Abstract: phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol cardiolipin phosphatidylserine lipopolysaccharide monosialoganglioside Although there has been significant progress in our understanding of how water-soluble proteins fold (1Fenton W.A. Horwich A. Protein Sci. 1997; 6: 743-760Crossref PubMed Scopus (329) Google Scholar, 2Ellis R.J. Hartl F.U. Curr. Opin. Struct. Biol. 1999; 9: 102-110Crossref PubMed Scopus (269) Google Scholar), the factors and mechanism driving correct folding of integral membrane proteins are largely unknown. The folding of membrane proteins, like their soluble counterparts, is dictated by their amino acid sequence and their environment (Fig. 1). Integral membrane proteins can also interact with other proteins within the membrane and with the hydrophobic and hydrophilic components of the lipid bilayer itself during and after attainment of native structure. The role of lipids as an important structure-forming environment was elucidated during the last decade (3Marsh D. Watts A. New Comprehensive Biochemistry: Protein-Lipid Interactions. 25. Elsevier Science Publishers B.V., Amsterdam1993: 41-66Google Scholar). However, the role individual lipids play as part of the protein folding machinery has been largely ignored. Are individual lipids mobilized to protect and guide the nascent polypeptide chain during its membrane assembly? Do lipids act as specific molecular chaperones or transient ligands during the assembly of a membrane protein? The main experimental approach to study soluble protein folding has been to investigate refolding of denatured (unfolded) protein back to the native state (4Anfinsen C.B. Sheraga H.A. Adv. Protein Chem. 1975; 29: 205-300Crossref PubMed Scopus (812) Google Scholar). The discovery of protein molecular chaperones indicated that in vivo folding of proteins is a more complex process than initially thought (5Pelham H.R. Cell. 1986; 46: 959-961Abstract Full Text PDF PubMed Scopus (1146) Google Scholar). Similarly, the folding of membrane proteins during in vitro renaturation may greatly differ from in vivo folding, which involves interaction with the phospholipid bilayer. Therefore, detergent/lipid micelles and lipid vesicles have been included in the renaturation solution used to dilute the denaturant to non-denaturating conditions (6Huang K.S. Bayley H. Liao M.-J. London E. Korana H. J. Biol. Chem. 1981; 256: 3802-3809Abstract Full Text PDF PubMed Google Scholar) because they mimic the properties of the lipid bilayer. Using this approach, the α-helical protein bacteriorhodopsin was the first membrane protein refolded from the denatured state in detergent to a folded state in detergent/lipid micelles (7London E. Korana H.G. J. Biol. Chem. 1982; 257: 7003-7011Abstract Full Text PDF PubMed Google Scholar) and lipid vesicles (8Curran A.R. Templer R.H. Booth P.J. Biochemistry. 1999; 38: 9328-9336Crossref PubMed Scopus (111) Google Scholar, 9Booth P.J. Curran A.R. Curr. Opin. Struct. Biol. 1999; 9: 115-121Crossref PubMed Scopus (96) Google Scholar). α-Helix formation (the rate-limiting step in folding) is much slower for bacteriorhodopsin relative to that of soluble proteins. Folding, as well as membrane insertion, is slowed even more as the proportion of dimyristoyl-PC1 is increased in dimyristoyl-PC/dihexanoyl-PC mixed vesicles or as the non-bilayer-forming lipid phosphatidylethanolamine (PE) is added to PC vesicles. These results indicate that an increase in the radius of curvature of the bilayer (or departure from a flat bilayer) may slow α-helix formation within or inhibit insertion of polypeptide into the membrane bilayer. The kinetics of refolding of the Escherichia coli membrane protein, OmpA, in lipid vesicles has also been studied (10Kleinschmidt J.H. Tamm L.K. Biochemistry. 1996; 35: 12993-13000Crossref PubMed Scopus (149) Google Scholar, 11Surrey T. Jahnig F. J. Biol. Chem. 1995; 270: 28199-28203Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar). Surprisingly, the yield in renaturation of this β-barrel membrane protein was considerably higher in dimyristoyl-PC/dimyristoyl-PG (80/20) mixed bilayers than in mixtures of 1-palmitoyl 2-oleoyl-PE/1-palmitoyl 2-oleoyl-PG (80/20) mixed bilayers that more closely mimic the lipid composition of the E. coli inner cytoplasmic membrane (13Jahnig F. Surrey T. von Heijne G. Membrane Protein Assembly. R. G. Landes Co., Austin, TX1997: 83-98Google Scholar). This protein resides in the outer membrane of E. coli but must pass through the inner membrane where presumably it must maintain a more unfolded state. In these studies the folding of integral membrane proteins has been investigated as a function of the collective properties of lipid bilayers, i.e. as a function of the hydrophobicity (14Carpenter K.A. Wilkes B.C. De Lean A. Fournier A. Schiller P.W. Biopolymers. 