©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphatidylethanolamine Is Required for in Vivo Function of the Membrane-associated Lactose Permease of Escherichia coli(*)

(Received for publication, July 25, 1994; and in revised form, October 24, 1994)

Mikhail Bogdanov William Dowhan (§)

From the Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Experiments with mutant Escherichia coli cells lacking phosphatidylethanolamine (PE) as a membrane component (DeChavigny, A., Heacock, P. N., and Dowhan, W.(1991) J. Biol. Chem. 266, 5323-5332) were carried out to establish whether or not PE is necessary for full function of the lac permease in vivo. The V(max) for active transport of both lactose (in cells lacking beta-galactosidase, lacZ) and the unhydrolyzable lactose analog, methyl-beta-D-galactopyranoside (TMG), by mutant cells lacking PE was reduced 5-10-fold relative to cells containing PE, while the K for the uptake of both substrates was the same in both types of cells. The low rate of TMG and lactose uptake by PE-deficient cells was unaffected by the presence of a protonophore (uncoupler) and for TMG uptake was on the order of the greatly reduced rate of uptake in uncoupler-treated cells containing PE. The rate of entry of lactose into lacZ derivatives of both types of cells, as a measure of facilitated diffusion, was nearly the same. The K for lactose (lacZ cells) and TMG transport in PE-deficient cells was unaffected by the presence of an uncoupler which had a small effect on V(max). In PE-containing cells these kinetic parameters for TMG transport were reduced by an uncoupler to the level found with PE-deficient cells while an uncoupler reduced lactose uptake by PE-containing (lacZ) cells to below measureable levels. Inverted membrane vesicles made from both types of cells could be loaded with TMG, but energizing TMG-loaded vesicles by ATP only induced rapid, uphill, permease-dependent efflux of TMG from PE-containing vesicles. The decrease in apparent active transport activity of cells with no PE was not due to a change in membrane permeability, to a reduced Deltaµ (proton electrochemical gradient) across the cell membrane, or to a reduced level of membrane-associated lac permease protein. These results suggest that in the absence of PE the lac permease cannot couple substrate uptake to Deltaµ in order to effect accumulation of substrate and as a result only carries out facilitated diffusion.


INTRODUCTION

Transport of beta-galactosides into Escherichia coli is catalyzed by the product of the lacY gene, the lac permease. In the presence of a proton electrochemical gradient (Deltaµ

The function, membrane topology, and assembly of the lac permease has been the subject of numerous detailed investigations utilizing biochemical, physical chemical, and molecular genetic approaches(2, 4) . However, only a limited number of reports address the role of the native phospholipid environment in the assembly and function of this transporter. In most studies on the lipid requirement of transport systems, the carrier has been extracted from the membrane (5) followed by reconstitution of the transport system into liposomes of different phospholipid compositions. Reconstituted lac permease showed a near absolute dependence of active transport, but not downhill facilitated diffusion, on proteoliposomes containing the amino-based zwitterionic phospholipids PE or phosphatidylserine(6, 7) . However, limitations of this in vitro approach are that differences observed between the various phospholipids employed might be due to variability in the incorporation efficiency of the transport protein into proteoliposomes of different compositions, in the ability of proteoliposomes to maintain an artificially imposed Deltaµ, in the ability to form sealed proteoliposomes, and in proteoliposomal internal volumes. The extraction and reconstitution procedures can also affect native conformation of reconstituted proteins. Even mild approaches to altering phospholipid composition such as fusion of membrane vesicles and liposomes (8) and nonspecific lipid transfer protein-based methods (9) have the disadvantage that endogenous lipids remain present in the membranes and the natural lipid asymmetry of the membrane may not be maintained. Finally, in all in vitro reconstituted systems influence of dynamic metabolism on regulation and function is not present or testable. What is generally lacking in this area are extensive studies of these carriers in vivo in membranes with systematically controlled variations in phospholipid composition.

Limited data on the effect of alterations in phospholipid metabolism on the in vivo properties of the lac permease system have been reported. Accumulation of phosphatidylserine to about 20% of the total phospholipid of E. coli in a temperature-sensitive mutant of the psd gene (encoding phosphatidylserine decarboxylase) had little effect on the properties of the endogenous lac permease(10) , which is consistent with the in vitro data(6, 7) . The use of the existing conditional lethal mutants in E. coli phospholipid metabolism is complicated by the necessity of separating the pleiotrophic effects brought about by cell growth arrest or cell death from the specific effects on transport systems. The recent development of a series of mutants in which dramatic changes in phospholipid composition can be made under conditions which allow for cell growth and viability has opened up the possibility of more systematic studies on the role of specific phospholipids and phospholipid composition in cell processes in vivo(9, 11, 12, 13) .

