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J. Biol. Chem., Vol. 280, Issue 28, 26032-26038, July 15, 2005
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-AMINOBUTYRIC ACID PERMEASE (GabP) OF ESCHERICHIA COLI*
¶**
From the
Department of Biochemistry and Molecular Biology, University of Texas-Houston, Medical School and Graduate School of Biomedical Sciences, Houston, Texas, 77030 and the ||Department of Integrative Biosciences, Oregon Health and Science University, Portland, Oregon 97239
Received for publication, May 4, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-aminobutyric acid (GABA) permease (GabP) of Escherichia coli has been found in this study to be affected both topologically and kinetically by membrane lipids. Using single cysteine accessibility methods with viable E. coli strains of natural lipid composition and those lacking phosphatidylethanolamine (PE), we have shown that the N-terminal hairpin of GabP is inverted relative to the membrane in PE-lacking cells, with a hinge point in transmembrane domain III. The rate of GABA transport is reduced by more than 99% in PE-lacking cells. The Michaelis constant for GABA transport is not greatly affected nor is the dependence of transport on energy. However, "transport specificity ratio" analysis demonstrated a clear transition state stability difference for GABA and nipecotic acid between the protein in PE-containing and PE-lacking cells. The patterns of observed effects are similar to those seen with the phenylalanine transporter of E. coli (Zhang, W., Bogdanov, M. Pi, J. Pittard, A. J., and Dowhan, W. (2003) J. Biol. Chem. 278, 50128–50135), also an amino acid/polyamine/organocation family member but quite distinct from those observed with lactose permease (Bogdanov, M., Heacock, P. N., and Dowhan, W. (2002) EMBO J. 21, 2107–2116), a major facilitator superfamily member. Therefore, by extending the studies of similarities and differences in lipid responses among and between family groups, we may identify elements within the proteins that facilitate lipid responsiveness. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Initial studies were carried out on lactose permease (LacY), which is the most widely studied member of the large major facilitator superfamily. Major facilitator superfamily transporters are 12 TM domain spanning single component uni-, sym-, or antiporters of sugars, amino acids, drugs, and other low molecular weight solutes; LacY belongs to a six-member subfamily of oligosaccharide:H+ symporters (6). LacY either reconstituted in proteoliposomes containing PE or expressed in E. coli with wild type phospholipid composition carries out active transport of substrate against a concentration gradient. However, LacY in either whole cells (7) or proteoliposomes lacking PE (8, 9) only carries out energy-independent facilitated transport of substrate. The bioenergetic properties of cells and proteoliposomes are independent of the presence or absence of PE (7, 9). The level of functional LacY and total amount of LacY, as measured by the initial rate of facilitated transport and Western blotting, respectively, are also the same in both cases (7).
Even more dramatic than the alteration in functional properties is the change in the organization of LacY when assembled in cells lacking PE. A major topological inversion of the N-terminal six-TM helical bundle of LacY occurs with respect to the membrane bilayer, whereas the C-terminal helical bundle, excepting TM domain VII, retains wild type topological organization (4). Reintroduction of PE after assembly of LacY triggers a conformational change resulting in a lipid-dependent restoration of active transport function and recovery of normal topology of at least one LacY subdomain (4).
The dependence on PE for proper topological organization and function is not restricted to LacY. A similar requirement for PE was observed with the phenylalanine permease (PheP) of E. coli (3). Assembly in the absence of PE resulted in an inversion with respect to the membrane bilayer of the first two N-terminal TM domains and their adjacent cytoplasmic and periplasmic extramembrane domains. PheP expressed in cells lacking PE exhibited a 5-fold increase in Km and 5-fold decrease in Vmax for active phenylalanine transport. Because both Vmax and Km were affected, one can rule out that lack of PE simply causes loss of activity for the majority of PheP molecules. Introduction of PE after assembly of PheP in PE-lacking cells restored full active transport and the topology of the inverted N-terminal hairpin.
These results demonstrate that membrane phospholipid composition is a determinant of the topological organization of some TM domains and that once TM domain orientation is established during assembly, a change in phospholipid composition can effect a change in topological organization. Although in vitro evidence for several secondary transport proteins has shown a dependence on PE for function (8, 10, 11), the above results are the first to demonstrate a dependence of function on in vivo membrane lipid composition. LacY and PheP structural and functional dependence on PE are similar, but the details of structural organization and function are significantly different. LacY contains a large N-terminal helical bundle, and PheP contains a small N-terminal hairpin structure that depends on PE for proper topological organization. LacY is absolutely dependent on PE for active transport, whereas the lack of PE adversely affects the kinetics of PheP active transport.