1997; 42: 37-48Crossref PubMed Google Scholar), electrostatics (15Rankin S.E. Watts A. Pinheiro T.J.T. Biochemistry. 1998; 37: 12588-12595Crossref PubMed Scopus (48) Google Scholar), rigidity (16Booth P.J. Riley M.L. Flitsch S.L. Templer R.H. Faroog A. Curran A.R. Chadborn N. Wright P. Biochemistry. 1997; 36: 197-203Crossref PubMed Scopus (105) Google Scholar), and intrinsic radius of curvature (9Booth P.J. Curran A.R. Curr. Opin. Struct. Biol. 1999; 9: 115-121Crossref PubMed Scopus (96) Google Scholar). No systematic investigation of the possible role of specific lipids or the chemical properties of membrane lipids on the yield of refolding, formation of refolding intermediates, or alteration of the refolding pathway was included in these studies. However, these reports suggest that membrane protein folding is more complex than interaction with the lipid bilayer approximated as a simple hydrophobic core bounded by water interfaces. The role of protein molecular chaperones in directing protein folding, preventing protein misfolding (17Frydman J. Hartl F.U. Science. 1996; 272: 1497-1502Crossref PubMed Scopus (219) Google Scholar), and even unfolding proteins (18Shtilerman M. Lorimer G.H. Englander S.W. Science. 1999; 284: 822-825Crossref PubMed Scopus (265) Google Scholar) is now well established for the conformational maturation of soluble and membrane proteins (19Martin J. Hartl F.U. Curr. Opin. Struct. Biol. 1997; 7: 41-52Crossref PubMed Scopus (164) Google Scholar, 20Tatu U. Helenius A. J. Cell Biol. 1997; 136: 555-565Crossref PubMed Scopus (187) Google Scholar). The presence of protein molecular chaperones in refolding protocols can significantly increase the yield of native structure. Evidence is now accumulating that specific lipids may also participate as molecular chaperones in the folding and possibly the unfolding of integral membrane proteins. The development of mutants of E. coli (21Dowhan W. Annu. Rev. Biochem. 1997; 66: 199-232Crossref PubMed Scopus (786) Google Scholar, 22Dowhan W. Biochim. Biophys. Acta. 1998; 1376: 446-455Crossref Scopus (24) Google Scholar) in which the membrane phospholipid composition can be readily altered has made it possible to separate phospholipid-assisted and -unassisted folding events and study the role of individual phospholipids in protein folding. The absence of the major phospholipid PE in one such mutant results in the misfolding of the polytopic membrane protein lactose permease (LacY) without affecting its insertion into the membrane. Refolding dependent on phospholipid of unfolded LacY was monitored using the Eastern-Western blotting procedure (23Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). This procedure involves the transfer of proteins from a SDS-polyacrylamide gel by standard Western blotting methodology to a solid support coated with phospholipids that interact with partially denatured proteins as they refold. These refolding experiments have uncovered a role for PE as a non-protein molecular chaperone. LacY initially assembled in the presence of PE but not in its absence has full biological function (25Bogdanov M. Dowhan W. J. Biol. Chem. 1995; 270: 732-739Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and displays a continuous epitope (4B1) recognized by the conformation-dependent monoclonal antibody 4B1 (23Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). This epitope is in a periplasmic loop near the middle of LacY between transmembrane helices VII and VIII. The proper conformation of this epitope is required for full function of LacY as a transporter (26Sun J. Wu J. Carrasco N. Kaback H.R. Biochemistry. 1996; 35: 990-998Crossref PubMed Scopus (85) Google Scholar) and depends on exposure to PE during assembly (23Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar,24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). Sufficient conformational information was retained to detect epitope 4B1 after partial unfolding of LacY by SDS, complete removal of PE, and refolding in the absence of PE. Therefore, once information is imparted by PE during LacY folding in vivo, the presence of PE is no longer required to maintain proper conformation of this epitope consistent with the primary definition of a molecular chaperone (27Ellis R.J. Biochem. Biophys. Res. Commun. 