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Manipulating conformational equilibria in the lactose permease of Escherichia coli1
doi:10.1006/jmbi.2001.5289 available online at http://www.idealibrary.com on
J. Mol. Biol. (2002) 315, 561±571
Manipulating Conformational Equilibria in the Lactose Permease of Escherichia coli Adam B. Weinglass, Melissa Sondej and H. Ronald Kaback* Howard Hughes Medical Institute, Departments of Physiology and Microbiology & Molecular Genetics, Molecular Biology Institute, University of California Los Angeles Los Angeles, CA 900951662, USA
A mechanism proposed for lactose/H symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding. Moreover, in the ground state, the symported H is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX). Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H to Glu325 and reorientation of the binding site to the inner surface. After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The conservative mutation Glu269 ! Asp causes a 50-100-fold decrease in substrate binding af®nity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and ef¯ux. Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269 ! Asp in an attempt to rescue function, and two mutants with increased activity are identi®ed and characterized. Mutant Thr266 ! Gly/Met267 ! Gly/Glu269 ! Asp binds ligand with increased af®nity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in ef¯ux or equilibrium exchange is observed. In contrast, mutant Gly262 ! Ala/ Glu269 ! Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as ef¯ux and equilibrium exchange is observed. Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease. The ®ndings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H symport. Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H transfer is enhanced from one side of the membrane or the other. When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H transfer are restored. # 2002 Academic Press
*Corresponding author
Keywords: bioenergetics; transport; membrane proteins; substrate binding; conformational change
Introduction The lactose permease (LacY), encoded by the lacY gene of Escherichia coli,1 catalyzes galactoside/ H symport and is a paradigm for transport proteins from Archaea to the mammalian central ner-
vous system that transduce free energy stored in electrochemical ion gradients into solute concentration gradients (reviewed by Kaback2 ± 4). LacY has been solubilized and puri®ed in a completely active state (reviewed by Viitanen et al.5) and func-
Abbreviations used: LacY, lactose permease; TDG, b,D-galactopyranosyl-1-thio-b, RSO, right-side-out; KPi, potassium phosphate; MalNEt, N-ethyl maleimide; CCCP, carbonylcyanide-m-chlorophenylhydrazone. E-mail address of the corresponding author: [email protected]
D-galactopyranoside;
0022-2836/02/040561±11 $35.00/0
# 2002 Academic Press
562 tions as a monomer.6 The molecule contains 12 transmembrane helices connected by hydrophilic loops with both the N and C termini on the cytoplasmic face of the membrane (Figure 1(a)).7 ± 9 In a functional mutant devoid of native Cys residues, each residue has been replaced with Cys.9 Analysis of the mutant library in conjunction with a battery of biochemical and biophysical techniques has led to the following developments:10,11 (a) the great majority of the mutants are expressed normally in the membrane, exhibit signi®cant activity, and only six side-chains are clearly irreplaceable for active transport; Glu126 (helix IV) and Arg144 (helix V), which are indispensable for substrate binding, and Glu269 (helix VIII), Arg302 (helix IX), His322 and Glu325 (helix X), which are critical for coupling sugar and H translocation. (b) Helix packing, tilts and ligand-induced conformational changes have been determined. (c) Positions that are accessible to solvent have been revealed. (d) Positions where the reactivity of the Cys replacement is increased or decreased by ligand binding have been identi®ed. (e) LacY has been shown to be a highly ¯exible molecule. (f) A working model describing a mechanism for lactose/H symport has been described. Site-directed studies utilizing excimer ¯uorescence and engineered divalent metal binding sites provide a strong indication that the four irreplaceable residues involved in H translocation and/or coupling, Glu269 (helix VIII), Arg302 (helix IX) His322 and Glu325 (helix X), are within close proximity.12 ± 15 Furthermore, since interaction between Glu269 and His322 appears to be essential for high-af®nity ligand binding,16,17 and mutations in these residues alter the pH-dependency of ligand binding,17 it is likely that in the ground state, the symported H is shared between Glu269 and His322, and Arg302 is charge paired with Glu325.16,17 In addition, Glu126 (helix IV) and Arg144 (helix V), which are also charge paired, play a critical role in substrate binding.18 ± 21 Glu269 is probably involved directly in protonation and coupling to substrate binding and translocation. Extensive mutagenesis and functional characterization reveal that neutral replacements for Glu269 lead to LacY that is defective in all translocation reactions.22,23 Replacement of Glu269 with Asp yields a permease that hardly catalyzes active lactose transport, ef¯ux down a concentration gradient or equilibrium exchange, but accumulates b,D-galactopyranosyl 1-thio-b,D-galactopyranoside (TDG) with an increase in H:TDG stoichiometry. Mutant E269D{ also exhibits markedly decreased af®nity for TDG.16,17 Gly-scanning mutagenesis has been used to probe conformational ¯exibility and/or helix packing at the substrate binding site of LacY.24 When conserved Gly residues lying one or two turns of
Role of Glu269 in LacY
an a-helix from Arg144 are replaced with Ala in mutant E126D, substrate binding and transport are abolished and remarkably, in two mutants, signi®cant activity is recovered when Ala residues at approximately parallel positions in helix IV are replaced with Gly. In a more recent study,25 conformational ¯exibility and/or helix packing between helices IX and X of mutant E325D was manipulated by Gly-scanning mutagenesis, and replacement of Val316 at the periplasmic end of helix X rescues the activity of mutant E325D, presumably by allowing closer approximation between the carboxylic acid and Arg302, resulting in more ef®cient deprotonation after release of substrate and relaxation of LacY to the ground-state conformation. More recent experiments (A.B.W. & H.R.K., unpublished results) show that Ala replacement for Val316 is equally effective, but replacements that are more bulky than Val completely inactivate both the wild-type and the E325D mutant. Thus, rescue may be due to a change in helix packing at the periplasmic end of helix X that alters the tilt of the helix so that the Asp side-chain at position 325 can more closely re-approximate Arg302 and allow more ef®cient deprotonation of the carboxylic acid. In this study, a similar approach is applied to mutant E269D. In an effort to compensate for the decrease in length of the carboxyl-containing sidechain of mutant E269D, 25 non-essential residues in helix VIII were replaced systematically with Gly, and the two native Gly residues were replaced with Ala (Figure 1(a) and (b)). Two mutants with increased transport activity were identi®ed and characterized. One mutant exhibits increased af®nity for ligand and an increased rate of active transport with no improvement in ef¯ux or equilibrium exchange. The other mutant exhibits only small increases in af®nity and in the rate of active transport, but marked improvement in the steadystate level of accumulation, ef¯ux and equilibrium exchange. When the mutations are combined, all translocation reactions return towards wild-type levels. The results indicate that the precise positioning of a carboxyl group at position 269 is critical for transport, and the mutations that rescue E269D LacY appear to alter the equilibrium between outwardly and inwardly facing conformations.
Results Wild-type LacY versus mutant E269D E. coli T184 (lacZÿYÿ) expressing wild-type LacY catalyzes lactose accumulation at a high rate to a steady-state level of ca 120 nmol/mg of protein in approximately ten minutes, which corresponds to about a 100-fold concentration gradient
{ Site-directed mutants are designated as follows: the one-letter amino acid code is used followed by a number indicating the position of the residue which is followed by the desired mutation at the position.
