusion consistent with the finding that PE acts as a molecular chaperone in the folding of LacY into its final native conformation (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar, 37Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar). Most inner membrane proteins in Escherichia coli are targeted to the membrane by the signal recognition particle (SRP)1 pathway and insert into the membrane via the Sec machinery (1Herskovits A.A. Bochkareva E.S. Bibi E. Mol. Microbiol. 2000; 38: 927-939Crossref PubMed Scopus (97) Google Scholar, 2Dalbey R.E. Chen M.Y. Jiang F.L. Samuelson J.C. Curr. Opin. Cell Biol. 2000; 12: 435-442Crossref PubMed Scopus (17) Google Scholar, 3de Gier J.W. Luirink J. Mol. Microbiol. 2001; 40: 314-322Crossref PubMed Scopus (82) Google Scholar, 4Bernstein H.D. Curr. Opin. Microbiol. 2000; 3: 203-209Crossref PubMed Scopus (40) Google Scholar). It has been suggested that bacterial SRP (Ffh protein and 4.5 S RNA) binds to the hydrophobic region of nascent membrane proteins, and subsequently, the ribosome-nascent chain complex and SRP interact with the SRP receptor (FtsY) at the membrane surface. Recently, it has been proposed that FtsY is a primary membrane-docking site for the ribosome, and SRP binds to nascent protein after FtsY-ribosome binding (5Herskovits A.A. Shimoni E. Minsky A. Bibi E. J. Cell Biol. 2002; 159: 403-410Crossref PubMed Scopus (57) Google Scholar). The Sec machinery is comprised of SecY, SecE, and SecG proteins in the cytoplasmic membrane (6Driessen A.J.M. Manting E.H. van der Does C. Nat. Struct. Biol. 2001; 8: 492-498Crossref PubMed Scopus (177) Google Scholar, 7Mori H. Ito K. Trends Microbiol. 2001; 9: 494-500Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 8Müller M. Koch H.G. Beck K. Schafer U. Prog. Nucleic Acid Res. Mol. Biol. 2001; 66: 107-157Crossref PubMed Google Scholar, 9Van Voorst F. De Kruijff B. Biochem. J. 2000; 347: 601-612Crossref PubMed Scopus (63) Google Scholar) and contributes to the topology of some membrane proteins (10Prinz W.A. Boyd D.H. Ehrmann M. Beckwith J. J. Biol. Chem. 1998; 273: 8419-8424Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). It has been shown (11Scotti P.A. Urbanus M.L. Brunner J. de Gier J.W. von Heijne G. van der Does C. Driessen A.J. Oudega B. Luirink J. EMBO J. 2000; 19: 542-549Crossref PubMed Scopus (298) Google Scholar, 12Samuelson 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 (417) Google Scholar) that the Sec machinery and YidC exist as a complex, and several inner membrane proteins interact with YidC during insertion. Thus, YidC appears to play a key role in inner membrane protein insertion (12Samuelson 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 (417) Google Scholar). SecD, SecF, and YajC form another heterotrimeric complex that binds to the SecYEG complex (13Duong F. Wickner W. EMBO J. 1997; 16: 4871-4879Crossref PubMed Scopus (165) Google Scholar). In addition, it has been reported (14Nouwen N. Driessen A.J.M. Mol. Microbiol. 2002; 44: 1397-1405Crossref PubMed Scopus (134) Google Scholar) that YidC interacts directly with SecDF rather than SecYEG, which forms an even larger complex with SecYEG. Furthermore, SecA, a cytosolic protein with ATPase activity, is also important for insertion of inner membrane proteins with large periplasmic loops (15Andersson H. von Heijne G. EMBO J. 1993; 12: 683-691Crossref PubMed Scopus (125) Google Scholar, 16Koch H.G. Hengelage T. Neumann-Haefelin C. MacFarlane J. Hoffschulte H.K. Schimz K.L. Mechler B. Müller M. Mol. Biol. Cell. 1999; 10: 2163-2173Crossref PubMed Scopus (128) Google Scholar). Although some details regarding the mechanism of integral membrane protein insertion have been elucidated, there are still many issues that require resolution, particularly those related to insertion of polytopic membrane proteins. Examples include folding of intermediates, interaction between helices during translation/insertion, the order and timing of helices exiting from the translocon into the lipid bilayer, and topological determinants. In an effort to study some of these issues we have chosen to use the lactose permease of E. coli (LacY) as a model system. LacY is a member of the Major Facilitator Superfamily (17Saier Jr., M.H. Mol. Microbiol. 