In order to initiate studies on the in vivo role of phospholipids in lac permease assembly and function, we utilize in this report strains carrying the pss93::kan null allele (normally encodes phosphatidylserine synthase) which renders the cell devoid of the zwitterionic amino-based phospholipids phosphatidylserine and PE (14) . Our results support the previously reported in vitro requirement for PE in the functioning of the lac permease particularly in proton-coupled active transport. A preliminary report of this work has appeared(15) .


EXPERIMENTAL PROCEDURES

Materials

Lactose, TMG, TDG, ONPG, nigericin, valinomycin, ATP, and phosphoenolpyruvate were purchased from Sigma. IPTG and pyruvate kinase were purchased from Boehringer Mannheim. TONPG was obtained from Aldrich. [methyl-^14C]TMG was obtained from DuPont NEN, and lactose (labeled at [1-^14C]]glucose) and S-labeled Protein A came from Amersham Corp. [^14C]DMO and rabbit polyclonal antiserum directed against the C-terminal dodecapeptide of lac permease were generous gifts of Dr. H. R. Kaback (University of California, Los Angeles).

Bacterial Strains and Plasmids

Table 1summarizes the strains and plasmids used in this work. Strains carrying the pss93::kan null allele require either a functional plasmid-borne copy of the pss gene (plasmid pDD72, temperature sensitive for replication) or growth media containing 30-50 mM MgCl(2) for viability(14) . The wild type control strain (PE-containing) was a pss93::kan null allele-containing strain (PE-deficient) carrying plasmid pDD72; strains lacking PE had the expected phospholipid composition (14) of 65% phosphatidylglycerol, 13% cardiolipin, and 18% phosphatidic acid. Strains with mutant lacY or lacZ genes were constructed using phage P1vir transduction (16) to transfer the lacY328 allele from strain M7044 to strain AD90/pDD72. Transductants defective in the lac operon were selected for by their resistance to the metabolic poison TONPG when grown on succinate minimal medium(17) ; the phenotypes were confirmed by the lack of ONPG hydrolysis by whole cells (lacY strain) and the lack of beta-galactosidase activity in cell extracts (lacZ strain). Strains were made recA by P1 transduction of the closely linked srl::Tn10 marker of strain GE2046 followed by screening for UV sensitivity(18) . Strains were cured of plasmid pDD72 as described previously(14) .



Growth of Bacteria

Cells were grown in either liquid medium or on agar plates composed of LB rich medium (10 g of Bactopeptone, 5 g of Bacto-yeast extract, and 5 g of NaCl/liter). Antibiotics were included in the growth medium where necessary for strain selection or maintenance of plasmids(18) ; only ampicillin selection was utilized for PE-deficient strains. Since replication of plasmid pDD72 is temperature-sensitive and mutants lacking PE require MgCl(2) for viability, all strains (mutant and wild type) were grown at 30 °C in 50 mM MgCl(2) unless otherwise noted. For transport assays and membrane preparations fresh overnight cultures were diluted 50-fold and allowed to grow in LB medium to an A of 0.25 before induction with either 0.2 mM IPTG for lac permease expression from the chromosomal copy of the gene or 1 mM IPTG for the expression from the copy of the gene carried by plasmid pT7-5/lacY unless otherwise noted. After additional growth in exponential phase for one generation, cells were harvested by centrifugation.

Preparation of IMV

The pellet of cells grown in exponential phase in LB medium was resuspended to a concentration of A = 15 in 100 mM HEPES/KOH (pH 7.5) containing 25 mM MgCl(2), 1 mM dithiothreitol, and 0.5 mM phenylmethanesulfonyl fluoride. Total cell lysates were prepared by subjecting the cell suspensions to a single passage through a motor-driven French pressure cell to form IMV(20) ; undisrupted cells and outer membrane vesicles were removed from the lysate by centrifugation at 30,000 times g for 20 min. The inner membrane fraction (as IMV) was then collected by centrifugation at 200,000 times g for 1 h and resuspended in the original buffer containing 250 mM sucrose to a protein concentration of 15-20 mg/ml; aliquots were stored at -80 °C for subsequent use.