563
Role of Glu269 in LacY
(Figure 2(a)). In contrast, as reported previously,22,23 E. coli T184 expressing E269D LacY catalyzes lactose transport at less than 5 % the rate of wild-type, and by one hour, the level of accumulation is only 10-15 % of wild-type. Cells transformed with plasmid pT7-5 with no lacY insert exhibit negligible activity. Immunoblotting demonstrates that permease expression is comparable in cells expressing wild-type LacY or the E269D mutant and undetectable in cells harboring plasmid pT7-5 with no lacY insert (data not shown). Kinetic analysis reveals that while the apparent Km (Kapp for the wild-type is 0.65( 0.05) mM, m ) of approximately mutant E269D exhibits a Kapp m 7.5(0.9) mM (Figure 2(b)) and a reduced Vmax (810(90) nmol/min per mg and 106(15) nmol/ min per mg, repectively). Cells expressing wild-type LacY catalyze lactose ef¯ux down a concentration gradient ca. 50-fold more rapidly than cells expressing mutant E269D (t1/2 0.17 minute and 8.8 minutes, respectively (Figure 2(c)). Ef¯ux from cells expressing either wild-type LacY or mutant E269D after inactivation with N-ethyl maleimide (MalNEt) is extremely slow, re¯ecting a very low rate of passive diffusion. Moreover, equilibrium exchange with cells expressing E269D LacY is also approximately 65-fold slower than that with cells expressing wild-type LacY (t1/2 0.08 minute and 6.7 minute, respectively (Figure 2(d)). Again, exchange from cells expressing either wild-type LacY or mutant E269D after inactivation with MalNEt is extremely slow, indicating that virtually all the activity is LacY-mediated. Scanning mutagenesis of helix VIII In an effort to rescue activity in mutant E269D, each non-essential residue in helix VIII was replaced individually with Gly (Figure 3). In
Figure 1. Secondary structure and proposed mechanism of LacY. (a) Secondary structure model. Putative transmembrane helices are shown in boxes. Residues that are irreplaceable with respect to active transport are enlarged; Glu126 (helix IV) and Arg144 (helix V) are critical for substrate binding, and Glu269 (helix VIII), Arg302 (helix IX), His322 (helix X) and Glu325 (helix X) are essential for H translocation and coupling. The charge pairs Asp237 (helix VII)/Lys358 (helix XI) and Asp240 (helix VII)/Lys319 (helix X), which do not play an essential role in the mechanism, are shown. (b) Helices VIII and X are ampli®ed, with Glu269, His322 and Glu325 enlarged and emboldened, and positions where Gly replacement results in a signi®cant increase in activity in mutant E269D are emboldened. (c) Mechanism of lactose/H symport by wild-type lactose permease. For simplicity, six of the 12 helices that comprise LacY are viewed from the outer (periplasmic) surface of the membrane. The grey area designates the low-dielec-
tric environment of the lipid bilayer. In the ground-state conformation, the relevant H is shared by His322 (helix X) and Glu269 (helix VIII), whereas Arg302 (helix IX) is charge paired with Glu325 (helix X). In this conformation, lactose permease binds substrate at the outer surface (So). Glu126 (helix IV) and Arg144 (helix V) are charge paired and, together with Cys148 (helix V), comprise the main components of the substrate-binding site. Also shown is the charge pair between Asp240 (helix VII) and Lys319 (helix X), which is not essential for the mechanism. Substrate binding induces a conformational change that disrupts the E269-H322 and R302-E325 charge pairs, and leads to the transfer of the H to Glu325, which is stabilized by the low dielectric environment. At the same time, the substrate binding site becomes exposed to the inner surface of the membrane. After the substrate dissociates, Glu325 becomes deprotonated at the inner surface, as a result of the rejuxtapositioning of Glu325 with Arg302 as LacY relaxes back to the ground-state conformation.
564
Role of Glu269 in LacY
addition, the two native Gly residues in helix VIII (Gly262 and Gly268) were independently replaced with Ala. Of the 25 mutants, seven exhibit rates of lactose transport ranging from 0-30 % of E269D, 16 exhibit rates ranging from 30-130 % of E269D and two exhibit rates of over 130 % of E269D (Figure 3; *). Furthermore, a complete time-course (up to one hour) was measured for each mutant (data not shown). To test whether the variations in activity are due to differences in LacY expression, immunoblots were carried out on membrane preparations from the same cells for which transport was measured. All of the mutants are expressed in amounts comparable to E269D LacY (data not shown). For both mutants that exhibit enhanced transport activity in mutant E269D, active lactose transport was measured in the wild-type background. While G262A/E269D exhibits a threefold increase in the steady-state level of lactose accumulation in the E269D background, there is a minor reduction in the rate and extent of lactose accumulation in the wild-type background (Figure 4(a) and (b)). Similarly, with mutant T266G/M267G, a fourfold increase in rate is observed in the E269D background, while in the wild-type background, little or no effect on active lactose transport is observed (Figure 4(c) and (d)). Mutants T266G/M267G/E269D and G262A/E269D Kinetic analysis of mutant T266G/M267G/ and Vmax are improved E269D reveals that Kapp m signi®cantly with respect to mutant E269D (2.59(0.22) mM and 450(46) nmol/min per mg versus 7.5(0.9) mM and 106(15) nmol/min per mg, repectively). In contrast, with mutant G262A/E269D, there is little change in kinetic par-
Figure 2. Wild-type permease versus mutant E269D. (a) Time-courses of active transport by E. coli T184 expressing wild-type permease (WT), no permease (pT75 with no lacY insert) or the mutant E269D. Aliquots (50 ml) of cell suspensions containing 35 mg of protein in 100 mM KPi (pH 7.5), 10 mM MgSO4 were assayed at 0.4 mM ®nal external concentration of lactose as
described in Materials and Methods. (b) Concentrationdependent lactose transport by E. coli T184 expressing wild-type permease or the mutant E269D. Initial rates of transport were determined at concentrations ranging from 40 mM to 5 mM [1-14C]lactose as described in Materials and Methods. For each datum point, nonspeci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted. Data were ®tted to the Michaelis-Menten equation. (c) Time-course of lactose ef¯ux by E. coli T184 expressing WT or the mutant E269D. Aliquots of cells equilibrated with 10 mM [1-14C]lactose in 100 mM KPI (pH 7.5), 10 mM MgSO4 and 20 mM CCCP were diluted 100-fold into the same solution without lactose as described in Materials and Methods. Where indicated, MalNEt (NEM, 10 mM, ®nal concentration) was used to inactivate LacY. (d) Time-course of lactose equilibrium exchange by E. coli T184 expressing WT or the mutant E269D. Cells were prepared as described for lactose ef¯ux, except they were diluted into the same solution with 10 mM lactose as described in Materials and Methods. Where indicated, MalNEt (NEM, 10 mM, ®nal concentration) was used to inactivate LacY.
Role of Glu269 in LacY
565
Figure 3. Active transport by E. coli T184 expressing E269D and individual Gly or Ala replacement mutants. The single-letter amino-acid code is used along the horizontal axis to denote the original residues from Arg259 to Ile283 in helix VIII. Complete times-courses of transport up to one hour were measured, and the rates of transport measured at two minutes as described in Materials and Methods. The results are expressed as a percentage of E269D activity.
ameters relative to E269D (Kapp m 9.3 mM and Vmax 160(12) mmol/min per mg) (Figure 5(a)). Since mutant E269D exhibits about a 100-fold decrease in af®nity relative to the wild-type,17 the increased activity of mutants T266G/M267G/ E269D and G262A/E269D may be due to increased substrate af®nity. Therefore, the effect of the mutations on the af®nity of mutant E269D for
TDG was appproximated by measuring the inhibition constant (Ki) of TDG with the wild-type and mutants E269D, G262A/E269D or T266G/M267G/ E269D at a lactose concentration around the Kapp m of each mutant. As reported previously,17 the Ki of the wild-type and mutant E269D are 48(4) mM and 3250(250) mM, respectively (Figure 5(b)). Interestingly, mutant T266G/M267G/E269D exhi-
Figure 4. Effects of Ala replacement at Gly262 and Gly replacement at Thr266/Met267. Timecourses of active transport by E. coli T184 expressing wild-type (WT) permease or E269D mutants with Ala in place of the native Gly residue at (a) and (b) position 262 or (c) and (d) Gly in place of the native Thr and Met at positions 266 and 267. Experiments were carried out as described in Materials and Methods.
566
Role of Glu269 in LacY
relative to E269D (t1/2 5.88 minutes versus 6.7 minutes (Figure 6(d)) is observed. Mutant G262A/T266G/M267G/E269D
Figure 5. Kinetics of lactose transport and effect of TDG. (a) Concentration-dependent lactose transport by E. coli T184 expressing WT permease, mutant T266G/ M267G/E269D, mutant G262A/E269D or mutant E269D. Initial rates of transport were determined at concentrations ranging from 40 mM to 5 mM [1-14C]lactose as described in Materials and Methods. For each datum point, non-speci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted. Data were ®tted to the Michaelis-Menten equation. (b) Concentration-dependent inhibition of lactose transport by TDG in E. coli T184 expressing WT permease, mutant T266G/M267G/E269D, mutant G262A/E269D or mutant E269D. Using a concentration of [1-14C]lactose equal to the Km of WT or a given mutant, initial rates of transport were determined in the presence of varying concentrations of TDG. For each datum point, nonspeci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted.
bits about a tenfold increase in af®nity for TDG with respect to mutant E269D (Ki 322 mM), while mutant G262A/E269D exhibits no change in af®nity (Ki 6389 mM). Lactose ef¯ux and equilibrium exchange measurements with mutant G262A/E269D reveal marked increases in the rate of ef¯ux (t1/2 2.3 versus 8.8 minutes (Figure 6(a)), as well as equilibrium exchange (t1/2 0.16 minutes versus 6.7 minutes (Figure 6(b)). Remarkably, with mutant T266G/M267G/E269D, only minor improvement in the rate of ef¯ux (t1/2 6.6 versus 8.8 minutes (Figure 6(c)) or equilibrium exchange
Since mutant G262A/E269D exhibits improved ef¯ux and exchange, while mutant T266G/ M267G/E269D exhibits improved af®nity and an increased rate of accumulation, both sets of mutants were combined to produce mutant G262A/T266G/M267G/E269D. Remarkably, the time-course of lactose active transport for this mutant exhibits a rate of accumulation similar to that of mutant T266G/M267G/E269D, and the same steady-state level of accumulation as G262A/ E269D (Figure 7(a)). Kinetic analysis reveals that and Vmax are improved signi®cantly with Kapp m respect to E269D (2.7 mM and 408(35) mmol/min per mg versus 7.5 mM and 146(4) mmol/min per mg). (Figure 7(b)). Furthermore, mutant G262A/ T266G/M267G/E269D exhibits a Ki of around 200 mM for TDG, demonstrating more than a tenfold increase in af®nity relative to mutant E269D (Figure 7(c)). In addition, lactose ef¯ux and equilibrium exchange are both improved dramatically in mutant G262A/T266G/M267G/E269D relative to mutant E269D (t1/2 0.45 versus 8.8 minutes, respectively, for ef¯ux (Figure 7(d)); t1/2 0.08 versus 6.7 minutes, respectively, for equilibrium exchange (Figure 7(e)).