Because LacY and PheP belong to different families of secondary transport proteins, we wished to address whether, first, the above general dependence on PE is exhibited by other transport proteins and, second, whether the details of the dependence are similar within members of the same subfamily of transporters. The -aminobutyric acid (GABA) permease (GabP) of E. coli belongs to the same amino acid/polyamine/organocation family of transporters as PheP (12). We report herein that lack of PE during assembly of GabP results in an inversion of the N-terminal TM hairpin similar to that observed for PheP. In the absence of PE, transport activity remained active with a dramatically reduced Vmax but a similar Km for GABA. By determining a difference in the "transport specificity ratio" (TSR) for GABA and nipecotic acid (NA) as substrates (13) for GabP between PE-containing and PE-lacking cells, we established that lack of PE did not simply inactivate the majority of permease molecules, but affected intrinsic kinetic characteristics. These results further broaden the number of secondary transporters whose topological organization and function are dependent on PE and suggest that closely related members of the same subfamily of transporters may share common topogenic signals that respond to membrane lipid composition.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Bacterial Strains, Plasmids, and Growth Conditions—E. coli strains AD93 and AL95 (see Table I), derived from the same host strain W3899, lack the ability to synthesize PE (pss93::kan) unless they carry pssA-containing plasmids pDD72 or pDD72GM, respectively (4). All growth media were supplemented with 50 mM MgCl2, which is required for viability of PE-lacking strains (5). Single cysteine replacements in putative extramembrane domains of a Cys-less GabP (native cysteines at positions 158, 251, 300, and 443 replaced by alanine) were constructed using site-directed mutagenesis of single-stranded DNA from phage-mid, pSCK-GP11 (encoding Cys-less GabP), as described previously (14). These derivatives (see Table I and Fig. 1 for more details) were expressed under PtacOP regulation in the host plasmid pSCK-380-Hx10, which added a His10 tag to the C terminus. The nomenclature for these plasmids is pSCK-380-X#Y-Hx10 where X denotes the amino acid at position # that is replaced by Y (in all cases, Y = Cys, and in the case of A300C this represents a reintroduction of the native cysteine). Unless otherwise indicated, cells carrying these plasmids were grown in LB medium containing ampicillin (100 µg/ml). Expression of plasmid-borne gabP was induced during growth in exponential phase by the addition of 0.24 mM isopropyl 1-thio-
-D-galactopyranoside (IPTG) for 2.5 h unless otherwise indicated.
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0.2 A600, resuspended in 0.1 volumes of 10% (w/v) either polyethylene glycol-3350 or polyethylene glycol-8000, 5% (v/v) dimethyl sulfoxide, and 50 mM MgCl2 in LB medium. Aliquots were frozen at –80 °C. Cells were transformed with plasmid DNA by a 5–60-min incubation on ice, followed by a 1 h recovery in LB supplemented with 50 mM MgCl2 and 20 mM glucose. Transport Assays—Transport of [3H]GABA was measured in intact E. coli cells by standard methods (16), with the following modifications. E. coli strains expressing the Cys-less GabP from pSCK-380-CL-Hx10 were cultured at either 30 °C (PE-containing: AL95/pDD72GM) or 37 °C (PE-lacking: AL95) in LB medium supplemented with ampicillin (100 µg/ml) and 5 mM glucose. IPTG (1.0 mM) was added during the final 2 h of logarithmic growth to induce GabP expression. The cells were washed twice and resuspended to an A600 of 6.0 in ice-cold assay buffer, 20 mM MOPS, 120 mM KCl, 50 mM MgCl2,5mM glucose (pH 7.0) (MKM buffer), and supplemented with 80 µg/ml chloramphenicol. Washed cells (100 µl) at room temperature were rapidly combined with substrate mixture (11 µl) containing varying amounts of [3H]GABA in MKM buffer. Reactions were stopped by transfer of 100 µl of cell mixture into 1 ml of ice-cold MKM buffer supplemented with 20 mM HgCl2. Reactions were terminated at 15 s for PE-containing cells and 2 min for PE-lacking cells, conditions that were established to reflect initial velocities. The sample was applied to a GF/F filter, which was then washed with 3 ml of ice-cold MKM supplemented with 5 mM HgCl2. Filters were dissolved in Ultima GoldTM scintillation mixture, and 3H radioactivity was quantified using a liquid scintillation spectrometer. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone was added to a final concentration of 50 µM by 1:100 dilution from an ethanol stock solution 1–2 min prior to beginning the assay, where indicated. Vmax and Km values were determined by direct fit to the Michaelis-Menten equation using the program Kaleidagraph. Activity over a range of GABA concentrations was measured at least three times, each a separate culture from freshly transformed cells. Parallel experiments performed with PE-containing and PE-lacking strains carrying empty vector plasmids were used to correct for the background uptake of [3H]GABA in all cases.