Sugar Transport Assays

Transport of radiolabeled lactose (0.1-0.4 µCi/ml) and radiolabeled TMG (0.1-0.4 µCi/ml) at final concentrations ranging from 0.1 to 2.0 mM was assayed in intact cells as described previously (21) except 30 mM MgCl(2) was added to the assay mixture and stop solution. Under conditions where uninduced cells retained about 200 dpm of substrate (10-fold over background) the amount of radiolabel retained by IPTG-induced cells ranged from 1200 to 50,000 dpm/assay. The initial rates of transport were determined from time points taken during the first 5 min of the assay where the initial rate of transport was linear. Kinetic constants (V(max) and K(m)) derived from the Michaelis-Menton equation were the average of three determinations utilizing DeltaGraph Pro version 3.0 (DeltaPoint, Inc.) for curve fitting; the graphical representation reported in each case was of one typical experiment. Cells, grown as described above, were harvested by centrifugation and resuspended in ice-cold 100 mM HEPES/KOH (pH 7.5) or in LB medium containing both 30 mM MgCl(2) and chloramphenicol (50 µg/ml) to an A of 10 (about 0.8-1.0 mg of protein/ml) and kept on ice until used. Individual aliquots (0.1 ml) of the cell suspension were used for each time point and reactions were carried out at room temperature in plastic tubes. The assay was initiated by addition of radioactive substrate and stopped by addition of 3 ml of 100 mM HEPES/KOH (pH 7.5), 100 mM LiCl, and 30 mM MgCl(2) followed by rapid filtration through glass fiber filters (Whatman GF/F). The filters were washed once with stop solution (10 ml) and radioactivity trapped on the filters was determined by liquid scintillation spectrometry. De-energized cells were obtained by pretreating the cells with 50 µM of the uncoupler, FCCP, for 10 min prior to assaying transport in the presence of uncoupler (22) .

Transport by IMV was determined as described previously (23) with minor modifications. For assay, IMV were diluted into 0.5 ml of 100 mM HEPES/KOH buffer (pH 7.5) containing 15 mM MgCl(2), 250 mM sucrose, 20 mM KCl to make a final concentration of 2.5 mg of protein/ml. The assay mixture was supplemented with 10 mM phosphoenolpyruvate and 0.05 mg/ml phosphoenolpyruvate kinase. The IMV were incubated for 10 min at room temperature with 0.4 mM radioactive TMG (5 µCi/ml). ATP (3 mM final concentration) was then added and samples (50 µl) were transferred to stop solution (0.15 ml of 10 mM HgCl(2)) at various times followed immediately by the addition of 1 ml of ice-cold wash buffer (100 mM HEPES/KOH (pH 7.5) containing 20 mM MgCl(2) and 0.2 mg of poly-L-lysine (M(r) = 10, 500)). The resulting flocculant suspensions were collected on 0.1-µm Gelman Super-100 filters, washed with 5 ml of ice-cold wash buffer (no polylysine), and dried. The radioactivity trapped on the filters was counted by liquid scintillation spectrometry. In control experiments with P-labeled IMV, it was found that IMV do not pass through the filters under the above conditions.

Alkali Fractionation

Total French pressure cell lysates were mixed with an equal volume of cold 0.1 or 0.2 N NaOH, vortexed, and separated into a pellet and supernatant fraction by centrifugation in a microcentrifuge at 18,000 times g for 15 min at 4 °C(24) . Aliquots of whole lysate, pellet (suspended in water), and supernatant were adjusted to a final concentration of 10% trichloroacetic acid and the resulting protein precipitates washed with acetone followed by solubilization in SDS sample buffer and subjected to SDS-PAGE and immunoblot analysis as described below.

DeltapH Determination

Cells induced for expression of the lac operon were harvested during mid-log phase of growth by centrifugation at 1000 times g for 7 min and resuspended in the 100 mM MES/NaOH buffer (pH 5.5) containing 30 mM MgCl(2) to an A of 10-15 (1.0-1.5 mg of cell protein/ml). The difference in pH across the membrane of the cells was measured by determining the difference in the amount of radiolabeled DMO associated with cells before (energized cells) and after (de-energized cells) treatment with 1.7% toluene; this approach provides a correction for nonspecific binding of DMO. At 25 °C steady-state distribution of DMO was obtained within 2 min and remained constant for at least 10 min. After 3 min of incubation, 0.5-ml samples (1 mg of cell protein) were removed from the reaction mixtures (cells suspended in 100 mM MES/KOH (pH 5.5), 150 mM KCl, 16 mM succinate, [^14C]DMO (0.25 µCi/ml, 60 µM final concentration), 30 mM MgCl(2)) and layered onto 0.15 ml of 22% perchloric acid and 0.50 ml of silicone oil (d = 1.05, Aldrich) in microcentrifuge tubes, and centrifugated through the silicone oil in an Eppendorf microcentrifuge at 14,000 times g for 5 min at room temperature; a de-energized sample was treated in the same way. After centrifugation, 0.05-ml samples of the perchloric acid phase and the supernatant remaining above the silicon oil were removed for scintillation counting. The internal pH (pH) was calculated from the Nernst equation (25) as follows:

where pK(a) is the negative logarithm of the apparent ionization constant of DMO (approximately 6.32 at 25 °C), pH is the extracellular pH, DMO is the intracellular concentration of DMO, DMO is the extracellular concentration of DMO. The intracellular water volume was taken as 6.34 µl/mg of cell protein(26) . The DeltapH is the difference between pH and pH (alkaline inside). The above method was also used to verify that 50 µM FCCP fully de-energized cells at extracellular pH 5.5 and 150 mM potassium ion.