Discussion Recent studies in which ligand binding af®nity was measured as a function of pH in single-Cys148 permease with various replacements for Glu269, Arg302, His322 and Glu325 have led to a working model describing a mechanism for lactose/H symport17 (Figure 1(c)). In the ground state, LacY is protonated, the H is shared between His322 and Glu269, Glu325 is charge paired with Arg302, and substrate is bound with high af®nity between helices IV (Glu126) and V (Arg144 and Cys148) from the outside surface of the membrane. Substrate binding induces a conformational change that leads to transfer of H from His322/Glu269 to Glu325 and reorientation of the binding site to the inner surface and dissociation of substrate. Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The His322/Glu269 complex is then protonated from the outside to re-initiate the cycle. This scheme can be described kinetically, as shown in Figure 8. The only replacement for Glu269 that catalyzes transport to any extent whatsoever is Asp, and even with the shortened carboxylate-containing side-chain, a tenfold reduction in both the Km and Vmax for active lactose transport is observed with a 50-fold reduction in af®nity for the highaf®nity analog TDG. Similarly, both lactose ef¯ux down a concentration gradient (which occurs in symport with H) and equilibrium exchange (which is thought to occur with an H
Role of Glu269 in LacY
567
Figure 6. Time-course of lactose ef¯ux by E. coli T184 expressing WT, mutant E269D, (a) mutant G262A/E269D or (c) mutant T266G/M267G/E269D. Aliquots of cells equilibrated with 10 mM [1-14C]lactose in 100 mM KPI (pH 7.5), 10 mM MgSO4 and 20 mM CCCP were diluted 100-fold into the same solution without lactose as described in Materials and Methods. MalNEt (10 mM, ®nal concentration) was used to inactivate LacY (not shown). Timecourse of lactose equilibrium exchange by E. coli T184 expressing WT, mutant E269D, (b) mutant G262A/E269D or (d) mutant T266G/M267G/E269D. Cells were prepared as described for lactose ef¯ux, except they were diluted into the same solution with 10 mM lactose as described in Materials and Methods. MalNEt (10 mM, ®nal concentration) was used to inactivate LacY.
shared between Glu269 and His322 or oscillating between Glu269/His322 and His322/Glu325)26 are also at least 50-fold slower with mutant E269D LacY relative to wild-type. Mechanistically, the primary defect in E269D seems to be a major decrease in protonation of either the outwardly or inwardly facing conformation of LacY, which leads to a decrease in substrate af®nity, as well as a defect in all translocation reactions that require protonation of the protein. For lactose translocation (Figure 8), shortening the carboxyl-containing side-chain at position 269 seems to effect the following steps in the kinetic cycle. (1) Interaction between Asp269 and His322 is reduced, and protonation is less ef®cient (i.e. state 1 is preferred over state 2). (2) The conformational change promoted by protonation of Glu269 is compromised leading to low-af®nity substrate binding. (3) Poor positioning of the H between Asp269 and His322 leads to inef®cient transfer to His322/Glu325 and ®nally to Glu325 (i.e. the transition between conformations 3 through 5), leading to inef®cient H translocation. As shown previously,24,25 manipulation of conformational dynamics and/or helix packing may be used to rescue function of inactive or partially inactive mutants. Here, each non-essential residue in helix VIII, which contains the E269D mutation, was replaced with Gly or both of the native Gly residues in helix VIII were replaced with Ala in an effort to compensate for the shortened carboxyl-
containing side-chain of the Asp residue in place of Glu269. Two mutants are identi®ed that exhibit improved transport, T266G/M267G/E269D and G262A/E269D, and it should be emphasized that replacement of the native residues with Cys in Cys-less LacY does not increase activity,27 nor do the mutations have a signi®cant effect on the activity of wild-type LacY. Mutant T266G/M267G/E269D exhibits an increase in af®nity for TDG, and the rate of lactose transport is improved markedly without signi®cant rescue of ef¯ux or equilibrium exchange. Possibly, reduction in the volume of the amino acid side-chains at positions 266 and 267 creates a packing surface that allows an adjacent helix to alter the tilt angle of helix VIII. Alternatively, replacement of Thr266 and Met267 with Gly results in looser helix packing in the immediate environment, thereby permitting greater mobility of the Asp269 side-chain. Either of these possibilities might result in a closer approximation between Asp269 and His322, which improves protonation and leads to improved substrate af®nity. Thus, the equilibrium between states 1 and 2 is shifted towards state 2 (Figure 8). That is, the outwardly facing conformation is favored, and the mutant is protonated more ef®ciently than with E269D alone. The observation that ef¯ux and equilibrium exchange are not rescued in this mutant is consistent with the interpretation that the ability of the inwardly facing conformation of the mutant to transfer H to
568
Role of Glu269 in LacY
Figure 8. Kinetic model describing lactose translocation reactions catalyzed by LacY. Also indicated are the steps at which residues Glu269, His322 and Glu325 are thought to be involved. C, LacY; H, transported proton; S, sugar.
Glu325/His322 and/or His322/Asp269 remains impaired (i.e. the equilibrium between steps 5 and 4 and/or 4 and 3 lie towards steps 5 and 4). Although H movements can occur in both directions, in the presence of a m H (interior negative and/or alkaline), in¯ux would be favored. However, during ef¯ux or equilibrium exchange that are measured in the absence of
Figure 7. Properties of G262A/T266G/M267G/ E269D. (a) Time-course of active transport by E. coli T184 expressing WT permease, no permease (pT7-5 with no lacY insert), mutant E269D or mutant G262A/ T266G/M267G/E269D. Aliquots (50 ml) of cell suspensions containing 35 mg of protein in 100 mM KPi (pH 7.5), 10 mM MgSO4 were assayed at 0.4 mM ®nal external concentration of lactose as described in Materials and Methods. (b) Concentration-dependent
lactose transport by E. coli T184 expressing wild-type permease, mutant E269D or mutant G262A/T266G/ M267G/E269D. Initial rates of transport were determined at concentrations ranging from 40 mM to 5 mM [1-14C]lactose as described in Materials and Methods. For each datum point, non-speci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted. Data were ®tted to the Michaelis-Menten equation. (c) Concentration-dependent inhibition of lactose transport by TDG in E. coli T184 expressing WT permease, mutant G262A/T266G/M267G/E269D or the mutant E269D. Using a concentration of [1-14C]lactose equal to the Km of the sample, initial rates of transport were determined in the presence of varying concentrations of TDG. For each data point, nonspeci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted. (d) Time-course of lactose ef¯ux by E. coli T184 expressing WT, mutant E269D or mutant G262A/T266G/M267G/E269D. Aliquots of cells equilibrated with 10 mM [1-14C]lactose in 100 mM KPI (pH 7.5), 10 mM MgSO4 and 20 mM CCCP were diluted 100-fold into the same solution without lactose as described in Materials and Methods. (e) Time-course of lactose equilibrium exchange by E. coli T184 expressing WT, mutant E269D or mutant G262A/T266G/M267G/ E269D. Aliquots of cells equilibrated with 10 mM [1-14C]lactose in 100 mM KPI (pH 7.5), 10 mM MgSO4 and 20 mM CCCP were diluted 100-fold into the same solution with lactose as described in Materials and Methods.
569
Role of Glu269 in LacY
m H , there is inef®cient transfer of H from Glu325 to His322/Asp269 and therefore poor ef¯ux and exchange. Mutant G262A/E269D leads to a reciprocal phenotype where ef¯ux and equilibrium exchange are rescued with only marginal effects on af®nity or the rate of active transport. Since Gly residues in many membrane proteins form notches, allowing surfaces to pack tightly,28,29 and the tertiary structure model of LacY indicates that the periplasmic ends of helices VIII and X are adjacent 11, it is conceivable that the greater volume of the side-chain at position 262 in mutant G262A (which lies on the same face of helix VIII as Asp269, separated by two turns of an a-helix) leads to a subtle change in the tilt of helix VIII, thereby allowing Asp269 to approximate His322 more closely. Improved ef¯ux and exchange in this mutant suggest that transfer of H from Glu325 to His322/Asp269 is more ef®cient than in the E269D mutant alone (i.e. the inwardly facing conformation is favored). However, the lack of improvement in active transport argues that His322/Asp269 is unable to protonate ef®ciently from the outside. Clearly, subtle manipulations in the precise positioning of the carboxyl side-chain at position 269 elicit drastic changes in the transport properties of mutants T266G/M267G/E269D and G262A/ E269D by improving protonation of either the outwardly or inwardly facing conformations of LacY, respectively. Remarkably, by combining the two sets of mutations, all translocation reactions are rescued signi®cantly in mutant G262A/T266G/ M267G/E269D. In this case, the equilibrium between the outwardly and inwardly facing conformations approximates that of wild-type LacY more closely, and the mutant can then be protonated more ef®ciently from either side of the membrane.