2000; 35: 699-710Crossref PubMed Scopus (231) Google Scholar) and catalyzes the coupled stoichiometric translocation of galactosides and H+(symport) (18Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics: Transport Processes in Eukaryotic and Prokaryotic Organisms. II. Elsevier Science Publishers B. V., Amsterdam1996Google Scholar). The protein has been solubilized from the membrane and purified to homogeneity in a completely functional state (19Viitanen P. Garcia M.L. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1629-1633Crossref PubMed Scopus (74) Google Scholar, 20Viitanen P. Newman M.J. Foster D.L. Wilson T.H. Kaback H.R. Methods Enzymol. 1986; 125: 429-452Crossref PubMed Scopus (180) Google Scholar) and is a 12-helix bundle with the N and C termini on the cytoplasmic face of the membrane (21Foster D.L. Boublik M. Kaback H.R. J. Biol. Chem. 1983; 258: 31-34Abstract Full Text PDF PubMed Google Scholar, 22Calamia J. Manoil C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4937-4941Crossref PubMed Scopus (231) Google Scholar, 23Kaback H.R. Wu J. Q. Rev. Biophys. 1997; 30: 333-364Crossref PubMed Scopus (121) Google Scholar). In addition, LacY is physiologically (24Sahin-Tóth M. Lawrence M.C. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5421-5425Crossref PubMed Scopus (137) Google Scholar) and structurally a monomer in the membrane (25Costello M.J. Escaig J. Matsushita K. Viitanen P.V. Menick D.R. Kaback H.R. J. Biol. Chem. 1987; 262: 17072-17082Abstract Full Text PDF PubMed Google Scholar, 26Sun J. Kaback H.R. Biochemistry. 1997; 36: 11959-11965Crossref PubMed Scopus (54) Google Scholar, 27Guan L. Murphy F.D. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3475-3480Crossref PubMed Scopus (47) Google Scholar). Analysis of an extensive library of mutants, particularly Cys replacement mutants (28Frillingos S. Sahin-Tóth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (318) Google Scholar), in conjunction with a battery of site-directed biochemical and biophysical techniques has led to the formulation of a tertiary-structure model (29Sorgen P.L. Hu Y. Guan L. Kaback H.R. Girvin M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14037-14040Crossref PubMed Scopus (82) Google Scholar) as well as a hypothesis for the mechanism of lactose/H+ symport (30Kaback H.R. Sahin-Tóth M. Weinglass A.B. Nat. Rev. Mol. Cell Biol. 2001; 2: 610-622Crossref PubMed Scopus (249) Google Scholar). Like most inner membrane proteins, LacY is inserted into the membrane co-translationally (31Stochaj U. Ehring R. Eur. J. Biochem. 1987; 163: 653-658Crossref PubMed Scopus (11) Google Scholar, 32Ahrem B. Hoffschulte H.K. Müller M. J. Cell Biol. 1989; 108: 1637-1646Crossref PubMed Scopus (31) Google Scholar), and insertion involves SRP and FtsY (33MacFarlane J. Müller M. Eur. J. Biochem. 1995; 233: 766-771Crossref PubMed Scopus (99) Google Scholar,34Seluanov A. Bibi E. J. Biol. Chem. 1997; 272: 2053-2055Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) as well as SecY (35Ito K. Akiyama Y. Mol. Microbiol. 1991; 5: 2243-2253Crossref PubMed Scopus (77) Google Scholar). Phosphatidylethanolamine (PE) is also important for the late maturation of LacY and required for proper assembly and function (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). PE can even reverse misfolding of LacY topology after insertion into membranes devoid of PE (37Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar). However, the mechanism by which LacY is inserted into the membrane and folds into its final tertiary conformation is far from clear. Remarkably, co-expression of LacY in two contiguous, non-overlapping fragments with a covalent discontinuity in either cytoplasmic or periplasmic loops leads to complementation resulting in resistance of the fragments to proteolysis and functional LacY (38Bibi E. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4325-4329Crossref PubMed Scopus (166) Google Scholar, 39Wrubel W. Stochaj U. Sonnewald U. Theres C. Ehring R. J. Bacteriol. 1990; 172: 5374-5381Crossref PubMed Google Scholar, 40Zen K.H. McKenna E. Bibi E. Hardy D. Kaback H.R. Biochemistry. 1994; 33: 8198-8206Crossref PubMed Scopus (80) Google Scholar, 41Sahin-Tóth M. Kaback H.R. Friedlander M. Biochemistry. 1996; 35: 2016-2021Crossref PubMed Scopus (15) Google Scholar). Furthermore, a salt bridge between Asp-237 (Helix VII) and Lys-358 (Helix XI) plays an important role in membrane insertion (42Dunten R.L. Sahin-Tóth M. Kaback H.R. Biochemistry. 