Immunoblotting

Membrane fractions prepared as described above, protein precipitates, or whole cell lysates were resuspended in SDS-PAGE loading buffer containing 2.8% SDS, 10% glycerol, 100 mM dithiothreitol and heated at 37 °C for 20 min prior to being subjected to PAGE in 12.5% polyacrylamide(27) . Protein was transferred from the gel to nitrocellulose sheets (Schleicher & Schuel) by electroblotting for 90 min using a semi-dry electroblotting system (Milliblot-SDE Electroblotting apparatus). The nitrocellulose sheets were then blocked overnight with 5% bovine serum albumin in 50 mM potassium phosphate buffer (pH 7.5). Rabbit polyclonal antiserum directed against the C-terminal dodecapeptide of lac permease was added at a final dilution of 1:2500 in the same blocking solution and incubated for an additional hour. The sheets were washed 3 times with TBS buffer (10 mM Tris-HCl (pH 7.4), 0.9% NaCl) for 15 min each and then 3 times with TBS buffer containing 0.05% Nonidet P-40 for 15 min each. The washed sheets were incubated in 50 mM phosphate buffer (pH 7.5) containing 1% bovine serum albumin and S-labeled Protein A (0.1 µCi/ml, 320 Ci/mmol) for 1 h followed by six 15-min washes with TBS and TBS/Nonidet P-40 buffers. The dried sheets were scanned for radiolabel and quantified using a Betagene Imager.

Miscellaneous Procedures

Protein was determined by the BCA assay method according to the manufacturers suggestions, and beta-galactosidase was assayed using cells pretreated with toluene, as described by Miller(16) . Labeling of phospholipids in vivo with PO(4) was carried out as described previously(14) .


RESULTS

Lactose Utilization

The ability of PE-deficient cells to import and metabolize lactose was assessed qualitatively by growing the cells on MacConkey indicator plates containing 25 mM lactose(16) . Cells expressing functional lacY and lacZ gene products can import and hydrolyze lactose and subsequent metabolism of the monosaccharides causes acidification of the surrounding agar which results in purple-red colonies. Strains carrying the null pss93 allele and either lacking (PE-deficient) or containing (PE-containing) plasmid pDD72 form purple-red colonies on the above plates. These indicator plates only establish downhill lactose transport and give no indication as to whether or not cells catalyze active accumulation against a concentration gradient(28) .

Active Transport

In order to examine the ability of the cells lacking PE to actively transport beta-galactosides against a concentration gradient, their ability to accumulate lactose and its non-metabolizable analog TMG was investigated. As shown in Fig. 1, fully energized PE-containing and PE-deficient cells can both take up TMG at subsaturating concentrations (0.1-0.5 mM); however, the initial rate of uptake by PE-deficient cells was 8- to 10-fold lower than that for wild type cells whether the lac permease was expressed from the single chromosome copy (Fig. 1A) or from multiple copies of the gene (Fig. 1B). When the transport assay was performed directly in growth medium to eliminate any perturbations resulting from cell isolation as a variable, the results were the same. The V(max) for apparent active transport in whole cells was 4 ± 0.5 nmol/min mg of protein for the strain lacking PE (Fig. 1D) while the same strain carrying plasmid pDD72 and containing PE had a V(max) of 40 ± 5 nmol/min mg of protein (Fig. 1C); the K(m) (0.8 ± 0.3 mM) for transport was the same for both strains although the steady-state level of uptake by PE-deficient cells was significantly reduced (Fig. 1B), consistent with the lack of significant accumulation of substrate against a concentration gradient. Apparent active transport was also measured using entry of the natural substrate lactose into cells lacking a functional beta-galactosidase. Again cells lacking PE were not able to rapidly take up lactose to high levels when compared to cells containing PE (Fig. 2A). The V(max) for lactose uptake (PE-deficient versus PE-containing cells, respectively) was reduced 6-fold (2.1 ± 0.13 versus 12 ± 0.7 nmol/min mg of protein) for cells expressing the lac permease from a single copy of the gene (Fig. 2B), and 5-fold (40 ± 5 versus 200 ± 30 nmol/min mg of protein) for cells carrying plasmid pT7-5/lacY (data not shown); the K(m) (0.5 ± 0.06 mM) was the same for all strains.


Figure 1: Uptake of TMG by PE-containing and PE-deficient cells. The uptake of TMG (0.1 mM) was determined as a function of time by either strain AD93/pDD72 (box, pss) or strain AD93 (circle, pss), without (A) or with (B) plasmid pT7-5/lacY as described under ``Experimental Procedures.'' Dependence of the reciprocal of the initial rate of TMG uptake (V) on the reciprocal of the TMG concentration was determined for strain AD93/pDD72 (C) and strain AD93 (D).