Materials and Methods Materials Oligodeoxynucleotides were synthesized by SigmaGenosys (The Woodland, TX). Restriction endonucleases, phage T4 DNA ligase and Vent polymerase were from New England Biolabs (Beverly, MA). All other materials were of reagent grade and were obtained from commercial sources.
Growth of bacteria E. coli T184 [lacIOZÿYÿ(A), rpsL, metÿ, thr, recA, hsdM, hsdR/F0 , lacIqOZD118(YA)]30 transformed with plasmid pT7-5/cassette lacY encoding given LacY mutants was grown aerobically at 37 C in Luria-Bertani broth with ampicillin (100 mg/ml). Fully grown cultures were diluted tenfold and grown for two hours at 37 C before induction with 1 mM isopropyl 1-thio-b,D-galactopyranoside. After additional growth for two hours at 37 C, cells were harvested by centrifugation.
Construction of LacY mutants Using plasmid pT7-5/cassette lacY encoding mutant E269D permease, oligonucleotide-directed site-speci®c mutagenesis by two-step PCR31 was used to replace each of 25 non-essential residues in helix VIII with Gly individually. In a similar manner, each native Gly in helix VIII was replaced with Ala. Following restriction endonuclease digestion with KpnI and SpeI, the PCR products were sub-cloned back into the similarly treated parental vector. The identical procedure was used to replace given residues in the same region with Gly or Ala in the wild-type background. The KpnI-SpeI region of the lacY gene in all mutants was fully sequenced through the restriction sites by using the dideoxynucleotide method32 on an ABI 373A automatic sequencer. Transport assays E. coli T184 expressing given LacY mutants was grown at 37 C, subsequently washed once with 100 mM KPi (pH 7.5), 10 mM MgSO4 and adjusted to an A420 of 10 (0.7 mg of protein/ml). Transport was initiated by addition of [1-14C]lactose (5 mCi/mmol) to a ®nal concentration of 0.4 mM. Samples were quenched at given times with 100 mM KPi (pH 5.5), 100 mM LiCl and assayed by rapid ®ltration.33 To determine kinetic parameters, cells were concentrated to an A420 of 20, and 50 ml of cells was mixed with 50 ml of [1-14C]lactose (0.06-10 mM ®nal concentrations). Initial rates were measured over one minute for cells expressing wild-type LacY and over two minutes for cells expressing E269D LacY or derived mutants. Values were corrected for lactose uptake by cells carrying the pT7-5 vector with no lacY insert. To measure the Ki, cells were adjusted to an A420 of 10, and 50 ml of cells was mixed with 1 ml of given concentrations of TDG for ®ve minutes. Initial rates of [1-14C]lactose transport were measured over one minute for cells expressing wild-type permease and over two minutes for cells expressing E269D LacY or derived mutants. Values were corrected for lactose uptake by cells carrying the pT7-5 vector with no lacY insert. To measure lactose ef¯ux, E. coli T184 cells expressing given LacY mutants were re-suspended at 3-4 mg of protein/ml in 100 mM KPi (pH 7.5), EDTA was added to a ®nal concentration of 10 mM, the suspensions were incubated at 37 C for two minutes and placed on ice. Cells were then washed with 100 mM KPi (pH 7.5) and re-suspended in 100 mM KPi (pH 7.5), 10 mM MgSO4 before adjusting to an A420 of 40 (2.8 mg protein/ml). [1-14C]Lactose (10 mM; 10 mCi/mmol) and 20 mM carbonylcyanide-m-chlorophenylhydrazone (CCCP) were added to each sample, and the suspensions were equilibriated on ice overnight. Aliquots (10 ml) of each sample were diluted 1:100 (v/v) in KPi (pH 7.5) containing 20 mM CCCP, and at given times the reactions were quenched with 100 mM KPi (pH 5.5), 100 mM LiCl and ®ltered immediately.33 Where indicated, preloaded cells were diluted 1:100 (v/v) in 100 mM KPi (pH 7.5), 20 mM CCCP and 10 mM MalNEt to inactivate LacY. Zerotime values were determined by dilution of 10 ml; aliquots directly into quench buffer followed by rapid ®ltration. For equilibrium exchange, cells were treated identically; however, the samples were diluted 1:100 (v/v) in KPi (pH 7.5) containing 20 mM CCCP and an
570 equal concentration of unlabeled lactose prior to quenching the reactions as described. Western blotting Crude membranes from the same cells utilized for active transport assays were prepared by osmotic lysis and sonication.27 Total membrane protein was assayed by a modi®ed Lowry procedure.34 A sample containing 60 mg of membrane protein from each sample was subjected to electrophoresis in SDS/12 % (w/v) polyacylamide gels.35 Proteins were electroblotted on to polyvinylidene ¯uoride membranes (Immobilon-PVDF; Millipore) and probed with site-directed antibody against the C terminus of LacY (residues 402-417)36 prior to treatment with anti-mouse IgG-conjugated horse radish peroxidase.
Acknowledgments We thank MikloÂs Sahin-ToÂth, Irina Smirnova, Vladimir Kasho and Jose Luis Vazquez Ibar for helpful discussions. This work was supported, in part, by NIH grant DK51131:06 to H.R.K.
References 1. MuÈller-Hill, B. (1996). The lac Operon: A Short History of a Genetic Paradigm, Walter de Gruyter, Berlin, New York. 2. Kaback, H. R. (1976). Molecular biology and energetics of membrane transport. J. Cell Physiol. 89, 575593. 3. Kaback, H. R. (1983). The lac carrier protein in Escherichia coli: from membrane to molecule. J. Membr. Biol. 76, 95-112. 4. Kaback, H. R. (1989). Molecular biology of active transport: from membranes to molecules to mechanism. Harvey Lectures, 83, 77-103. 5. Viitanen, P., Newman, M. J., Foster, D. L., Wilson, T. H. & Kaback, H. R. (1986). Puri®cation, reconstitution, and characterization of the lac permease of Escherichia coli. Methods Enzymol. 125, 429-452. 6. Sahin-ToÂth, M., Lawrence, M. C. & Kaback, H. R. (1994). Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli. Proc. Natl Acad. Sci. USA, 91, 5421-5425. 7. Kaback, H. R. (1996). The lactose permease of Escherichia coli: past, present and future. In Handbook of Biological Physics: Transport Processes in Eukaryotic and Prokaryotic Organisms (Konings, W. N., Kaback, H. R. & Lolkema, J. S., eds), vol. 2, pp. 203-227, Elsevier, Amsterdam. 8. Kaback, H. R. & Wu, J. (1997). From membrane to molecule to the third amino acid from the left with the lactose permease of Escherichia coli. Quart. Rev. Biophys. 30, 333-364. 9. Frillingos, S., Sahin-ToÂth, M., Wu, J. & Kaback, H. R. (1998). Cys-scanning mutagenesis: a novel approach to structure-functions relationships in polytopic membrane proteins. FASEB J. 12, 1281-1299. 10. Kaback, H. R. & Wu, J. (1999). What to do while awaiting crystals of a membrane transport protein and thereafter. Accts Chem. Res. 32, 805-813.
