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Permease from Escherichia Coli Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli Mikhail Bogdanova,1, Philip Heacocka, Ziqiang Guanb, and William Dowhana,1 aDepartment of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, TX, 77030; and bDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710 Edited by William T. Wickner, Dartmouth Medical School, Hanover, NH, and approved July 15, 2010 (received for review May 9, 2010) Phosphatidylcholine (PC) has been widely used in place of naturally N-terminal two-TM helical hairpin of PheP and GabP are in- occurring phosphatidylethanolamine (PE) in reconstitution of verted with respect to their orientation in PE-containing cells bacterial membrane proteins. However, PC does not support native and the remaining TMs. These permeases do not carry out pro- structure or function for several reconstituted transport proteins. ton-coupled energy-dependent uphill transport of substrate in Lactose permease (LacY) of Escherichia coli, when reconstituted cells lacking PE, but still display energy-independent downhill in E. coli phospholipids, exhibits energy-dependent uphill and transport. LacY reconstituted into total E. coli phospholipids car- energy-independent downhill transport function and proper con- ries out uphill transport with domains C6 and P7 on opposite formation of periplasmic domain P7, which is tightly linked to sides of the membrane bilayer as observed in wild-type cells uphill transport function. LacY expressed in cells lacking PE and (9). Leaving out PE during reconstitution results in only downhill containing only anionic phospholipids exhibits only downhill trans- transport with domains C6 and P7 residing on the same side of port and lacks native P7 conformation. Reconstitution of LacY in the bilayer as observed in PE-lacking cells (9). Reconstitution E. coli the presence of -derived PE, but not dioleoyl-PC, results in into proteoliposomes where dioleoyl-PC (diC18∶1PC) replaces uphill transport. We now show that LacY exhibits uphill transport PE results in wild-type topology and downhill transport (9) but and native conformation of P7 when expressed in a mutant of not uphill transport (9–11). An ionizable amine or hydrogen bond BIOCHEMISTRY E. coli in which PC completely replaces PE even though the structure donor capacity is also required for uphill transport because is not completely native. E. coli-derived PC and synthetic PC species methylation of the amine of PE progressively reduces activity containing at least one saturated fatty acid also support the native (10, 11). PE but not diC18∶1PC or eukaryotic-derived PC also sup- conformation of P7 dependent on the presence of anionic phos- ports uphill transport by the multidrug transporter (LmrP) of pholipids. Our results demonstrate that the different effects of Lactococcus lactis (12, 13), the leucine permease of Pseudomonas PE and PC species on LacY structure and function cannot be ex- aeruginosa (14), the branched chain amino acid transporter of plained by differences in the direct interaction of the lipid head Streptococcus cremoris (15), the ABC transporter HorA from groups with specific amino acid residues alone but are due to more Lactococcus lactis (16), and the Ca2þ ATPase of the sarcoplasmic complex effects of the physical and chemical properties of the lipid reticulum (17). environment on protein structure. This conclusion is supported by Another feature of LacY that is strongly correlated with uphill the effect of different lipids on the proper folding of domain P7, transport is the structure of the extramembrane domain P7 (3) which indirectly influences uphill transport function. that is exposed to the periplasm and connects TMs VII and VIII. The conformation-specific monoclonal antibody (mAb) 4B1 ver 35% of all proteins are integrated into cell membranes, recognizes this domain in membranes and on Western blots of Oand many others interact at the membrane surface. There- LacY that has been assembled in PE-containing but not in fore, determining how lipid-protein interactions govern protein PE-lacking E. coli membranes (7). Binding of mAb 4B1 also in- structure and function is fundamental to understanding cellular hibits uphill transport by LacY (2, 3). Epitope recognition is re- processes at the molecular level. Although reconstitution of stored to LacY from PE-lacking membranes by exposure to PE purified membrane proteins with lipids is necessary to study but not diC18∶1PC blotted to the same solid support prior to lipid-protein interactions, the complexity of lipid molecular forms Western blot analysis (8). Recognition by mAb 4B1 appears to that make up the lipidome has largely been ignored in most be a reliable indicator of the proper topological and structural studies, which have employed lipid mixtures that rarely reflect organization of LacY in the vicinity of domain P7 (Fig. 1) and the composition of host membranes. of