Figure 2: Uptake of lactose by PE-containing and PE-deficient cells in a lacZ background. A, strain AD932 (lacZ) harboring either both plasmids pDD72 and pT7-5/lacY (box, pss) or plasmid pT7-5/lacY alone (circle, pss) was assayed for its ability to take up lactose (0.4 mM) as a function of time as described under ``Experimental Procedures.'' B, strain AD932 either with (box, pss) or without (circle, pss) plasmid pDD72 was assayed for its ability to take up lactose as a function of lactose concentration and displayed as in Fig. 1, C and D.



Maintenance of membrane impermeability to lac permease substrates was verified by measuring ONPG hydrolysis by whole cells. Cells of strain AD93 (lacY lacZ) with and without plasmid pDD72 showed similar high rates of ONPG hydrolysis by internal beta-galactosidase. However, cells of strain AD931 (lacY lacZ) showed the same low rate of ONPG hydrolysis either with or without plasmid pDD72, thus ruling out an increase in membrane permeability resulting in exit of substrate through passive diffusion as an explanation of the low rate of active transport and low level of accumulation by PE-deficient cells.

The addition of the uncoupler FCCP had no effect on either the low rate of uptake or the level of accumulation of TMG by PE-deficient cells and was on the order of the residual FCCP-independent uptake by PE-containing cells (Fig. 3); the level of uptake observed in the presence of FCCP was significant over the control level in uninduced cells. In PE-containing cells FCCP reduced the V(max) for TMG uptake to 8 nmol/min mg of protein (versus 40 nmol/min mg of protein, Fig. 1C) with no effect on K(m) while neither V(max) nor K(m) were affected by FCCP in PE-deficient cells (data not shown). Similarly, the kinetics of lactose uptake in PE-deficient lacZ cells was little affected by FCCP (data not shown) which reduced the V(max) from 2.1 ± 0.13 to 0.83 ± 0.06 nmol/min mg of protein with no change in K(m) (0.5 ± 0.1 mM). The steady-state level of lactose accumulation in PE-deficient cells was also unaffected by FCCP (Fig. 4) but was drastically reduced in PE-containing cells; cells carrying a plasmid borne copy of lacY gene were used to rapidly reach a steady-state level of accumulation. Unlike the measurement of TMG kinetic parameters in the presence of FCCP, a quantitative determination of these parameters for lactose uptake in FCCP-treated PE-containing cells (lac Z) was not possible in the 0.1-2 mM range probably consistent with the high K(m) (>10 mM) characteristic of facilitated diffusion in wild type de-energized cells(22, 29) ; no similar data is available on the effect of de-energization on the K(m) for TMG uptake, but our data indicates there is no effect on K(m). De-energization of both strains by FCCP under the above conditions was confirmed by measurement of the chemical gradient of protons across the membrane with the weak acid DMO at pH 5.5 and 150 mM potassium ion(30) .


Figure 3: Effect of FCCP on TMG uptake by PE-containing and PE-deficient cells. Strain AD93 either with (pss, box, ) or without (pss, circle, bullet) plasmid pDD72 was used to measure uptake of TMG (0.4 mM) either in the absence (circle, box) or presence (bullet, ) of 50 µM FCCP as described under ``Experimental Procedures.'' As a control, TMG uptake was monitored in strain AD93 (up triangle) without prior induction by IPTG.




Figure 4: Effect of FCCP on lactose uptake by PE-containing and PE-deficient cells in a lacZ background. Strain AD932/pT7-5/lacY either with (pss, box, ) or without (pss, circle, bullet) plasmid pDD72 was used to measure uptake of lactose (0.4 mM) as a function of time either in the absence (circle, box) or presence (bullet, ) of 50 µM FCCP as described under ``Experimental Procedures.''



Proton Gradient in PE-deficient Cells

As the magnitude of the Deltaµ across the membrane is decreased in wild type cells, the K(m) for lactose transport increases to that of facilitated diffusion with little change in V(max)(22, 29, 31) . Since PE-deficient cells displayed a significantly lower V(max) with no difference in K(m) when lactose transport was compared to PE-containing cells, it is unlikely that the low V(max) for cells lacking PE was due to an alteration in the Deltaµ; however, the steady-state level of substrate accumulation is also proportional to the Deltaµ. In order to rule out a reduction in the Deltaµ or membrane leakiness to protons as the basis for the reduction in either the rate or extent of beta-galactoside uptake in the absence of PE, the ability of cells lacking PE to maintain a normal DeltapH gradient across their membranes was investigated. The magnitude of the DeltapH was measured by determining the partitioning of the weak acid DMO between the internal volume of intact cells and the medium (Table 2). The experiment showed an equal ability of cells both with and without PE to maintain a nearly 2 pH unit difference across their respective cytoplasmic membranes. These results are consistent with an earlier report showing that IMV from PE-deficient cells were normal in their ability to form and maintain a DeltapH upon being energized(32) .