Role of Glu269 in LacY 11. Kaback, H. R., Sahin-ToÂth, M. & Weinglass, A. B. (2001). The kamikaze approach to membrane transport. Nature Rev. Mol. Cell Biol. 2, 610-622. 12. Jung, K., Jung, H., Wu, J., PriveÂ, G. G. & Kaback, H. R. (1993). Use of site-directed ¯uorescence labeling to study proximity relationships in the lactose permease of Escherichia coli. Biochemistry, 32, 1227312278. 13. Jung, K., Voss, J., He, M., Hubbell, W. L. & Kaback, H. R. (1995). Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli. Biochemistry, 34, 6272-6277. 14. He, M. M., Voss, J., Hubbell, W. L. & Kaback, H. R. (1995). Use of designed metal binding sites to study helix proximity in the lactose permease of Escherichia coli. 2. proximity of helix IX (Arg302) with helix X (His322 and Glu325). Biochemistry, 34, 15667-15670. 15. He, M., Voss, J., Hubbell, W. L. & Kaback, H. R. (1997). Arginine 302 (helix X) in the lactose permease of Escherichia coli is in close proximity to glutamate 269 (helix VIII) as well as glutamate 325 (helix X). Biochemistry, 36, 13682-13687. 16. He, M. & Kaback, H. R. (1997). Interaction between residues Glu269 (Helix VIII) and His322 (Helix X) of the lactose permease of Escherichia coli is essential for substrate binding. Biochemistry, 36, 13688-13692. 17. Sahin-ToÂth, M., Karlin, A. & Kaback, H. R. (2000). Unraveling the mechanism of lactose permease of Escherichia coli. Proc. Natl Acad. Sci. USA, 97, 1072910732. 18. Venkatesan, P. & Kaback, H. R. (1998). The substrate binding site in the lactose permease of Escherichia coli. Proc. Natl Acad. Sci. USA, 95, 9802-9807. 19. Zhao, M., Zen, K.-C., Hubbell, W. & Kaback, H. R. (1999). Proximity between Glu126 and Arg144 in the lactose permease of Escherichia coli. Biochemistry, 38, 7407-7412. 20. Sahin-ToÂth, M., le Coutre, J., Kharabi, D., le Maire, G., Lee, J. C. & Kaback, H. R. (1999). Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli. Biochemistry, 38, 813-819. 21. Sahin-ToÂth, M., Akhoon, K. M., Runner, J. & Kaback, H. R. (2000). Ligand recognition by the lactose permease of Escherichia coli: speci®city and af®nity are de®ned by distinct structural elements of galactopyranosides. Biochemistry, 39, 5907-5103. 22. Ujwal, M. L., Sahin-ToÂth, M., Persson, B. & Kaback, H. R. (1994). Role of glutamate-269 in the lactose permease of Escherichia coli. Mol. Membr. Biol. 1, 9-16. 23. Franco, P. J. & Brooker, R. J. (1994). Functional roles of Glu-269 and Glu-325 within the lactose permease of Escherichia coli. J. Biol. Chem. 269, 7379-7386. 24. Weinglass, A. B. & Kaback, H. R. (1999). Conformational ¯exibility at the substrate binding site in the lactose permease of Escherichia coli. Proc. Natl Acad. Sci. USA, 96, 11178-11182. 25. Weinglass, A. B., Smirnova, I. N. & Kaback, H. R. (2001). Engineering conformational ¯exibility in the lactose permease of Escherichia coli: use of glycinescanning mutagenesis to rescue mutant Glu325 Asp. Biochemistry, 40, 769-776. 26. Zhang, W. & Kaback, H. R. (2000). Effect of the lipid phase transition on the lactose permease from Escherichia coli. Biochemistry, 47, 14538-14542. 27. Sahin-ToÂth, M. & Kaback, H. R. (1993). Cysteine scanning mutagenesis of putative transmembrane
571
Role of Glu269 in LacY
28.
29.
30.
31.
helices IX and X in the lactose permease of Escherichia coli. Protein Sci. 2, 1024-1033. MacKenzie, K. R. & Engelman, D. M. (1998). Structure-based prediction of the stability of transmembrane helix-helix interactions: the sequence dependence of glycophorin A dimerization. Proc. Natl Acad. Sci. USA, 95, 3583-3590. Javadpour, M. M., Eiler, M., Groesbeek, M. & Smith, S. O. (1999). Helix packing in polytopic membrane proteins: role of glycine residues in transmembrane helix association. Biophys. J. 77, 1609-1618. Teather, R. M., Bramhall, J., Riede, I., Wright, J. K., Furst, M., Aichele, G. et al. (1980). Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y-gene of the lac operon. Eur. J. Biochem. 108, 223-231. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. (1989). Site-directed mutagenesis by
32. 33. 34. 35.
36.
overlap extension using the polymerase chain reaction. Gene, 77, 51-59. Sanger, F., Nicklen, S. & Coulsen, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA, 74, 5463-5467. Kaback, H. R. (1974). Transport in isolated bacterial membrane vesicles. Methods Enzymol. 31, 698-709. Peterson, G. L. (1977). A simpli®cation of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346-356. Newman, M. J., Foster, D. L., Wilson, T. H. & Kaback, H. R. (1981). Puri®cation and reconstitution of functional lactose carrier from Escherichia coli. J. Biol. Chem. 256, 11804-11808. Carrasco, N., Herzlinger, D., Mitchell, R., DeChiara, S., Danho, W., Gabriel, T. F. & Kaback, H. R. (1984). Intramolecular dislocation of the COOH terminus of the lac carrier protein in reconstituted proteoliposomes. Proc. Natl Acad. Sci. USA, 81, 4672-4676.
Edited by G. von Heijne (Received 4 September 2001; received in revised form 23 November 2001; accepted 23 November 2001)
J. Mol. Biol. (2002) 315, 561±571
Manipulating Conformational Equilibria in the Lactose Permease of Escherichia coli Adam B. Weinglass, Melissa Sondej and H. Ronald Kaback* Howard Hughes Medical Institute, Departments of Physiology and Microbiology & Molecular Genetics, Molecular Biology Institute, University of California Los Angeles Los Angeles, CA 900951662, USA
A mechanism proposed for lactose/H symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding. Moreover, in the ground state, the symported H is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX). Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H to Glu325 and reorientation of the binding site to the inner surface. After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The conservative mutation Glu269 ! Asp causes a 50-100-fold decrease in substrate binding af®nity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and ef¯ux. Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269 ! Asp in an attempt to rescue function, and two mutants with increased activity are identi®ed and characterized. Mutant Thr266 ! Gly/Met267 ! Gly/Glu269 ! Asp binds ligand with increased af®nity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in ef¯ux or equilibrium exchange is observed. In contrast, mutant Gly262 ! Ala/ Glu269 ! Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as ef¯ux and equilibrium exchange is observed. Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease. The ®ndings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H symport. Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H transfer is enhanced from one side of the membrane or the other. When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H transfer are restored. # 2002 Academic Press
*Corresponding author
Keywords: bioenergetics; transport; membrane proteins; substrate binding; conformational change
Introduction The lactose permease (LacY), encoded by the lacY gene of Escherichia coli,1 catalyzes galactoside/ H symport and is a paradigm for transport proteins from Archaea to the mammalian central ner-
vous system that transduce free energy stored in electrochemical ion gradients into solute concentration gradients (reviewed by Kaback2 ± 4). LacY has been solubilized and puri®ed in a completely active state (reviewed by Viitanen et al.5) and func-
Abbreviations used: LacY, lactose permease; TDG, b,D-galactopyranosyl-1-thio-b, RSO, right-side-out; KPi, potassium phosphate; MalNEt, N-ethyl maleimide; CCCP, carbonylcyanide-m-chlorophenylhydrazone. E-mail address of the corresponding author: [email protected]
D-galactopyranoside;
0022-2836/02/040561±11 $35.00/0
# 2002 Academic Press
562 tions as a monomer.6 The molecule contains 12 transmembrane helices connected by hydrophilic loops with both the N and C termini on the cytoplasmic face of the membrane (Figure 1(a)).7 ± 9 In a functional mutant devoid of native Cys residues, each residue has been replaced with Cys.9 Analysis of the mutant library in conjunction with a battery of biochemical and biophysical techniques has led to the following developments:10,11 (a) the great majority of the mutants are expressed normally in the membrane, exhibit signi®cant activity, and only six side-chains are clearly irreplaceable for active transport; Glu126 (helix IV) and Arg144 (helix V), which are indispensable for substrate binding, and Glu269 (helix VIII), Arg302 (helix IX), His322 and Glu325 (helix X), which are critical for coupling sugar and H translocation. (b) Helix packing, tilts and ligand-induced conformational changes have been determined. (c) Positions that are accessible to solvent have been revealed. (d) Positions where the reactivity of the Cys replacement is increased or decreased by ligand binding have been identi®ed. (e) LacY has been shown to be a highly ¯exible molecule. (f) A working model describing a mechanism for lactose/H symport has been described. Site-directed studies utilizing excimer ¯uorescence and engineered divalent metal binding sites provide a strong indication that the four irreplaceable residues involved in H translocation and/or coupling, Glu269 (helix VIII), Arg302 (helix IX) His322 and Glu325 (helix X), are within close proximity.12 ± 15 Furthermore, since interaction between Glu269 and His322 appears to be essential for high-af®nity ligand binding,16,17 and mutations in these residues alter the pH-dependency of ligand binding,17 it is likely that in the ground state, the symported H is shared between Glu269 and His322, and Arg302 is charge paired with Glu325.16,17 In addition, Glu126 (helix IV) and Arg144 (helix V), which are also charge paired, play a critical role in substrate binding.18 ± 21 Glu269 is probably involved directly in protonation and coupling to substrate binding and translocation. Extensive mutagenesis and functional characterization reveal that neutral replacements for Glu269 lead to LacY that is defective in all translocation reactions.22,23 Replacement of Glu269 with Asp yields a permease that hardly catalyzes active lactose transport, ef¯ux down a concentration gradient or equilibrium exchange, but accumulates b,D-galactopyranosyl 1-thio-b,D-galactopyranoside (TDG) with an increase in H:TDG stoichiometry. Mutant E269D{ also exhibits markedly decreased af®nity for TDG.16,17 Gly-scanning mutagenesis has been used to probe conformational ¯exibility and/or helix packing at the substrate binding site of LacY.24 When conserved Gly residues lying one or two turns of
Role of Glu269 in LacY
an a-helix from Arg144 are replaced with Ala in mutant E126D, substrate binding and transport are abolished and remarkably, in two mutants, signi®cant activity is recovered when Ala residues at approximately parallel positions in helix IV are replaced with Gly. In a more recent study,25 conformational ¯exibility and/or helix packing between helices IX and X of mutant E325D was manipulated by Gly-scanning mutagenesis, and replacement of Val316 at the periplasmic end of helix X rescues the activity of mutant E325D, presumably by allowing closer approximation between the carboxylic acid and Arg302, resulting in more ef®cient deprotonation after release of substrate and relaxation of LacY to the ground-state conformation. More recent experiments (A.B.W. & H.R.K., unpublished results) show that Ala replacement for Val316 is equally effective, but replacements that are more bulky than Val completely inactivate both the wild-type and the E325D mutant. Thus, rescue may be due to a change in helix packing at the periplasmic end of helix X that alters the tilt of the helix so that the Asp side-chain at position 325 can more closely re-approximate Arg302 and allow more ef®cient deprotonation of the carboxylic acid. In this study, a similar approach is applied to mutant E269D. In an effort to compensate for the decrease in length of the carboxyl-containing sidechain of mutant E269D, 25 non-essential residues in helix VIII were replaced systematically with Gly, and the two native Gly residues were replaced with Ala (Figure 1(a) and (b)). Two mutants with increased transport activity were identi®ed and characterized. One mutant exhibits increased af®nity for ligand and an increased rate of active transport with no improvement in ef¯ux or equilibrium exchange. The other mutant exhibits only small increases in af®nity and in the rate of active transport, but marked improvement in the steadystate level of accumulation, ef¯ux and equilibrium exchange. When the mutations are combined, all translocation reactions return towards wild-type levels. The results indicate that the precise positioning of a carboxyl group at position 269 is critical for transport, and the mutations that rescue E269D LacY appear to alter the equilibrium between outwardly and inwardly facing conformations.