1997; 238: 687-692Crossref PubMed Scopus (53) Google Scholar). LacY lacking recognition by monoclonal antibody 4B1, because of initial assembly in the absence of PE, can be induced to form this epitope if partially denatured and then renatured in the presence of PE or phosphatidylserine (PS) specifically; other phospholipids such as PC, PG, or cardiolipin (CL) do not support the regain of epitope 4B1 during refolding. Thus, the misfolded conformation of LacY can be corrected by specific lipids (absent during in vivoassembly) by interaction with non-native LacY, also consistent with an important criterion in the definition of a molecular chaperone (27Ellis R.J. Biochem. Biophys. Res. Commun. 1997; 238: 687-692Crossref PubMed Scopus (53) Google Scholar). Refolding of denatured full-length protein is not an ideal model because the folding of the nascent peptide in vivo begins during translation (28Fedorov A.N. Baldwin T.O. J. Biol. Chem. 1997; 272: 32715-32718Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Moreover, molecular chaperones were shown to interact differently with denatured proteins than with their newly translated counterparts (17Frydman J. Hartl F.U. Science. 1996; 272: 1497-1502Crossref PubMed Scopus (219) Google Scholar). Molecular chaperones can bind transiently to nascent proteins and prevent premature folding (28Fedorov A.N. Baldwin T.O. J. Biol. Chem. 1997; 272: 32715-32718Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) or can assist in folding by binding post-translationally at a later stage of assembly (29Hendrick J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1470) Google Scholar). The liposome-induced conformational transition of colicin E1 occurs concomitantly with insertion into membranes (30Mel S.F. Stroud R.M. Biochemistry. 1993; 32: 2082-2089Crossref PubMed Scopus (37) Google Scholar, 31van der Goot F.G. Gonzalez-Manas J.M. Lakey J.H. Pattus F. Nature. 1991; 354: 408-410Crossref PubMed Scopus (421) Google Scholar). The rate of unfolding of acetylcholine esterase from Torpedo californica was greatly enhanced in the presence of PC vesicles with concomitant insertion of the protein into the lipid bilayer (32Shin I. Kreimer D. Silman I. Weiner L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2848-2852Crossref PubMed Scopus (40) Google Scholar). For β-barrel membrane proteins, folding and membrane insertion are coupled processes that involve kinetically distinguishable steps (10Kleinschmidt J.H. Tamm L.K. Biochemistry. 1996; 35: 12993-13000Crossref PubMed Scopus (149) Google Scholar,33Kleinschmidt J.H. den Blaauwen T. Driessen A.J. Tamm L.K. Biochemistry. 1999; 38: 5006-5016Crossref PubMed Scopus (123) Google Scholar). However, by using mutants lacking PE coupled with the ability to add PE post-translationally to LacY already assembled in the membrane, the process of membrane insertion was shown to be separate from a late step of conformational maturation requiring specific PE assistance (24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). In situ synthesis of LacY in the presence of membranes originally lacking PE either with co- or post-translational expression of PE resulted in the appearance of the PE-dependent epitope 4B1. In situ synthesis of PE in isolated membranes originally lacking PE and containing in vivo synthesized LacY also resulted in the appearance of epitope 4B1. Thus PE appears to be required in a late step of conformational maturation occurring after membrane insertion and involving final adjustments for the transition from near native to the native state. This late stage involvement is similar to the temporal point of action of most protein molecular chaperones (34Clark A.C. Frieden C. J. Mol. Biol. 1999; 285: 777-788Google Scholar) and is consistent with the observation that LacY in the absence of PE is partially functional as a facilitating transporterin vivo and in situ (25Bogdanov M. Dowhan W. J. Biol. Chem. 1995; 270: 732-739Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). LacY synthesized and assembled in the absence of PE also regained both full transport function and epitope 4B1 after initiation of PE synthesis in vivo. 2M. Bogdanov, P. Heacock, and W. Dowhan, unpublished data. Therefore PE can act as a non-protein molecular chaperone (tentatively named a "lipochaperone") that specifically mediates the late stage folding of LacY. If the general argument for phospholipids as molecular chaperones is correct, there should exist molecular species of lipids other than PE that are involved transiently in the folding of integral membrane proteins. Several reports on the unfolding, refolding, and assembly of outer membrane porins of E. coli strongly suggest a molecular chaperone role for lipopolysaccharide (LPS). Refolding of denatured OmpA occurred exclusively in the presence of LPS and not with other lipids (35Schweizer M. Hindennach I. Garten W. Henning U. Eur. J. Biochem. 