Some transport systems are driven by the DeltapH component of the Deltaµ at an external pH of 5.5 while at an external pH of 7.5 all transport systems are driven by the membrane potential component of Deltaµ(33) . The measurement of TMG uptake by cells under the conditions of the DeltapH determination (i.e. at pH of 5.5) showed a similar reduction in TMG uptake by PE-deficient cells (data not shown); therefore, the potential to utilize DeltapH in place of the membrane potential at pH 5.5 did not alter transport in cells lacking PE.

Facilitated Diffusion in PE-deficient Cells

In lacZ cells metabolizable substrates such as lactose are immediately hydrolyzed upon import and do not accumulate against a concentration gradient; therefore, their uptake occurs by carrier mediated facilitated diffusion or downhill transport and is not limited by the internal concentration of substrate or the Deltaµ. As shown in Fig. 5, both PE-deficient and PE-containing cells showed similar levels of downhill lactose transport. The lactose entry measured in these experiments was via the lactose transport system as evidenced by blockage with the specific inhibitor TDG. Consistent with measuring facilitated diffusion was the lack of a significant affect of FCCP on lactose uptake (lacZ cells) by PE-deficient cells and the similarity in the uptake by both cells in the presence of FCCP (Fig. 6).


Figure 5: Downhill lactose transport by PE-containing and PE-deficient cells. Strain AD93 (lacZ) containing both plasmids pDD72 and pT7-5/lacY (box, , pss) or plasmid pT7-5/lacY alone (circle, bullet, pss) was assayed for its ability to transport lactose (0.4 mM) in the absence (box, circle) or presence (, bullet) of 5 mM TDG as described under ``Experimental Procedures.''




Figure 6: Downhill lactose transport by PE-containing and PE-deficient cells in the presence of FCCP. Strain AD93 (lacZ) containing both plasmids pDD72 and pT7-5/lacY (box, , pss) or plasmid pT7-5/lacY alone (circle, bullet, pss) was assayed for its ability to transport lactose (0.4 mM) in the absence (box, circle) or presence (, bullet) of 50 µM FCCP as described under ``Experimental Procedures.''



Active Transport by IMV

Closed proton-impermeant IMV will carry out both active and facilitated carrier-dependent transport; the exterior of such vesicles is the cytoplasmic face of the membrane and contains the exposed functional ATPase activity which allows for energizing the membrane by addition of ATP(23) . The bioenergetic parameters of IMV made from cells lacking PE were verified for their ability to form a normal Deltaµ coupled to ATP hydrolysis as has previously been shown(32) . The IMV from both PE-containing and PE-deficient cells showed a time-dependent facilitated equilibration of TMG with the medium reaching a steady-state level of about 200-250 pmol/mg of protein (Fig. 7) associated with the vesicles. The addition of ATP induced the uncoupler-sensitive efflux of internal TMG against a concentration gradient from PE-containing vesicles but did not induce TMG efflux from PE-deficient vesicles. The inhibition of efflux by valinomycin and nigericin indicated that the ATP-induced efflux from PE-containing vesicles resulted from electrogenic pumping of protons inward by the ATPase. The results verify that TMG efflux driven by Deltaµ occurs in PE-containing IMV, but such efflux cannot be supported in PE-deficient IMV. Since the orientation of these vesicles is inside-out, the TMG efflux corresponds to uptake in whole cells.


Figure 7: ATP-driven efflux of internal TMG from IMV prepared from PE-containing and PE-deficient cells. IMV were prepared from strain AD93 harboring either both plasmids pDD72 and pT7-5/lacY (box, , pss) or plasmid pT7-5/lacY alone (circle, pss) and assayed for TMG efflux from preloaded vesicles as described under ``Experimental Procedures.'' ATP (3 mM, final concentration) was added at the arrow. Valinomycin and nigericin (4 µg each per mg of membrane protein) were added prior to ATP in one experiment for IMV prepared from pss cells ().



Lac Operon Expression and Gene Product Levels in PEdeficient Cells

To exclude the possibility that lac operon expression was suppressed in the absence of PE, the level of beta-galactosidase in fully IPTG-induced cells was measured. This level was the same in cells with and without PE (900 and 1100 Miller units, respectively). The possibility that membranes with different phospholipid composition might contain different levels of lac permease protein was also considered. Immunoblot analysis using lac permease-specific antibody and S-labeled Protein A for quantification (Fig. 8) indicated that membranes isolated from cells with and without PE have the same amount of lac permease protein/mg of membrane protein (5210 ± 370 dpm and 5840 ± 480 dpm/mg of protein, respectively). The possibility that lac permease produced in PE-deficient cells fails to be integrated into the membrane and exists as an unintegrated protein aggregate was investigated by alkaline fractionation of lysates(24) . The bilayer structure of biological membranes is largely preserved at high pH and consequently proteins integrated into the lipid phase (integral membrane proteins) are not solubilized by 0.1 N NaOH. In contrast, proteins peripherally bound to the membrane surface as well as protein aggregates are usually solubilized by alkali treatment. Thus centrifugation following alkali treatment of cell lysates should selectively pellet integral membrane proteins(24) . When whole cell French press lysates (Fig. 9) were subjected to alkali fractionation, the pellet fraction (containing the membrane protein fraction) from cells with and without PE was found to contain the same amount of lac permease protein (2060 dpm ± 215 and 2890 ± 320 dpm/mg of protein, respectively); only trace amounts of lac permease were found in the supernatant fractions. Therefore, all of the lac permease protein appears to be associated with the membrane as an integral protein independent of the presence of PE. Treatment of membranes with 0.2 N NaOH (Fig. 9) resulted in considerable release of the permease from membranes containing PE, but resulted in no release from membranes lacking PE possibly suggesting an alteration in the normal organization of the permease in membranes lacking PE.