Results Wild-type LacY versus mutant E269D E. coli T184 (lacZÿYÿ) expressing wild-type LacY catalyzes lactose accumulation at a high rate to a steady-state level of ca 120 nmol/mg of protein in approximately ten minutes, which corresponds to about a 100-fold concentration gradient
{ Site-directed mutants are designated as follows: the one-letter amino acid code is used followed by a number indicating the position of the residue which is followed by the desired mutation at the position.
563
Role of Glu269 in LacY
(Figure 2(a)). In contrast, as reported previously,22,23 E. coli T184 expressing E269D LacY catalyzes lactose transport at less than 5 % the rate of wild-type, and by one hour, the level of accumulation is only 10-15 % of wild-type. Cells transformed with plasmid pT7-5 with no lacY insert exhibit negligible activity. Immunoblotting demonstrates that permease expression is comparable in cells expressing wild-type LacY or the E269D mutant and undetectable in cells harboring plasmid pT7-5 with no lacY insert (data not shown). Kinetic analysis reveals that while the apparent Km (Kapp for the wild-type is 0.65( 0.05) mM, m ) of approximately mutant E269D exhibits a Kapp m 7.5(0.9) mM (Figure 2(b)) and a reduced Vmax (810(90) nmol/min per mg and 106(15) nmol/ min per mg, repectively). Cells expressing wild-type LacY catalyze lactose ef¯ux down a concentration gradient ca. 50-fold more rapidly than cells expressing mutant E269D (t1/2 0.17 minute and 8.8 minutes, respectively (Figure 2(c)). Ef¯ux from cells expressing either wild-type LacY or mutant E269D after inactivation with N-ethyl maleimide (MalNEt) is extremely slow, re¯ecting a very low rate of passive diffusion. Moreover, equilibrium exchange with cells expressing E269D LacY is also approximately 65-fold slower than that with cells expressing wild-type LacY (t1/2 0.08 minute and 6.7 minute, respectively (Figure 2(d)). Again, exchange from cells expressing either wild-type LacY or mutant E269D after inactivation with MalNEt is extremely slow, indicating that virtually all the activity is LacY-mediated. Scanning mutagenesis of helix VIII In an effort to rescue activity in mutant E269D, each non-essential residue in helix VIII was replaced individually with Gly (Figure 3). In
Figure 1. Secondary structure and proposed mechanism of LacY. (a) Secondary structure model. Putative transmembrane helices are shown in boxes. Residues that are irreplaceable with respect to active transport are enlarged; Glu126 (helix IV) and Arg144 (helix V) are critical for substrate binding, and Glu269 (helix VIII), Arg302 (helix IX), His322 (helix X) and Glu325 (helix X) are essential for H translocation and coupling. The charge pairs Asp237 (helix VII)/Lys358 (helix XI) and Asp240 (helix VII)/Lys319 (helix X), which do not play an essential role in the mechanism, are shown. (b) Helices VIII and X are ampli®ed, with Glu269, His322 and Glu325 enlarged and emboldened, and positions where Gly replacement results in a signi®cant increase in activity in mutant E269D are emboldened. (c) Mechanism of lactose/H symport by wild-type lactose permease. For simplicity, six of the 12 helices that comprise LacY are viewed from the outer (periplasmic) surface of the membrane. The grey area designates the low-dielec-
tric environment of the lipid bilayer. In the ground-state conformation, the relevant H is shared by His322 (helix X) and Glu269 (helix VIII), whereas Arg302 (helix IX) is charge paired with Glu325 (helix X). In this conformation, lactose permease binds substrate at the outer surface (So). Glu126 (helix IV) and Arg144 (helix V) are charge paired and, together with Cys148 (helix V), comprise the main components of the substrate-binding site. Also shown is the charge pair between Asp240 (helix VII) and Lys319 (helix X), which is not essential for the mechanism. Substrate binding induces a conformational change that disrupts the E269-H322 and R302-E325 charge pairs, and leads to the transfer of the H to Glu325, which is stabilized by the low dielectric environment. At the same time, the substrate binding site becomes exposed to the inner surface of the membrane. After the substrate dissociates, Glu325 becomes deprotonated at the inner surface, as a result of the rejuxtapositioning of Glu325 with Arg302 as LacY relaxes back to the ground-state conformation.
564
Role of Glu269 in LacY
addition, the two native Gly residues in helix VIII (Gly262 and Gly268) were independently replaced with Ala. Of the 25 mutants, seven exhibit rates of lactose transport ranging from 0-30 % of E269D, 16 exhibit rates ranging from 30-130 % of E269D and two exhibit rates of over 130 % of E269D (Figure 3; *). Furthermore, a complete time-course (up to one hour) was measured for each mutant (data not shown). To test whether the variations in activity are due to differences in LacY expression, immunoblots were carried out on membrane preparations from the same cells for which transport was measured. All of the mutants are expressed in amounts comparable to E269D LacY (data not shown). For both mutants that exhibit enhanced transport activity in mutant E269D, active lactose transport was measured in the wild-type background. While G262A/E269D exhibits a threefold increase in the steady-state level of lactose accumulation in the E269D background, there is a minor reduction in the rate and extent of lactose accumulation in the wild-type background (Figure 4(a) and (b)). Similarly, with mutant T266G/M267G, a fourfold increase in rate is observed in the E269D background, while in the wild-type background, little or no effect on active lactose transport is observed (Figure 4(c) and (d)). Mutants T266G/M267G/E269D and G262A/E269D Kinetic analysis of mutant T266G/M267G/ and Vmax are improved E269D reveals that Kapp m signi®cantly with respect to mutant E269D (2.59(0.22) mM and 450(46) nmol/min per mg versus 7.5(0.9) mM and 106(15) nmol/min per mg, repectively). In contrast, with mutant G262A/E269D, there is little change in kinetic par-
Figure 2. Wild-type permease versus mutant E269D. (a) Time-courses of active transport by E. coli T184 expressing wild-type permease (WT), no permease (pT75 with no lacY insert) or the mutant E269D. Aliquots (50 ml) of cell suspensions containing 35 mg of protein in 100 mM KPi (pH 7.5), 10 mM MgSO4 were assayed at 0.4 mM ®nal external concentration of lactose as
described in Materials and Methods. (b) Concentrationdependent lactose transport by E. coli T184 expressing wild-type permease or the mutant E269D. Initial rates of transport were determined at concentrations ranging from 40 mM to 5 mM [1-14C]lactose as described in Materials and Methods. For each datum point, nonspeci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted. Data were ®tted to the Michaelis-Menten equation. (c) Time-course of lactose ef¯ux by E. coli T184 expressing WT or the mutant E269D. Aliquots of cells equilibrated with 10 mM [1-14C]lactose in 100 mM KPI (pH 7.5), 10 mM MgSO4 and 20 mM CCCP were diluted 100-fold into the same solution without lactose as described in Materials and Methods. Where indicated, MalNEt (NEM, 10 mM, ®nal concentration) was used to inactivate LacY. (d) Time-course of lactose equilibrium exchange by E. coli T184 expressing WT or the mutant E269D. Cells were prepared as described for lactose ef¯ux, except they were diluted into the same solution with 10 mM lactose as described in Materials and Methods. Where indicated, MalNEt (NEM, 10 mM, ®nal concentration) was used to inactivate LacY.