LacY uphill transport function. Phosphatidylcholine (PC) has been extensively used in recon- The inability of PC to support the full function of several trans- stitution studies because it spontaneously organizes into bilayers. port systems as well as the native structure of domain P7 of LacY However, use of PC for reconstitution of membrane proteins raises several questions of broad significance to studies of from bacterial sources that do not contain PC may not provide membrane protein structure, function, and lipid interaction in re- reliable information, because there are no data on the in vivo constituted systems. Is lack of functional and structural support function of these proteins in membranes where PC replaces the major phospholipid phosphatidylethanolamine (PE). Therefore, a distinction between a biological preference for PE over PC and Author contributions: M.B., P.H., and W.D. designed research; M.B., P.H., and Z.G. an artifact in a reconstituted system cannot be made. performed research; P.H. and Z.G. contributed new reagents/analytic tools; M.B. and W.D. analyzed data; and M.B. and W.D. wrote the paper. The topological orientation and function of the 12 transmem- brane (TM) domain-spanning membrane integrated permeases The authors declare no conflict of interest. for lactose (LacY), phenylalanine (PheP), and γ-aminobutyrate This article is a PNAS Direct Submission. (GabP) of Escherichia coli are dependent on membrane lipid 1To whom correspondence may be addressed at: 6431 Fannin Street, Suite 6.200, Department of Biochemistry and Molecular Biology, University of Texas Medical School composition (1). In mutants lacking PE (normally ca. 70% of at Houston, Houston, TX, 77030. E-mail: [email protected] or William. total phospholipid) and containing only anionic phospholipids [email protected]. [primarily phosphatidylglycerol (PG) and cardiolipin (CL)], the This article contains supporting information online at www.pnas.org/lookup/suppl/ N-terminal six TM helical bundle of LacY (see Fig. 1) and the doi:10.1073/pnas.1006286107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1006286107 PNAS Early Edition ∣ 1of6 Downloaded by guest on October 2, 2021 H205 Legionella pneumophila pcsA gene (22) under induction control NT CT by arabinose of the promoter P . PC is synthesized from Q340 araB L5 C4 C6 R285 endogenous CDP-diacylglycerol and choline from the growth G71 C2 R135 C10 medium. The L. pneumophila gene was selected because the Si- G13 C8 L14 norhizobium meliloti or Bradyrhizobium japonicum genes (22) were unstable in E. coli. Full induction by arabinose and supple- mentation of the medium with choline resulted in a level of PC I II III IV V VIVII VIII IX X XI XII (70%, see Fig. S1) equivalent to the level of PE (70%) in wild- type cells (1). The remaining lipids were primarily CL (27%) and F PG (3%) with the former being higher and the latter being lower F* R than that found in wild-type cells (usually ca. 20–25% PG and I103 T163 T T A312 G* P11 – P1 P3 P5 S P7 P9 5 10% CL). Cells lacking PE contain equal amounts of PG S41 250 F* Q FATG*E and CL (lane 1 of Fig. S1). Mass spectral analysis of total phos- þ − þ 4B1 pholipid extracts from PE and PE PC cells revealed that CT PE and PC of E. coli exhibit a near identical spectrum of species with respect to fatty acid composition (Fig. S2). PE-lacking cells P1 P3 P5 C10 C8 require supplementation with tryptophan for growth in minimal medium due to a defect in uphill transport by aromatic amino acid transporters (23). This defect was corrected by introduction of GlcDAG (23) into −PE mutants and was also corrected by I II III IV V VI VIII IX X XI XII introduction of PC. However, PC did not relieve the requirement for Mg2þ in the growth medium. P9 Transport Function of LacY in −PE þ PC Cells. VII P11 We analyzed transport C2 function of wild-type LacY expressed from a plasmid in strain C4 AL95 without additional plasmids (−PE) or with either plasmid NT pssAþ þ − þ P7 pDD72 ( , PE) or plasmid pAC-PCSlp-Sp-Gm ( PE C6 PC). Choline and Mg2þ were present in all growth media. Con- − Fig. 1. Topological organization of LacY as a function of membrane lipid sistent with previous results (18), LacY assembled in PE cells composition. TMs (Roman numerals), extramembrane domains (P for peri- was unable to catalyze uphill transport of the nonmetabolizable plasmic and C for cytoplasmic as in þPE cells), N-terminus (NT) and C-terminus substrate methyl-β-D-galactopyranoside (TMG) (Fig. 2A). Sur- (CT) domains are indicated. The approximate positions and names of amino prisingly −PE þ PC cells showed the same rate and level of uphill acids substituted by cysteine and used for topological analysis are indicated transport as þPE cells (Fig. 2A), which were confirmed by inhibi- near the closed and open symbols in a cysteineless derivative of LacY used in tion of accumulation of TMG by a protonophore (Fig.
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