Competitive Uptake and Transport Specificity Ratio Analysis—Dual-label competitive uptake assays were performed as described previously (14) with the following modifications. E. coli strains expressing the Cys-less GabP from pSCK-380-CL-Hx10 were cultured at either 30 °C (PE-containing, AL95/pDD72GM) or 37 °C (PE-lacking, AL95) in LB medium supplemented with ampicillin (100 µg/ml) and glucose (0.4% w/v). IPTG was added during the final 2 h of logarithmic growth to induce GabP expression. The cells were washed by centrifugation, resuspended (A600 = 6.7) in ice-cold MKM buffer supplemented with chloramphenicol (40 µg/ml), and held on ice until used. Washed cells (90 µl), prewarmed to 30 °C, were rapidly combined with a prewarmed substrate mixture (10 µl) containing 70 µM [3H]NA (4.2 µCi/ml) and 30 µM [14C]GABA (0.6 µCi/ml) in MKM buffer and then stopped at times indicated in the figure legends by the addition of ice-cold MKM buffer supplemented with 20 mM HgCl2. The sample was applied to a type hemagglutinin filter, which was then washed with 3 ml of ice-cold MKM supplemented with 5 mM HgCl2. Filters were dissolved in Ultima Gold scintillation mixture, and 3H and 14C radioactivity (disintegrations) was analyzed with a Packard BioScience Tri-Carb 2900 TR liquid scintillation spectrometer using stored Ultima GoldTM quench curves and automatic quench compensation. Parallel experiments were performed with PE-containing and PE-lacking strains lacking the GabP expression plasmid and were used to correct for the background uptake of [14C]GABA and [3H]NA.
The TSR was obtained by analyzing the results of dual-label competitive uptake with Equation 1 (13),
 | (Eq. 1) |
 | (Eq. 2) |
Gb, is realized in the transition state transporter conformation. Thus, the TSR parameter (a constant, characteristic of a particular transporter molecule and a pair of substrates) quantifies the change in binding energy (Gb) that is because of the structural difference between two competing substrates (e.g. GABA versus NA). Therefore, any measured change in the TSR parameter requires an underlying change in the transition state binding affinity for at least one member of the substrate pair (13).
Competitive Inhibition—The PE-containing (AL95/pDD72GM) and PE-lacking (AL95) E. coli strains harboring pSCK-380-CL-Hx10 (encodes Cys-less GabP) were cultured and processed for transport as described in the previous section. The PE-containing and PE-lacking strains were induced, respectively, with 2 µM and 200 µM IPTG to obtain comparable initial transport velocities. Prewarmed cells were exposed for 45 s (initial rate) to 10 µM [14C]GABA (0.2 µCi/ml) plus 10–12 different inhibitor concentrations, spanning approximately a 500-fold range centered on the titration midpoint. Parallel experiments were performed with strains lacking the GabP expression plasmid to account for background [14C]GABA uptake. GraphPad PrismTM software was used to fit the resulting data by Equation 3 (17),
 | (Eq. 3) |
 | (Eq. 4) |
Chemical Labeling of Cys Residues—A 50-ml culture of cells carrying a pSCK-380-X#Y-Hx10 plasmid was induced for GabP expression, grown to a final A600 of 0.5–0.6, and resuspended after harvesting to an A600 of 25 in 1 ml of buffer A (100 mM K+-HEPES (pH 7.5), 250 mM sucrose, 50 mM MgCl2, 0.1 mM KCl). Exposed reactive groups on whole cells were biotinylated by adding MPEOB to a final concentration of 100 µM followed by incubation for 5 min at room temperature. The reaction was quenched by the addition of -mercaptoethanol to 20 mM, followed by two cycles of centrifugation and resuspension in buffer A containing 20 mM -mercaptoethanol. To expose Cys residues facing the cytoplasm to external solvent, cell suspensions were vortexed vigorously with 0.5% (v/v) toluene for 1 min and subjected to labeling as described above. Labeled cells were resuspended in 0.5 ml of 10 mM Tris-HCl (pH 8.0), 5 mM EDTA, 5 mM -mercaptoethanol and then solubilized and immunoprecipitated as described below.