Figure 8: Western blots of membranes from PE-containing and PE-deficient cells. Membranes were prepared from the cells of strain AD93 (induced with IPTG) either with (pss) or without plasmid pDD72 (pss) and were subjected to SDS-PAGE (12.5, 25, and 50 µg of protein/lane from left to right) and to Western blot analysis using anti-lacY permease antibody and S-labeled Protein A as described under ``Experimental Procedures.'' The uninduced lane represents membranes from strain AD93/pDD72 grown in the absence of IPTG. The major band at 33 kDa represents the lac permease.




Figure 9: Alkali fractionation of extracts from PE-containing and PE-deficient cells. Total French press lysates (T) from the same strains as described in Fig. 8were alkali-fractionated (0.1 N NaOH) into pellet (P) and supernatant (S) fractions as described under ``Experimental Procedures.'' Pellets were also retreated with 0.2 N NaOH and the remaining pellets (P*) examined. The samples were subjected to SDS-PAGE and Western blot analysis as described in Fig. 8. Analyses were carried out also on membranes isolated from French press lysates from strain AD93/pDD72 uninduced with IPTG(-) or strain AD93/pDD72/pT7-5/lacY induced (+) for lac permease expression from the T7 RNA polymerase promoter on plasmid pT7-5/lacY. The major band at 33 kDa represents the lac permease.




DISCUSSION

Wilson (6, 7) reported the first detailed observations on the phospholipid requirement for activity of the E. coli lac permease establishing that the transporter specifically requires a phospholipid with a free primary amine (such as PE or phosphatidylserine) for active transport (i.e. energy dependent) in reconstituted proteoliposomes. Phosphatidylcholine could not substitute for PE and monomethyl- and dimethyl-PE were progressively less effective. In a similar study Page et al.(34) showed that the phospholipid environment in which the lac permease is reconstituted greatly affects its activity. Binding of substrate (required for both facilitated and active transport) was not affected by the phospholipid environment but reactions involving net turnover of the permease (required for active transport) were dependent on both the nature of the phospholipid head group and the fluidity of the lipid core; again a strong dependence on PE was noted, but these authors could not verify the ability of phosphatidylserine to substitute for PE. We have extended these studies to determine whether a requirement exists for PE in the in vivo functioning of the lac permease by analyzing transport function in a mutant strain with a null allele of the pss gene either with (PE-containing) or without (PE-deficient) a plasmid-borne copy of the pss gene(14) .

Our results demonstrate that the rate of apparent active transport (V(max)) of lactose in a lacZ background and its analog TMG in a lacZ background is reduced at least 5-10-fold in PE-deficient cells relative to cells with a normal level of PE with no effect of the lipid composition on the K(m) for uptake. However, the entry of lactose (at subsaturating concentrations) into lacZ cells, as an index of facilitated (downhill) diffusion mediated by the lac permease under fully energized conditions, was not affected by the absence of PE. The sensitivity of active transport in PE-containing cells to an uncoupler and the resistance of transport activity to an uncoupler in PE-deficient cells confirms the Deltaµ-linked nature of active transport by lac permease in a wild type lipid environment and suggests that the carrier may only be able to mediate Deltaµ-independent facilitated diffusion in cells lacking PE. The level of uptake of TMG in PE-deficient cells (with or without FCCP) was on the order of the residual level of uptake by PE-containing cells in the presence of uncoupler, again consistent with lack of active transport in PE-deficient cells. Loading of inverted membrane vesicles with TMG, also as an indicator of carrier-mediated facilitated diffusion, was unaffected by the lack of PE, but TMG efflux driven by Deltaµ, as an indicator of active transport, only occurred with PE-containing membrane vesicles. The decreased rate of transport measured under conditions of active transport (absence of FCCP) in cells containing no PE was not due to the suppression of lac operon expression, to an increase proton permeability of the membrane, or to a reduction in the level or an alteration in subcellular location of the lac permease. These results indicate that the in vivo function of the lac permease in the active, energy-coupled mode is dependent on PE as observed in vitro(6, 7, 34) and that the permease in the absence of PE cannot effectively couple substrate uptake against a concentration gradient with the cotransport of a proton down a proton gradient. This conclusion is further supported by the lack of inhibition by an uncoupler of the low steady-state level of both TMG and lactose uptake by PE-deficient cells. However, even in the de-energized state, the apparent K(m) for lactose uptake in lacZ PE-deficient cells does not increase dramatically as has been reported for PE-containing cells (22, 31 and this work) which suggests a significant difference in the interaction of the permease with its second substrate, a proton or Deltaµ; our inability to measure significant lactose uptake in the presence of FCCP in PE-containing cells is consistent with a significant change in the kinetic parameters upon de-energizing of the membrane.