1993; 32: 3139-3145Crossref PubMed Scopus (114) Google Scholar, 43Frillingos S. Sahin-Tóth M. Lengeler J.W. Kaback H.R. Biochemistry. 1995; 34: 9368-9373Crossref PubMed Scopus (25) Google Scholar), indicating that Helix VII must interact with Helix XI before insertion of LacY into the bilayer. In addition, the relatively long middle cytoplasmic loop is important for functional expression, possibly acting as a time delay to allow the N-terminal six helices (N6) to clear the translocon before the last six helices are inserted (44Weinglass A.B. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8938-8943Crossref PubMed Scopus (38) Google Scholar). To analyze these phenomena more completely, as well as the insertion mechanism of LacY, an in vitro transcription/translation/insertion system is described in which LacY appears to be inserted in a native, functional conformation. In this study, we focus on insertion of the N-terminal half of LacY (N6). DISCUSSIONAlthough previous studies (32Ahrem B. Hoffschulte H.K. Müller M. J. Cell Biol. 1989; 108: 1637-1646Crossref PubMed Scopus (31) Google Scholar, 64Bochkareva E. Seluanov A. Bibi E. Girshovich A. J. Biol. Chem. 1996; 271: 22256-22261Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) indicate that LacY transcribed and translated in vitro can be inserted into ISO membrane vesicles with the correct topology (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar), questions remain as to whether or not the inserted protein is in a native conformation. Because LacY, like many other membrane proteins, is resistant to traditional means of structural analysis, alternative approaches have been developed to study topology and discern the overall three-dimensional fold (see Refs. 29Sorgen P.L. Hu Y. Guan L. Kaback H.R. Girvin M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14037-14040Crossref PubMed Scopus (82) Google Scholar and 30Kaback H.R. Sahin-Tóth M. Weinglass A.B. Nat. Rev. Mol. Cell Biol. 2001; 2: 610-622Crossref PubMed Scopus (249) Google Scholar). The studies presented here, which utilize some of these approaches, provide convincing evidence that LacY synthesized and inserted into the membrane in vitro is in a native conformation. (a) By using engineered Factor Xa protease sites in cytoplasmic or periplasmic loops, the topology of the polypeptide with respect to the membrane appears to be correct. (b) A mAb that binds to a discontinuous epitope comprised of residues in cytoplasmic loops VIII/IX and X/XI (61Sun J. Li J. Carrasco N. Kaback H.R. Biochemistry. 1997; 36: 274-280Crossref PubMed Scopus (27) Google Scholar) binds to LacY synthesized and inserted in vitro as well as it binds when LacY is synthesized and inserted in vivo. (c) Cys residues at positions 126 and 144 in the in vitro system undergo spontaneous cross-linking as observed in vivo (53Wolin C.D. Kaback H.R. Biochemistry. 2000; 39: 6130-6135Crossref PubMed Scopus (43) Google Scholar). (d) LacY synthesized and inserted in vitroexhibits lactose/Dns6-Gal counter-flow and significant ligand protection against alkylation of Cys-148. Therefore, transcription, translation, and insertion of LacY into ISO vesiclesin vitro represents a system in which the mechanism of insertion of a polytopic membrane protein and its folding into a tertiary conformation can be studied reliably.Despite conjecture regarding co-translational insertion of polytopic membrane proteins, it is unclear how many transmembrane helices are accommodated by the translocon before insertion into the bilayer. Furthermore, little information is available regarding whether folding into a tertiary conformation begins in the translocon or only after migration into the bilayer with the assistance of chaperones such as PE (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar, 37Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar, 70Bogdanov M. Dowhan W. J. Biol. Chem. 1999; 274: 36827-36830Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The observation that the first six helices of LacY are extracted with urea when the ribosome remains attached, whereas the first eight helices are not, suggests that the translocon can accommodate at least six transmembrane helices, a conclusion similar to that drawn from findings obtained with P-glycoprotein (71Borel A.C. Simon S.M. Biochemistry. 