Role of Glu269 in LacY
565
Figure 3. Active transport by E. coli T184 expressing E269D and individual Gly or Ala replacement mutants. The single-letter amino-acid code is used along the horizontal axis to denote the original residues from Arg259 to Ile283 in helix VIII. Complete times-courses of transport up to one hour were measured, and the rates of transport measured at two minutes as described in Materials and Methods. The results are expressed as a percentage of E269D activity.
ameters relative to E269D (Kapp m 9.3 mM and Vmax 160(12) mmol/min per mg) (Figure 5(a)). Since mutant E269D exhibits about a 100-fold decrease in af®nity relative to the wild-type,17 the increased activity of mutants T266G/M267G/ E269D and G262A/E269D may be due to increased substrate af®nity. Therefore, the effect of the mutations on the af®nity of mutant E269D for
TDG was appproximated by measuring the inhibition constant (Ki) of TDG with the wild-type and mutants E269D, G262A/E269D or T266G/M267G/ E269D at a lactose concentration around the Kapp m of each mutant. As reported previously,17 the Ki of the wild-type and mutant E269D are 48(4) mM and 3250(250) mM, respectively (Figure 5(b)). Interestingly, mutant T266G/M267G/E269D exhi-
Figure 4. Effects of Ala replacement at Gly262 and Gly replacement at Thr266/Met267. Timecourses of active transport by E. coli T184 expressing wild-type (WT) permease or E269D mutants with Ala in place of the native Gly residue at (a) and (b) position 262 or (c) and (d) Gly in place of the native Thr and Met at positions 266 and 267. Experiments were carried out as described in Materials and Methods.
566
Role of Glu269 in LacY
relative to E269D (t1/2 5.88 minutes versus 6.7 minutes (Figure 6(d)) is observed. Mutant G262A/T266G/M267G/E269D
Figure 5. Kinetics of lactose transport and effect of TDG. (a) Concentration-dependent lactose transport by E. coli T184 expressing WT permease, mutant T266G/ M267G/E269D, mutant G262A/E269D or mutant E269D. Initial rates of transport were determined at concentrations ranging from 40 mM to 5 mM [1-14C]lactose as described in Materials and Methods. For each datum point, non-speci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted. Data were ®tted to the Michaelis-Menten equation. (b) Concentration-dependent inhibition of lactose transport by TDG in E. coli T184 expressing WT permease, mutant T266G/M267G/E269D, mutant G262A/E269D or mutant E269D. Using a concentration of [1-14C]lactose equal to the Km of WT or a given mutant, initial rates of transport were determined in the presence of varying concentrations of TDG. For each datum point, nonspeci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted.
bits about a tenfold increase in af®nity for TDG with respect to mutant E269D (Ki 322 mM), while mutant G262A/E269D exhibits no change in af®nity (Ki 6389 mM). Lactose ef¯ux and equilibrium exchange measurements with mutant G262A/E269D reveal marked increases in the rate of ef¯ux (t1/2 2.3 versus 8.8 minutes (Figure 6(a)), as well as equilibrium exchange (t1/2 0.16 minutes versus 6.7 minutes (Figure 6(b)). Remarkably, with mutant T266G/M267G/E269D, only minor improvement in the rate of ef¯ux (t1/2 6.6 versus 8.8 minutes (Figure 6(c)) or equilibrium exchange
Since mutant G262A/E269D exhibits improved ef¯ux and exchange, while mutant T266G/ M267G/E269D exhibits improved af®nity and an increased rate of accumulation, both sets of mutants were combined to produce mutant G262A/T266G/M267G/E269D. Remarkably, the time-course of lactose active transport for this mutant exhibits a rate of accumulation similar to that of mutant T266G/M267G/E269D, and the same steady-state level of accumulation as G262A/ E269D (Figure 7(a)). Kinetic analysis reveals that and Vmax are improved signi®cantly with Kapp m respect to E269D (2.7 mM and 408(35) mmol/min per mg versus 7.5 mM and 146(4) mmol/min per mg). (Figure 7(b)). Furthermore, mutant G262A/ T266G/M267G/E269D exhibits a Ki of around 200 mM for TDG, demonstrating more than a tenfold increase in af®nity relative to mutant E269D (Figure 7(c)). In addition, lactose ef¯ux and equilibrium exchange are both improved dramatically in mutant G262A/T266G/M267G/E269D relative to mutant E269D (t1/2 0.45 versus 8.8 minutes, respectively, for ef¯ux (Figure 7(d)); t1/2 0.08 versus 6.7 minutes, respectively, for equilibrium exchange (Figure 7(e)).
Discussion Recent studies in which ligand binding af®nity was measured as a function of pH in single-Cys148 permease with various replacements for Glu269, Arg302, His322 and Glu325 have led to a working model describing a mechanism for lactose/H symport17 (Figure 1(c)). In the ground state, LacY is protonated, the H is shared between His322 and Glu269, Glu325 is charge paired with Arg302, and substrate is bound with high af®nity between helices IV (Glu126) and V (Arg144 and Cys148) from the outside surface of the membrane. Substrate binding induces a conformational change that leads to transfer of H from His322/Glu269 to Glu325 and reorientation of the binding site to the inner surface and dissociation of substrate. Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302. The His322/Glu269 complex is then protonated from the outside to re-initiate the cycle. This scheme can be described kinetically, as shown in Figure 8. The only replacement for Glu269 that catalyzes transport to any extent whatsoever is Asp, and even with the shortened carboxylate-containing side-chain, a tenfold reduction in both the Km and Vmax for active lactose transport is observed with a 50-fold reduction in af®nity for the highaf®nity analog TDG. Similarly, both lactose ef¯ux down a concentration gradient (which occurs in symport with H) and equilibrium exchange (which is thought to occur with an H
Role of Glu269 in LacY
567
Figure 6. Time-course of lactose ef¯ux by E. coli T184 expressing WT, mutant E269D, (a) mutant G262A/E269D or (c) mutant T266G/M267G/E269D. Aliquots of cells equilibrated with 10 mM [1-14C]lactose in 100 mM KPI (pH 7.5), 10 mM MgSO4 and 20 mM CCCP were diluted 100-fold into the same solution without lactose as described in Materials and Methods. MalNEt (10 mM, ®nal concentration) was used to inactivate LacY (not shown). Timecourse of lactose equilibrium exchange by E. coli T184 expressing WT, mutant E269D, (b) mutant G262A/E269D or (d) mutant T266G/M267G/E269D. Cells were prepared as described for lactose ef¯ux, except they were diluted into the same solution with 10 mM lactose as described in Materials and Methods. MalNEt (10 mM, ®nal concentration) was used to inactivate LacY.
shared between Glu269 and His322 or oscillating between Glu269/His322 and His322/Glu325)26 are also at least 50-fold slower with mutant E269D LacY relative to wild-type. Mechanistically, the primary defect in E269D seems to be a major decrease in protonation of either the outwardly or inwardly facing conformation of LacY, which leads to a decrease in substrate af®nity, as well as a defect in all translocation reactions that require protonation of the protein. For lactose translocation (Figure 8), shortening the carboxyl-containing side-chain at position 269 seems to effect the following steps in the kinetic cycle. (1) Interaction between Asp269 and His322 is reduced, and protonation is less ef®cient (i.e. state 1 is preferred over state 2). (2) The conformational change promoted by protonation of Glu269 is compromised leading to low-af®nity substrate binding. (3) Poor positioning of the H between Asp269 and His322 leads to inef®cient transfer to His322/Glu325 and ®nally to Glu325 (i.e. the transition between conformations 3 through 5), leading to inef®cient H translocation. As shown previously,24,25 manipulation of conformational dynamics and/or helix packing may be used to rescue function of inactive or partially inactive mutants. Here, each non-essential residue in helix VIII, which contains the E269D mutation, was replaced with Gly or both of the native Gly residues in helix VIII were replaced with Ala in an effort to compensate for the shortened carboxyl-
containing side-chain of the Asp residue in place of Glu269. Two mutants are identi®ed that exhibit improved transport, T266G/M267G/E269D and G262A/E269D, and it should be emphasized that replacement of the native residues with Cys in Cys-less LacY does not increase activity,27 nor do the mutations have a signi®cant effect on the activity of wild-type LacY. Mutant T266G/M267G/E269D exhibits an increase in af®nity for TDG, and the rate of lactose transport is improved markedly without signi®cant rescue of ef¯ux or equilibrium exchange. Possibly, reduction in the volume of the amino acid side-chains at positions 266 and 267 creates a packing surface that allows an adjacent helix to alter the tilt angle of helix VIII. Alternatively, replacement of Thr266 and Met267 with Gly results in looser helix packing in the immediate environment, thereby permitting greater mobility of the Asp269 side-chain. Either of these possibilities might result in a closer approximation between Asp269 and His322, which improves protonation and leads to improved substrate af®nity. Thus, the equilibrium between states 1 and 2 is shifted towards state 2 (Figure 8). That is, the outwardly facing conformation is favored, and the mutant is protonated more ef®ciently than with E269D alone. The observation that ef¯ux and equilibrium exchange are not rescued in this mutant is consistent with the interpretation that the ability of the inwardly facing conformation of the mutant to transfer H to
568
Role of Glu269 in LacY
Figure 8. Kinetic model describing lactose translocation reactions catalyzed by LacY. Also indicated are the steps at which residues Glu269, His322 and Glu325 are thought to be involved. C, LacY; H, transported proton; S, sugar.