There are several reports suggesting that other membrane-associated transport proteins show preference for PE when studied in vitro. PE had a stimulatory effect on the V(max) but not the K(m) for transport mediated by the reconstituted branched-chain amino acid carrier of E. coli(8) . A PE requirement has been shown for the melibiose transport protein of E. coli(35) as well for the sodium-dependent leucine transport system in Pseudomonas aeruginosa(36) . A similar requirement for amino acid carriers has been reported for Bacilli species (37) which, like E. coli and Pseudomonas species, have high levels of PE. The lac permease of E. coli catalyzes active beta-galactoside transport when expressed in the Gram-positive organism Corinebacterium glutamicum which lacks PE(38) . However, the membranes of this organism are rich in mono- and diglucosyldiglyceride glycolipids, the former of which shows stimulatory effects similar to those of PE on the in vitro function of amino acid carriers(8) .

On the basis of the distribution of glycolipids and PE among Gram-positive and Gram-negative bacteria, it has been suggested that these two types of lipids have similar structural functions in membranes(39) . These glycolipids share a common physical property with PE in their ability to form the nonbilayer structures referred to as the reversed hexagonal (H) phase(40) . Cardiolipin in the presence of divalent metal ions can also form such a phase which has been postulated as the reason mutants lacking PE require divalent cations, have increased cardiolipin levels, and have an absolute requirement for cardiolipin which is not shown by PE-containing cells (14, 41, 42) .

The molecular basis for the PE requirement of the above solute transporters is unknown. One might expect that such carriers would be dependent on the membrane environment first for proper assembly into the phospholipid bilayer and second for the conformational changes required for translocation of substrates. The insensitivity of the carrier to uncouplers and the inability to effect accumulation of substrate in energized PE-deficient cells might suggest that the carrier is defective in the release of a proton on the interior of the cell which would be necessary to carry out active transport coupled to Deltaµ. The permease in the absence of PE may be locked in a low K(m) form, in contrast to the normal case where the K(m) is inversely proportional to the Deltaµ(22, 29, 31) . The inability of the permease in the absence of PE to cycle between a low K(m) form (protonated) on the outside of the cell and a high K(m) form (unprotonated) on the inside of the cell would explain the lack of active transport with retention of high affinity for lactose and insensitivity to uncouplers.

The apparent defect in active transport may be related to misassembly of the permease in membranes lacking PE. Little is known about the mechanism or factors which govern the assembly of polytopic membrane proteins, except the observation that cytoplasmically oriented hydrophilic loops of such proteins tend to carry a net positive charge (43) . Preliminary experiments employing the expression of lacY-phoA chimeric fusions indicate that these fusion proteins do not properly orient into membranes lacking PE(44) . Future studies following the assembly of such polytopic protein chimers into membranes with different phospholipid composition may reveal a previously unrecognized dependence on specific phospholipids for both the assembly and function of membrane proteins as was observed in the dependence on anionic phospholipids of protein translocation across the cytoplasmic membrane of E. coli(9, 11, 12, 13) .


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant GM 20478 from the National Institutes of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 713-792-5600; Fax: 713-794-4150; wdowhan{at}utmmg.med.uth.tmc.edu.

(^1)
The abbreviations used are: Deltaµ, proton electrochemical gradient; PE, phosphatidylethanolamine; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; TMG, methyl-beta-D-galactopyranoside; TDG, thiodigalactoside (galactopyranosyl-1-thio-beta-D-galactopyranoside); ONPG, o-nitrophenyl-beta-D-galactopyranoside; TONPG, o-nitrophenyl-1-thio-beta-D-galactopyranoside; FCCP, carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone; MES, 4-morpholinethanesulfonic acid; DMO, 5,5-dimethyloxazolidine-2,4-dione; PAGE, polyacrylamide gel electrophoresis; IMV, inverted inner membrane vesicles.


ACKNOWLEDGEMENTS

We thank Dr. H. R. Kaback for his assistance and advice in carrying out this study and preparing this manuscript for publication. We are also indebted to Dr. Kaback for supplying us with antibody directed against the lac permease, radiolabeled DMO, and plasmid pT7-5/lacY. Phil Heacock and Beto Zunniga aided us in preparing the figures and Shao-Chun Chang helped in analyzing the kinetic data.


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