1996; 35: 10587-10594Crossref PubMed Scopus (30) Google Scholar). Similarly, an in vitro study of the polytopic membrane protein MtlA suggests more than one pair of transmembrane domains are assembled before the exit from translocon (72Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (136) Google Scholar). On the other hand, the N6 portion of LacY may not be fully accommodated by the translocon, and part of N6 may be in contact with lipid, which would explain why the polypeptide is not completely extracted with urea (Fig. 3).Photo cross-linking studies on certain membrane proteins indicate that the transmembrane domains may indeed interact with lipid during insertion (72Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (136) Google Scholar, 73Urbanus M.L. Scotti P.A. Froderberg L. Saaf A. de Gier J.W. Brunner J. Samuelson J.C. Dalbey R.E. Oudega B. Luirink J. EMBO Rep. 2001; 2: 524-529Crossref PubMed Scopus (153) Google Scholar). With LacY, the first or second transmembrane helix might interact with phospholipid, whereas helices III to VI are still in the translocon. However, if this is the case, it is unlikely that the first two helices migrate very far from the translocon. Indeed, it has been suggested that in some endoplasmic reticulum membrane proteins, a transmembrane helix can re-enter the translocon after its initial release into the lipid phase if it has affinity for other helices still within the translocon (74Mothes W. Heinrich S.U. Graf R. Nilsson I. von Heijne G. Brunner J. Rapoport T.A. Cell. 1997; 89: 523-533Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Taken as a whole, the data suggest that the N6 half of LacY must remain near the translocon after exit into the bilayer as the C6 half is translated. Once the C6 half exits the translocon, folding into a final tertiary structure likely occurs within the bilayer, resulting in a protease-resistant, functional molecule.The differential sensitivity of engineered Factor Xa protease sites in cytoplasmic loop IV/V to cleavage in N6 with attached ribosomes versus fully translated N6 suggests that the conformation of the two polypeptides differs. The observation that fully translated N6 is not extracted with urea whereas a significant amount of the translocation intermediate is urea-soluble (Fig. 4) implies that a folding event occurs after N6 exits the translocon. Cross-linking data with Cys residues at positions 126 (Helix IV) and 144 (Helix V) also support the interpretation that N6 folds after insertion into the bilayer (Fig. 5). However, it seems unlikely that N6 synthesized and insertedin vitro has the same tertiary structure as it does in native, full-length LacY since N6 is clearly sensitive to proteolysis by FtsH protease in the absence of C6 but stabilized in its presence (Fig. 7A). Furthermore, N6 with a single Cys residue at position 148 does not react with NEM (Fig. 7B).Unlike the two-dimensional projection map of OxlT (75Heymann J.A. Sarker R. Hirai T. Shi D. Milne J.L. Maloney P.C. Subramaniam S. EMBO J. 2001; 20: 4408-4413Crossref PubMed Scopus (55) Google Scholar), which indicates that each helix in the two halves of the protein occupy symmetry-related positions, neither the Na+/H+antiporter NhaA (76Williams R.W. Methods Enzymol. 1986; 130: 311-331Crossref PubMed Scopus (156) Google Scholar, 77Williams K.A. Geldmacher-Kaufer U. Padan E. Schuldiner S. Kuhlbrandt W. EMBO J. 1999; 18: 3558-3563Crossref PubMed Scopus (110) Google Scholar), the Na+/sugar symporter MelB (78Hacksell I. Rigaud J.L. Purhonen P. Pourcher T. Hebert H. Leblanc G. EMBO J. 2002; 21: 3569-3574Crossref PubMed Scopus (41) Google Scholar), nor LacY (29Sorgen P.L. Hu Y. Guan L. Kaback H.R. Girvin M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14037-14040Crossref PubMed Scopus (82) Google Scholar) exhibit such symmetry. Rather, in these transport proteins, helices from the N- and C-terminal halves of the polypeptides interdigitate. Thus, it is not surprising that N6 in LacY probably does not insert into the bilayer in a native conformation, although some of the structural features of the native, full-length protein are observed. This being the case, it seems clear that folding of LacY into a native, functional conformation