1978; 82: 211-217Crossref PubMed Scopus (156) Google Scholar). An immature form of OmpA was detected in the inner membrane that undergoes a conformational change upon interaction with LPS making the protein "outer membrane compatible" (36Freudl R. Schwarz H. Stierhof Y.-D. Gamon K. Hindennach I. Henning U. J. Biol. Chem. 1986; 261: 11355-11361Abstract Full Text PDF PubMed Google Scholar). No lipid was found tightly associated with either conformational variant of OmpA. Similarly, the glycolipid part of LPS appears to be required for folding outer membrane PhoE porin monomers of E. coli as they pass through the inner membrane where LPS molecules are also in transit to the outer membrane. LPS inhibits the assembly of PhoE into its functional trimeric units while in the inner membrane. Release of LPS very likely is required for efficient trimerization because no interaction was detected between LPS and the mature trimer that forms upon assembly of monomers in the outer membrane. The transient interaction of LPS with the PhoE monomer hydrophobic interfaces could prevent aggregation during passage through the inner membrane consistent with this glycophospholipid acting as a molecular chaperone (37de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5593Crossref PubMed Scopus (65) Google Scholar). DegP, a periplasmic protein of E. coli, undergoes PG-dependent conformational changes that may protect the unfolded protein from self-aggregation at relatively low temperatures keeping critical interactive sites apart, thus preventing misfolding (38Skorko-Glonek J. Lipinska B. Krzewski K. Zolese G. Bertoli E. Tanfani F. J. Biol. Chem. 1997; 272: 8974-8982Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This phenomenon was not observed in the presence of PE or CL, demonstrating again specificity of folding of DegP with the aid of the putative lipochaperone PG. A sulfated glycosphingolipid (sulfatide) appears to act as a molecular chaperone by transiently interacting in a functional manner with non-native reversibly aggregated crystals of insulin (39Osterbye T. Jørgensen K.H. Kaas A. Tranum-Jensen J. Fredman P. Buschard K. Diabetologia. 1999; 42 Suppl. 1 (Abstr. 110): A30Google Scholar). This interaction prevents the irreversible aggregation of insulin into an amorphous form while either promoting or facilitating monomerization into the biologically active form. This is the first example of molecular chaperone action to simultaneously preserve a reversibly aggregated form and to convert protein aggregates into a biologically active form. These observations add to a growing body of evidence that lipids may bind to certain areas of unfolded proteins, thus reducing the likelihood of misfolding or aggregation. Therefore, lipids act in an analogous manner to protein molecular chaperones, thereby adding them to the growing list of non-protein chaperones (27Ellis R.J. Biochem. Biophys. Res. Commun. 1997; 238: 687-692Crossref PubMed Scopus (53) Google Scholar), which includes RNA and oligosaccharides (40Pillarisetti S. Paka L. Sasaki A. Vanni-Reyes T. Yin B. Parthasarathy N. Wagner W.D. Goldberg I.J. J. Biol. Chem. 1997; 272: 15753-15759Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 41Kudlicki W. Coffman A. Kramer G. Hardesty B. Folding Design. 1997; 2: 101-108Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). For glycerophosphate-based lipids chemical diversity in the fatty acid chains combined with the major headgroup classes define individual molecular species ranging in the hundreds to thousands (42Raetz C.R. Dowhan W. J. Biol. Chem. 1990; 265: 1235-1238Abstract Full Text PDF PubMed Google Scholar). In addition lipids assume various physical organizations such as the bilayer, micellar, inverted hexagonal, or cubic phase (43Epand R.M. Biochim. Biophys. Acta. 1998; 1376: 353-368Crossref PubMed Scopus (324) Google Scholar). The collective physical properties of an overall bilayer organization of lipids will have different physical properties depending on the proportion of non-bilayer- and bilayer-forming lipids present. Using LacY synthesized and assembled in vivo in the absence of PE, the physical and chemical properties of a lipid required to assist proper refolding of epitope 4B1 of LacY in vitro were investigated using the Eastern-Western technique (44Bogdanov M. Umeda M. Dowhan W. J. Biol. Chem. 1999; 274: 12339-12345Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). A primary amine (either PE or PS) was most effective. PE derivatives of increasing degrees of methylation were progressively less effective in supporting refolding with PC being totally ineffective. Monoacyl phospholipids were not functional, and the diacyl phospholipids had to contain at least one saturated fatty acid with a preference for chain lengths above 14 carbons. The requirements within the hydrophobic domain correlated closely with an apparent requirement for phospholipid mixtures that assume a collective bilayer rather than non-bilayer organization under experimental conditions. Therefore, as with bacteriorhodopsin (8Curran A.R. Templer R.H. Booth P.J. Biochemistry. 