Glu325/His322 and/or His322/Asp269 remains impaired (i.e. the equilibrium between steps 5 and 4 and/or 4 and 3 lie towards steps 5 and 4). Although H movements can occur in both directions, in the presence of a m H (interior negative and/or alkaline), in¯ux would be favored. However, during ef¯ux or equilibrium exchange that are measured in the absence of
Figure 7. Properties of G262A/T266G/M267G/ E269D. (a) Time-course of active transport by E. coli T184 expressing WT permease, no permease (pT7-5 with no lacY insert), mutant E269D or mutant G262A/ T266G/M267G/E269D. Aliquots (50 ml) of cell suspensions containing 35 mg of protein in 100 mM KPi (pH 7.5), 10 mM MgSO4 were assayed at 0.4 mM ®nal external concentration of lactose as described in Materials and Methods. (b) Concentration-dependent
lactose transport by E. coli T184 expressing wild-type permease, mutant E269D or mutant G262A/T266G/ M267G/E269D. Initial rates of transport were determined at concentrations ranging from 40 mM to 5 mM [1-14C]lactose as described in Materials and Methods. For each datum point, non-speci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted. Data were ®tted to the Michaelis-Menten equation. (c) Concentration-dependent inhibition of lactose transport by TDG in E. coli T184 expressing WT permease, mutant G262A/T266G/M267G/E269D or the mutant E269D. Using a concentration of [1-14C]lactose equal to the Km of the sample, initial rates of transport were determined in the presence of varying concentrations of TDG. For each data point, nonspeci®c lactose uptake by cells expressing no permease (pT7-5 with no lacY insert) was subtracted. (d) Time-course of lactose ef¯ux by E. coli T184 expressing WT, mutant E269D or mutant G262A/T266G/M267G/E269D. Aliquots of cells equilibrated with 10 mM [1-14C]lactose in 100 mM KPI (pH 7.5), 10 mM MgSO4 and 20 mM CCCP were diluted 100-fold into the same solution without lactose as described in Materials and Methods. (e) Time-course of lactose equilibrium exchange by E. coli T184 expressing WT, mutant E269D or mutant G262A/T266G/M267G/ E269D. Aliquots of cells equilibrated with 10 mM [1-14C]lactose in 100 mM KPI (pH 7.5), 10 mM MgSO4 and 20 mM CCCP were diluted 100-fold into the same solution with lactose as described in Materials and Methods.
569
Role of Glu269 in LacY
m H , there is inef®cient transfer of H from Glu325 to His322/Asp269 and therefore poor ef¯ux and exchange. Mutant G262A/E269D leads to a reciprocal phenotype where ef¯ux and equilibrium exchange are rescued with only marginal effects on af®nity or the rate of active transport. Since Gly residues in many membrane proteins form notches, allowing surfaces to pack tightly,28,29 and the tertiary structure model of LacY indicates that the periplasmic ends of helices VIII and X are adjacent 11, it is conceivable that the greater volume of the side-chain at position 262 in mutant G262A (which lies on the same face of helix VIII as Asp269, separated by two turns of an a-helix) leads to a subtle change in the tilt of helix VIII, thereby allowing Asp269 to approximate His322 more closely. Improved ef¯ux and exchange in this mutant suggest that transfer of H from Glu325 to His322/Asp269 is more ef®cient than in the E269D mutant alone (i.e. the inwardly facing conformation is favored). However, the lack of improvement in active transport argues that His322/Asp269 is unable to protonate ef®ciently from the outside. Clearly, subtle manipulations in the precise positioning of the carboxyl side-chain at position 269 elicit drastic changes in the transport properties of mutants T266G/M267G/E269D and G262A/ E269D by improving protonation of either the outwardly or inwardly facing conformations of LacY, respectively. Remarkably, by combining the two sets of mutations, all translocation reactions are rescued signi®cantly in mutant G262A/T266G/ M267G/E269D. In this case, the equilibrium between the outwardly and inwardly facing conformations approximates that of wild-type LacY more closely, and the mutant can then be protonated more ef®ciently from either side of the membrane.
Materials and Methods Materials Oligodeoxynucleotides were synthesized by SigmaGenosys (The Woodland, TX). Restriction endonucleases, phage T4 DNA ligase and Vent polymerase were from New England Biolabs (Beverly, MA). All other materials were of reagent grade and were obtained from commercial sources.
Growth of bacteria E. coli T184 [lacIOZÿYÿ(A), rpsL, metÿ, thr, recA, hsdM, hsdR/F0 , lacIqOZD118(YA)]30 transformed with plasmid pT7-5/cassette lacY encoding given LacY mutants was grown aerobically at 37 C in Luria-Bertani broth with ampicillin (100 mg/ml). Fully grown cultures were diluted tenfold and grown for two hours at 37 C before induction with 1 mM isopropyl 1-thio-b,D-galactopyranoside. After additional growth for two hours at 37 C, cells were harvested by centrifugation.
Construction of LacY mutants Using plasmid pT7-5/cassette lacY encoding mutant E269D permease, oligonucleotide-directed site-speci®c mutagenesis by two-step PCR31 was used to replace each of 25 non-essential residues in helix VIII with Gly individually. In a similar manner, each native Gly in helix VIII was replaced with Ala. Following restriction endonuclease digestion with KpnI and SpeI, the PCR products were sub-cloned back into the similarly treated parental vector. The identical procedure was used to replace given residues in the same region with Gly or Ala in the wild-type background. The KpnI-SpeI region of the lacY gene in all mutants was fully sequenced through the restriction sites by using the dideoxynucleotide method32 on an ABI 373A automatic sequencer. Transport assays E. coli T184 expressing given LacY mutants was grown at 37 C, subsequently washed once with 100 mM KPi (pH 7.5), 10 mM MgSO4 and adjusted to an A420 of 10 (0.7 mg of protein/ml). Transport was initiated by addition of [1-14C]lactose (5 mCi/mmol) to a ®nal concentration of 0.4 mM. Samples were quenched at given times with 100 mM KPi (pH 5.5), 100 mM LiCl and assayed by rapid ®ltration.33 To determine kinetic parameters, cells were concentrated to an A420 of 20, and 50 ml of cells was mixed with 50 ml of [1-14C]lactose (0.06-10 mM ®nal concentrations). Initial rates were measured over one minute for cells expressing wild-type LacY and over two minutes for cells expressing E269D LacY or derived mutants. Values were corrected for lactose uptake by cells carrying the pT7-5 vector with no lacY insert. To measure the Ki, cells were adjusted to an A420 of 10, and 50 ml of cells was mixed with 1 ml of given concentrations of TDG for ®ve minutes. Initial rates of [1-14C]lactose transport were measured over one minute for cells expressing wild-type permease and over two minutes for cells expressing E269D LacY or derived mutants. Values were corrected for lactose uptake by cells carrying the pT7-5 vector with no lacY insert. To measure lactose ef¯ux, E. coli T184 cells expressing given LacY mutants were re-suspended at 3-4 mg of protein/ml in 100 mM KPi (pH 7.5), EDTA was added to a ®nal concentration of 10 mM, the suspensions were incubated at 37 C for two minutes and placed on ice. Cells were then washed with 100 mM KPi (pH 7.5) and re-suspended in 100 mM KPi (pH 7.5), 10 mM MgSO4 before adjusting to an A420 of 40 (2.8 mg protein/ml). [1-14C]Lactose (10 mM; 10 mCi/mmol) and 20 mM carbonylcyanide-m-chlorophenylhydrazone (CCCP) were added to each sample, and the suspensions were equilibriated on ice overnight. Aliquots (10 ml) of each sample were diluted 1:100 (v/v) in KPi (pH 7.5) containing 20 mM CCCP, and at given times the reactions were quenched with 100 mM KPi (pH 5.5), 100 mM LiCl and ®ltered immediately.33 Where indicated, preloaded cells were diluted 1:100 (v/v) in 100 mM KPi (pH 7.5), 20 mM CCCP and 10 mM MalNEt to inactivate LacY. Zerotime values were determined by dilution of 10 ml; aliquots directly into quench buffer followed by rapid ®ltration. For equilibrium exchange, cells were treated identically; however, the samples were diluted 1:100 (v/v) in KPi (pH 7.5) containing 20 mM CCCP and an
570 equal concentration of unlabeled lactose prior to quenching the reactions as described. Western blotting Crude membranes from the same cells utilized for active transport assays were prepared by osmotic lysis and sonication.27 Total membrane protein was assayed by a modi®ed Lowry procedure.34 A sample containing 60 mg of membrane protein from each sample was subjected to electrophoresis in SDS/12 % (w/v) polyacylamide gels.35 Proteins were electroblotted on to polyvinylidene ¯uoride membranes (Immobilon-PVDF; Millipore) and probed with site-directed antibody against the C terminus of LacY (residues 402-417)36 prior to treatment with anti-mouse IgG-conjugated horse radish peroxidase.
Acknowledgments We thank MikloÂs Sahin-ToÂth, Irina Smirnova, Vladimir Kasho and Jose Luis Vazquez Ibar for helpful discussions. This work was supported, in part, by NIH grant DK51131:06 to H.R.K.
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Edited by G. von Heijne (Received 4 September 2001; received in revised form 23 November 2001; accepted 23 November 2001)