Immunoprecipitation and Western Blot Analysis—After MPEOB labeling, cells were lysed by addition of an equal volume of 0.2 M NaOH, vortexed, incubated for 15 min on ice, and then centrifuged at 20,800 x g for 15 min at 4 °C. The pellets were washed once with 4 M KCl and once with 10 mM Tris-HCl (pH 8.0), and then solubilized by resuspension in 50 µl of 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% SDS, followed by vigorous vortexing for 40 min at room temperature and incubation for 15 min at 37 °C. Samples were diluted by adding 1.4 ml of cold 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 0.1 mM EDTA, 0.1% Lubrol (immunoprecipitation buffer 1; IP1), and immunoprecipitated with anti-GabP polyclonal antibody by incubation overnight at 4 °C. The antibody complex was isolated by addition of 50 µl of a suspension of Pansorbin reconstituted in IP1 according to the supplier's instructions. The samples were gently rocked at 4 °C for 60 min. After centrifugation the pellet was washed once with 1 ml of IP1, twice with 1 ml of 50 mM Tris-HCl (pH 8.0) containing 1 M NaCl, 0.1% Lubrol, and once with 10 mM Tris-HCl (pH 8.0). The final precipitates were solubilized in 30 µl of SDS sample buffer (50 mM Tris (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol) and incubated at 37 °C for 15 min. The samples were centrifuged, and the solubilized proteins were subjected to SDS-PAGE in 12% gels. The samples were transferred from the gels to nitrocellulose membranes as described previously (19). Avidin-horseradish peroxidase (1:50,000 dilution of 2 mg/ml stock solution) and conventional ECL reagents were used to visualize biotinylated proteins according to the manufacturer's instructions. A Bio-Rad Fluor-S Max imager was used to record the results.
Direct Western blots were also performed. Cells were lysed by the addition of SDS-PAGE sample buffer. Following SDS-PAGE on 12% gels and membrane transfer, the anti-GabP polyclonal antibody was used at a dilution of 1:2000, with detection by an anti-rabbit antibody conjugated with (horseradish peroxidase) and imaging as above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Activity and Expression of GabP—The completely Cys-less GabP expressed from plasmid pSCK-380-CL-Hx10 displays a Vmax that is 50-fold lower than wild type GabP (16) and, except for reintroduction of the native Cys at position 300 (pSCK380-A300C-Hx10), is on the same order as the other single Cys replacements used (Table II). Therefore, the effects of phospholipid composition on GabP kinetic properties were carried out on cells expressing pSCK380-CL-Hx10, which was assumed to be representative of the single Cys replacements. Whereas expression levels of GabP from plasmid pSCK-380-CL-Hx10 (Cys-less GabP) were comparable in PE-containing (AL95/pDD72GM) and PE-lacking cells (AL95) as determined by Western blot analysis (data not shown), transport function was greatly compromised in PE-lacking cells. Vmax values for GabP transport of GABA, corrected for activity observed in plasmid-lacking strains, were 3.0 ± 0.6 nmol/min/mg in PE-containing cells, and 0.020 ± 0.008 nmol/min/mg in PE-lacking cells, a decrease of more than 99%. On the other hand, Km values were largely unaffected, 12.2 ± 0.6 µM for PE-containing cells and 6.7 ± 1.3 µM for PE-lacking cells. The observed transport was primarily energy-dependent for both cell types, as the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone reduced transport in PE-containing cells by 99% and in PE-lacking cells by 90%.
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-alanine are diminished modestly (2-fold) in PE-lacking cells.
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-galactosidase as an indicator (3, 20).