1999; 38: 9328-9336Crossref PubMed Scopus (111) Google Scholar), insertion of LacY into a lipid domain and subsequent folding may require a bilayer structure, and these may precede the requirement of PE as a lipochaperone. Unnatural diastereoisomers of PS (at either chiral center) were ineffective unless in binary mixtures with natural isomers of PG that were unable in themselves to support proper refolding of LacY. Therefore, the molecular chaperone effect is highly selective with respect to lipid chemical composition, chirality, and physical properties supporting a role for lipid that goes well beyond simply providing a nonspecific detergent-like or two-phase environment for refolding of LacY. PC, which is not found in E. coli, promoted the unfolding of native epitope 4B1 (44Bogdanov M. Umeda M. Dowhan W. J. Biol. Chem. 1999; 274: 12339-12345Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), suggesting that lipochaperones could function as do protein chaperones to maintain or induce an unfolded state. Zardeneta and Horowitz (45Zardeneta G. Horowitz P.M. J. Biol. Chem. 1992; 267: 5811-5816Abstract Full Text PDF PubMed Google Scholar) investigated the interaction of the mitochondrial phospholipid CL with unfolded and native rhodanese, a nuclear-encoded mitochondrial matrix enzyme. They demonstrated that unfolded non-native rhodanese, but not native rhodanese, associates transiently with CL-containing micelles, and this interaction increases the amount of enzyme reactivation by preventing aggregation. The anionic lipid PS was not as effective as CL, indicating a structural preference in addition to simple charge. Formation of secondary and tertiary structure during refolding of the outer membrane protein OmpA of E. coli correlated with the micelle-forming properties of short chain phospholipids and detergents and did not depend on the polar headgroup or hydrophobic chain length of phospholipids and detergents that were examined (46Kleinschmidt J.H. Wiener M.C. Tamm L.K. Biophys. J. 1999; 76 (Su-Pos 435): A106Google Scholar). Monomeric detergent solutions were not active. Finally, negatively charged LPS derivatives that tend to organize in non-bilayer structures were better facilitators of folding of PhoE in situ than their bilayer-forming counterparts (47de Cock H. Brandenburg K. Wiese A. Holst O. Seydel U. J. Biol. Chem. 1999; 274: 5114-5119Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). All these data taken together demonstrate the importance of both individual chemical makeup and supramolecular organization in the function of lipochaperones. The binding of partially folded proteins to a molecular chaperone prevents their misfolding and the aggregation of folding intermediates. Similarly, lipochaperones interact with non-native conformations of proteins and have a low affinity for native proteins (23Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar, 37de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5593Crossref PubMed Scopus (65) Google Scholar). Interaction of protein chaperones with substrate proteins also displays an ionic component (48Perrett S. Zahn R. Stenberg G. Fersht A.R. J. Mol. Biol. 1997; 269: 892-901Crossref PubMed Scopus (57) Google Scholar). Both the properties of the ionic headgroup and the organization of the hydrophobic domain of PE (44Bogdanov M. Umeda M. Dowhan W. J. Biol. Chem. 1999; 274: 12339-12345Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and LPS (47de Cock H. Brandenburg K. Wiese A. Holst O. Seydel U. J. Biol. Chem. 1999; 274: 5114-5119Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) also mimic critical components of protein chaperones. Several examples support the view that some proteins require molecular chaperones to fold correctly not to avoid a potential aggregation problem but to reverse stable misfolded states (18Shtilerman M. Lorimer G.H. Englander S.W. Science. 