must occur in the bilayer after both halves of the protein have exited the translocon, a conclusion consistent with the finding that PE acts as a molecular chaperone in the folding of LacY into its final native conformation (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar, 37Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar). Although previous studies (32Ahrem B. Hoffschulte H.K. Müller M. J. Cell Biol. 1989; 108: 1637-1646Crossref PubMed Scopus (31) Google Scholar, 64Bochkareva E. Seluanov A. Bibi E. Girshovich A. J. Biol. Chem. 1996; 271: 22256-22261Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) indicate that LacY transcribed and translated in vitro can be inserted into ISO membrane vesicles with the correct topology (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar), questions remain as to whether or not the inserted protein is in a native conformation. Because LacY, like many other membrane proteins, is resistant to traditional means of structural analysis, alternative approaches have been developed to study topology and discern the overall three-dimensional fold (see Refs. 29Sorgen P.L. Hu Y. Guan L. Kaback H.R. Girvin M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14037-14040Crossref PubMed Scopus (82) Google Scholar and 30Kaback H.R. Sahin-Tóth M. Weinglass A.B. Nat. Rev. Mol. Cell Biol. 2001; 2: 610-622Crossref PubMed Scopus (249) Google Scholar). The studies presented here, which utilize some of these approaches, provide convincing evidence that LacY synthesized and inserted into the membrane in vitro is in a native conformation. (a) By using engineered Factor Xa protease sites in cytoplasmic or periplasmic loops, the topology of the polypeptide with respect to the membrane appears to be correct. (b) A mAb that binds to a discontinuous epitope comprised of residues in cytoplasmic loops VIII/IX and X/XI (61Sun J. Li J. Carrasco N. Kaback H.R. Biochemistry. 1997; 36: 274-280Crossref PubMed Scopus (27) Google Scholar) binds to LacY synthesized and inserted in vitro as well as it binds when LacY is synthesized and inserted in vivo. (c) Cys residues at positions 126 and 144 in the in vitro system undergo spontaneous cross-linking as observed in vivo (53Wolin C.D. Kaback H.R. Biochemistry. 2000; 39: 6130-6135Crossref PubMed Scopus (43) Google Scholar). (d) LacY synthesized and inserted in vitroexhibits lactose/Dns6-Gal counter-flow and significant ligand protection against alkylation of Cys-148. Therefore, transcription, translation, and insertion of LacY into ISO vesiclesin vitro represents a system in which the mechanism of insertion of a polytopic membrane protein and its folding into a tertiary conformation can be studied reliably. Despite conjecture regarding co-translational insertion of polytopic membrane proteins, it is unclear how many transmembrane helices are accommodated by the translocon before insertion into the bilayer. Furthermore, little information is available regarding whether folding into a tertiary conformation begins in the translocon or only after migration into the bilayer with the assistance of chaperones such as PE (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar, 37Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar, 70Bogdanov M. Dowhan W. J. Biol. Chem. 1999; 274: 36827-36830Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). The observation that the first six helices of LacY are extracted with urea when the ribosome remains attached, whereas the first eight helices are not, suggests that the translocon can accommodate at least six transmembrane helices, a conclusion similar to that drawn from findings obtained with P-glycoprotein (71Borel A.C. Simon S.M. Biochemistry. 