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To further substantiate the misorganization of the N-terminal hairpin domain of GabP in PE-lacking E. coli cells, both cell types expressing single cysteine derivatives were pretreated with toluene before MPEOB labeling. In PE-containing cells no additional biotinylation occurred for the P domains, but extensive biotinylation of C domains occurred (Fig. 3). These results are in agreement with the putative location of single cysteines in the current topology map of GabP (25) (Fig. 1). In PE-lacking cells, P1 was rendered accessible to MPEOB after toluene treatment, whereas the N terminus and C2 showed no increase in the accessibility, consistent with an inversion in the topological orientation of the N-terminal hairpin. The remainder of the domains behaved the same as in PE-containing cells. No biotinylation of GabP occurred in cells expressing the A300C (TM lane) or S144C variant (not shown) consistent with the TM domain location of the cysteine replacement. Although we provide no direct evidence for the disposition of domains C4, C10, and P11 in either cell type, they are most likely as shown in Fig. 1 based on the reactivity of single cysteines in the adjacent extramembrane domains.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Results of topological mapping using SCAMTM (20) of GabP expressed in PE-containing E. coli are consistent with previously determined organization of TM domains and adjacent extramembrane domains (25). The topological inversion of the N-terminal hairpin domain of GabP when expressed in PE-lacking cells is very similar to that observed for PheP (3), which belongs to the same amino acid/polyamine/organocation family of transporters (Fig. 4). Similarly, TM domain III appears to be the putative hinge point or transition domain wherein a large structural rearrangement must occur in order for the remaining downstream TM domains to properly orient with respect to the plane of the bilayer. For such a transition domain to exist, TM domain III in both proteins must be highly flexible, which is consistent with the presence of Gly residues that also populate bent and kinked flexible TM domains of LacY (26). The existence of mini-loops, as illustrated for TM domain III, that do not traverse the membrane has been observed in the crystal structures of aquaporin-1 (27) and the glycerol facilitator (GlpF) (28). Previously, we had postulated (3) that the transition between aberrant and native topological organization in PheP was made possible by the unrecognized unusual length (33 amino acids) of its TM domain III. However, easy access by thiol reagent to N111C within P3 and the three charged residues between position 82 and 89 of C2 (25) do not support an unusual length (about 22 amino acids) for TM domain III of GabP. Therefore, the property of the transition region that allows for partial topological rearrangement remains unresolved for these two permeases. In LacY the transition point lies at the center of the protein between the two six-TM domain helical bundles wherein lies the substrate binding site (26). We have postulated that TM domain VII and the long cytoplasmic loop C6 that connects TM domains VI and VII of LacY may form a flexible domain that allows for large structural rearrangement in this region (4).
What are the topogenic signals within the protein sequence that are responsive to membrane lipid environment? Overall the GabP and PheP (amino acid/polyamine/organocation family) exhibit a similar response to changes in membrane lipid environment that differs markedly from that of LacY (major facilitator superfamily). A common motif of many bacterial cytoplasmic membrane proteins is a net positive charge for the extramembrane domains facing the cytoplasm as compared with those facing the exterior of the inner membrane (29). LacY follows the "positive inside rule" for its extramembrane domains but contains negatively charged amino acids near the cytoplasm domain-TM domain interfaces for extramembrane loops C2–C6. Placing negatively charged residues near this interface has been shown to weaken the topological influence of the positive inside rule (30) possibly making these domains more prone to translocation, particularly where an anionic lipid environment would favor protonation of the amino acid side chain. In GabP and PheP, domains C8, C10, and the C terminus (Fig. 4) are all highly positive relative to the neighboring periplasmic domains, which would favor their retention on the cytoplasmic side of the membrane in cells containing only anionic phospholipids (2). Domains P1 through C6 are neutral to acidic in both GabP and PheP and therefore may be more influenced in their orientation when the negative charge density at the membrane surface is increased by a lack of PE. The high negative charge density would also increase the pKa of the acidic amino acids at the membrane surface favoring their protonation and translocation across the membrane. Because C4 and C6 of GabP and PheP are not translocated in PE-lacking cells, there must be additional constraints on their topological organization. Among these may be TM domain helix packing and nearest neighbor interactions. Although glycine residues can introduce flexibility into TM domains, they also appear to be points of close interaction between TM domains within the membrane (31). Changing a cysteine to glycine in the TM domain V of LacY reduces flexibility and increases the thermal stability of the protein. In this case the introduction of a smaller amino acid appears to allow closer contact between TM domains I and V (26).
Is topological misorganization directly related to changes in kinetic properties of these permeases, or are there subtler lipid requirements for function? Reconstitution of purified LacY into proteoliposomes containing PE and PG (phosphatidylglycerol) or PC (phosphatidylcholine) and PG results in normal topological organization of the two halves of LacY relative to each other (9); lack of either zwitterionic phospholipid results in aberrant topology. However, PC plus PG or PG alone will support only facilitated but not active transport by LacY (8, 9). Interestingly, introduction of a neutral glycolipid, 
-monoglucosyl diacylglycerol, into E. coli cells lacking PE partially restored active transport function by LacY (32) and normal topological organization.2 These results suggest that topological organization may be dependent on interaction of the protein topogenic signals with the charge nature of the lipid bilayer, whereas the kinetic properties may be sensitive to less dramatic changes in the packing between TM helices and the organization of extramembrane domains. The combined subtle and major changes in protein structure, function, and organization brought about by changes in lipid environment supports a functional role for the broad diversity of lipids found in native membranes (1).