1999; 284: 822-825Crossref PubMed Scopus (265) Google Scholar, 49Peralta D.D. Hartman D.J. Hoogenraad N.J. Hoj P.B. FEBS Lett. 1994; 339: 45-49Crossref PubMed Scopus (63) Google Scholar). Lipochaperones could participate at a late stage of protein folding in facilitating such interactions and conformational changes. PE appears to interact with late folding intermediates to assist in attaining native structure of LacY. In situ folding studies on LacY (24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar) and the fact that LacY in the absence of PE is partially functional (25Bogdanov M. Dowhan W. J. Biol. Chem. 1995; 270: 732-739Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) as a facilitating transporter indicate that considerable conformational maturation must occur prior to interaction with PE followed by the imprint of conformational information by exposure to PE. More extensive unfolding of LacY using urea-SDS eliminates epitope 4B1 in LacY and prevents its proper refolding in the presence of PE (23Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), also suggesting a requirement for existing structural organization prior to the involvement of PE. A similar concept of "protein memory" was used to describe the action of intramolecular chaperones represented by the propeptides of some proteases (50Shinde U.P. Liu J.J. Inouye M. Nature. 1997; 389: 520-522Crossref PubMed Scopus (141) Google Scholar). Subtilisin and α-lytic protease purified and denatured in vivo fail to refold into active enzymes until their own prosequences are added. Moreover prosequences are not interchangeable in this reaction. In vivo, the propeptide of subtilisin imparts steric information during folding of the protease domain that, after folding is complete, is no longer required to maintain the native conformation. The late folding steps usually correspond to rearrangements of the polypeptide chain within an already compact or near native conformation (34Clark A.C. Frieden C. J. Mol. Biol. 1999; 285: 777-788Google Scholar). The lipid bilayer also affects certain folding events in the final stage of α-helix formation, which appear to be slow and rate-limiting steps in folding. This would suggest that folding intermediates trapped by interaction with the membrane bilayer could undergo conformational changes leading to properly folded integral membrane proteins (16Booth P.J. Riley M.L. Flitsch S.L. Templer R.H. Faroog A. Curran A.R. Chadborn N. Wright P. Biochemistry. 1997; 36: 197-203Crossref PubMed Scopus (105) Google Scholar). PE appears not to be preventing protein aggregation but rescues LacY from or prevents a reversible misfolding event. The order of folding events in the native membrane containing PE is not known. However, in the absence of PE LacY appears to be trapped within an energy minimum off the native folding pathway. Therefore, a lipochaperone could either remove the energy barrier (see Fig. 2 A) or prevent the formation of the energy barrier (see Fig. 2 B), thereby allowing attainment of the final native structure. To fulfill a requirement for molecular chaperone action, lipid must be released from the protein or no longer interact in a manner necessary to maintain protein structure (52Ellis R.J. Cell Stress Chaperones. 1996; 1: 155-160Crossref PubMed Scopus (41) Google Scholar, 53Ellis R.J. Trends Biochem. Sci. 1998; 23: 43-45Abstract Full Text PDF PubMed Scopus (47) Google Scholar). For LacY this criterion is difficult to establish in the native membrane where PE comprises 70% of the phospholipid. However, it is clear that epitope 4B1 of LacY once formed maintains its conformation in the absence of PE, and formation of this epitope is dependent on late folding events requiring PE rather than exposure of the mature or totally unfolded protein to PE (24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar, 25Bogdanov M. Dowhan W. J. Biol. Chem. 1995; 270: 732-739Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The evidence for dissociation of lipid from protein after folding is clear for several other examples of lipochaperone action. DegP appears to require PG for proper folding during passage through the inner membrane of E. coli but is no longer associated with lipid after being released into the periplasm (38Skorko-Glonek J. Lipinska B. Krzewski K. Zolese G. Bertoli E. Tanfani F. J. Biol. Chem. 1997; 272: 8974-8982Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Similarly mitochondrial rhodanese is no longer associated with CL after import through the mitochondrial membrane to the matrix (45Zardeneta G. Horowitz P.M. J. Biol. Chem. 1992; 267: 5811-5816Abstract Full Text PDF PubMed Google Scholar). In the case of PhoE LPS chaperone activity appears to occur in the inner membrane to facilitate the formation of the monomers competent for trimerization in the outer membrane where interaction with LPS appears not to be required. An outer membrane protein chaperone and phospholipid may be required for final release of LPS and maturation into the native trimer (54de Cock H. Schafer U. Potgeter M. Demel R. Muller M. Tommassen J. Eur. J. Biochem. 