1996; 35: 10587-10594Crossref PubMed Scopus (30) Google Scholar). Similarly, an in vitro study of the polytopic membrane protein MtlA suggests more than one pair of transmembrane domains are assembled before the exit from translocon (72Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (136) Google Scholar). On the other hand, the N6 portion of LacY may not be fully accommodated by the translocon, and part of N6 may be in contact with lipid, which would explain why the polypeptide is not completely extracted with urea (Fig. 3). Photo cross-linking studies on certain membrane proteins indicate that the transmembrane domains may indeed interact with lipid during insertion (72Beck K. Eisner G. Trescher D. Dalbey R.E. Brunner J. Müller M. EMBO Rep. 2001; 2: 709-714Crossref PubMed Scopus (136) Google Scholar, 73Urbanus M.L. Scotti P.A. Froderberg L. Saaf A. de Gier J.W. Brunner J. Samuelson J.C. Dalbey R.E. Oudega B. Luirink J. EMBO Rep. 2001; 2: 524-529Crossref PubMed Scopus (153) Google Scholar). With LacY, the first or second transmembrane helix might interact with phospholipid, whereas helices III to VI are still in the translocon. However, if this is the case, it is unlikely that the first two helices migrate very far from the translocon. Indeed, it has been suggested that in some endoplasmic reticulum membrane proteins, a transmembrane helix can re-enter the translocon after its initial release into the lipid phase if it has affinity for other helices still within the translocon (74Mothes W. Heinrich S.U. Graf R. Nilsson I. von Heijne G. Brunner J. Rapoport T.A. Cell. 1997; 89: 523-533Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Taken as a whole, the data suggest that the N6 half of LacY must remain near the translocon after exit into the bilayer as the C6 half is translated. Once the C6 half exits the translocon, folding into a final tertiary structure likely occurs within the bilayer, resulting in a protease-resistant, functional molecule. The differential sensitivity of engineered Factor Xa protease sites in cytoplasmic loop IV/V to cleavage in N6 with attached ribosomes versus fully translated N6 suggests that the conformation of the two polypeptides differs. The observation that fully translated N6 is not extracted with urea whereas a significant amount of the translocation intermediate is urea-soluble (Fig. 4) implies that a folding event occurs after N6 exits the translocon. Cross-linking data with Cys residues at positions 126 (Helix IV) and 144 (Helix V) also support the interpretation that N6 folds after insertion into the bilayer (Fig. 5). However, it seems unlikely that N6 synthesized and insertedin vitro has the same tertiary structure as it does in native, full-length LacY since N6 is clearly sensitive to proteolysis by FtsH protease in the absence of C6 but stabilized in its presence (Fig. 7A). Furthermore, N6 with a single Cys residue at position 148 does not react with NEM (Fig. 7B). Unlike the two-dimensional projection map of OxlT (75Heymann J.A. Sarker R. Hirai T. Shi D. Milne J.L. Maloney P.C. Subramaniam S. EMBO J. 2001; 20: 4408-4413Crossref PubMed Scopus (55) Google Scholar), which indicates that each helix in the two halves of the protein occupy symmetry-related positions, neither the Na+/H+antiporter NhaA (76Williams R.W. Methods Enzymol. 1986; 130: 311-331Crossref PubMed Scopus (156) Google Scholar, 77Williams K.A. Geldmacher-Kaufer U. Padan E. Schuldiner S. Kuhlbrandt W. EMBO J. 1999; 18: 3558-3563Crossref PubMed Scopus (110) Google Scholar), the Na+/sugar symporter MelB (78Hacksell I. Rigaud J.L. Purhonen P. Pourcher T. Hebert H. Leblanc G. EMBO J. 2002; 21: 3569-3574Crossref PubMed Scopus (41) Google Scholar), nor LacY (29Sorgen P.L. Hu Y. Guan L. Kaback H.R. Girvin M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14037-14040Crossref PubMed Scopus (82) Google Scholar) exhibit such symmetry. Rather, in these transport proteins, helices from the N- and C-terminal halves of the polypeptides interdigitate. Thus, it is not surprising that N6 in LacY probably does not insert into the bilayer in a native conformation, although some of the structural features of the native, full-length protein are observed. This being the case, it seems clear that folding of LacY into a native, functional conformation must occur in the bilayer after both halves of the protein have exited the translocon, a conclusion consistent with the finding that PE acts as a molecular chaperone in the folding of LacY into its final native conformation (36Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar, 37Bogdanov M. Heacock P.N. Dowhan W. EMBO J. 2002; 21: 2107-2116Crossref PubMed Scopus (190) Google Scholar). We are indebted to Eitan Bibi, William Dowhan, and Mikhail Bogdanov for editorial suggestions, K. Nishiyama for advice regarding the in vitro system, and T. Ogura for providing strains AR796 and -797.
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