For secondary transporters vectoral transport and accumulation of substrate against a concentration gradient depend on the direction of and coupling to the H+ electrochemical potential and not the orientation of the transporter in the membrane. Because GabP, PheP, and LacY still carry out transport in the absence of PE, the functioning permeases retain a substrate binding site and are sufficiently compact structures to execute transport. For LacY it is clear that changes in the lipid environment alter the ability of the transporter to utilize the H+ electrochemical potential independent of topological organization as discussed above. However, for GabP and PheP (3) transport remains largely active. The TSR data for GabP show that lipids can have a selective effect on substrate specificity, which is more evident in the transition state than in initial binding. For PheP it is also clear that the lipid environment changes substrate specificity as indicated in the significant increase in Km in the absence of PE. This renders characteristics of the lipid-permease interface subject to natural selection on the basis of uptake specificity. In other words, characteristics of the lipid-protein interface may be expected to co-evolve with permease specificity. This co-evolution may be highly general, because unless transport-related conformational transitions leave the lipid-protein interface energetically unperturbed, there are thermodynamic constraints requiring the interface to function as a determinant of substrate specificity (14). Thus, selective pressures favoring the emergence of novel substrate specificities can be satisfied in part by structural alterations at the lipid-protein interface.
Evidence is emerging that protein structure and function have co-evolved with their lipid environment. There are several other examples that support the concept that membrane protein sequence is written for a specific lipid environment. The P-glycoprotein is localized to mammalian cytoplasmic membranes where the protein contains 12 TM domains with both the N and C terminus exposed to the cytoplasm. When expressed in E. coli, the N-terminal half of the protein assumes native topology but TM domain VII no longer spans the membrane, TM domains VIII–XII assume an inverted orientation, and the C terminus is exposed to the periplasmic side of the membrane (33). The Klebsiella pneumoniae, citrate transporter displays 11 TM domains when inserted into dog pancreas endoplasmic reticulum membranes but only 9 TM domains when expressed in E. coli (34), whose lipid composition closely mimics that of its host.
This study has broadened the number of membrane proteins whose topological organization and function are affected by their lipid environment. The observation that GabP and PheP, belonging to the same amino acid/polyamine/organocation family of permeases, respond to a lipid environment in a similar but distinctly different manner than LacY suggests that the distribution of lipid-sensitive topogenic signals may be similar within related protein families. Current work is now focusing on identifying the topogenic signals within protein sequences that are responsive to lipid environment.
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FOOTNOTES |
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¶ Current address: Dept. of Biology, Stanford University, Palo Alto, CA 94305-5430. E-mail: weiz{at}stanford.edu.
** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 6431 Fannin St., Suite 6.200, University of Texas-Houston, Medical School, Houston, TX, 77030. Tel.: 713-500-6051; Fax: 713-500-0652; E-mail: William.Dowhan{at}uth.tmc.edu.
1 The abbreviations used are: PE, phosphatidylethanolamine; C, cytoplasmic; GABA, -aminobutyric acid; GabP, -aminobutyric acid permease; LacY, lactose permease; MKM, MOPS/KCl/MgCl2 buffer; MOPS, 3-[N-morpholino]propanesulfonic acid; MPEOB, (+)-Biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine; NA, nipecotic acid; P, periplasmic; PC, phosphatidylethanolamine; PG, phosphatidylglycerol; PheP, phenylalanine permease; SCAMTM, substituted cysteine accessibility method of TM analysis; TM, transmembrane; TSR, transport specificity ratio; IPTG, isopropyl 1-thio--D-galactopyranoside.
2 J. Xie, M. Bogdanov, and W. Dowhan, unpublished observations.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) and [3H]NA dpm (
) were used in conjunction with 
), which may be read from the right ordinate. The initial rate data (out to 20 s) are considered to provide an accurate TSR = 5, whereas beyond the linear uptake range (dashed lines), TSR calculations become increasingly approximate. Panel PE–, using PE-lacking cells, the initial rate slopes (solid lines fit by linear least squares) for competitive uptake of [14C]GABA dpm (