1999; 259: 96-103Crossref PubMed Scopus (73) Google Scholar). A preference for PE in this late step of assembly was recently observed. 3H. de Cock, personal communication. Reconstitution experiments (55Torok Z. Horvath I. Goloubinoff P. Kovacs E. Glatz A. Balogh G. Vigh L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2192-2197Crossref PubMed Scopus (192) Google Scholar) demonstrated that active GroEL-GroES chaperonin heterooligomers are able to associate with the phospholipid bilayer and may work in concert forming "lipochaperonin" that could assist membrane protein folding as well as the folding of water-soluble proteins that transiently interact while in the molten globular state (55Torok Z. Horvath I. Goloubinoff P. Kovacs E. Glatz A. Balogh G. Vigh L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2192-2197Crossref PubMed Scopus (192) Google Scholar). Components of the protein import machinery, which are required for the efficient post-translational translocation of precursor proteins into the mitochondrial matrix (56Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (978) Google Scholar), could form a "lipochaperonin" complex with CL as suggested from the studies of lipid-dependent unfolding of proteins imported into mitochondria (57Endo T. Eilers M. Schatz G. J. 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Thus lipids can display anti-chaperone activity facilitating aggregation of proteins. It is likely that examples of lipid-assisted protein folding will increase in the near future as the role of individual lipids in the folding of different proteins is further documented. There is no doubt that interaction of hydrophobic proteins with lipids increases the solubility of folding intermediates (64von Heijne G. Mol. Microbiol. 1997; 24: 249-253Crossref PubMed Scopus (29) Google Scholar) and thus prevents irreversible aggregation and promotes proper folding. However, the results reviewed herein strongly suggest a more specific role for lipids than merely providing a solvent for proper membrane protein folding. Whether lipids remain in specific association with proteins after attainment of final structure or not simply defines their role as a structural element or chaperone, respectively. More important is that lipids can assist in folding by interacting with non-native intermediates consistent with the most fundamental definition of molecular chaperones. The limited but broad spectrum of examples of lipid-assisted folding suggests that this is a widespread phenomenon with many similarities to the role of protein molecular chaperones. The molten globular state can be induced upon interaction with lipids and very likely could be an intermediate in proper insertion or translocation across the membrane (30Mel S.F. Stroud R.M. Biochemistry. 1993; 32: 2082-2089Crossref PubMed Scopus (37) Google Scholar, 31van der Goot F.G. Gonzalez-Manas J.M. Lakey J.H. Pattus F. Nature. 1991; 354: 408-410Crossref PubMed Scopus (421) Google Scholar). In this case unfolding of protein is concomitant with membrane insertion or translocation. Alternatively, lipids can participate in the fine-tuning of structure already folded into a near native compact state. In this case insertion and folding are mechanistically distinct and uncoupled events (24Bogdanov M. Dowhan W. EMBO J. 1998; 17: 5255-5264Crossref PubMed Scopus (141) Google Scholar). The consequence of the latter is 2-fold; some proteins could be awakened from a "silent" form by this interaction whereas other proteins could became activated for degradation or to cause adverse effects associated with disease. Future research should now focus on a more detailed understanding of the specific mechanism underlying lipid-assisted folding. What structural features of membrane proteins account for their dependence on lipid assistance? How are lipochaperones released from inserted membrane proteins or excluded from the immediate domain of polypeptides residing in the membrane? Could lipids hold the nascent chain in a conformation that permits interactions with other components of the insertion, folding, and translocation machinery? We thank Drs. John A. DeMoss and Alexey Fedorov for valued input and review of the manuscript before submission. We thank Drs. Hans de Cock and Pam Fredman for sharing results before publication. Phil Heacock provided useful discussion and assistance in preparing the figures.