From the Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands
Received for publication, December 8, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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The carboxyl-terminal membrane-spanning
segment 8 of the glutamate transporter GltT of Bacillus
stearothermophilus was studied by cysteine-scanning mutagenesis.
21 single cysteine mutants were constructed in a stretch ranging from
Gly-374 to Gln-404. Two mutants were not expressed, four were
inactive, and two showed severely reduced glutamate transport activity.
Cysteine mutations at the other positions were well tolerated. Only the
two most amino- and carboxyl-terminal mutants (G374C, I375C, S399C, and Q404C) could be labeled with the large thiol reagent fluorescein maleimide, indicating unrestricted access and a location in a loop
structure outside the membrane. The labeling pattern of these mutants
using membrane- permeable and -impermeable thiol reagents showed that
the N and C termini of the mutated stretch are located extra- and
intracellularly, respectively. Thus, the location of the
membrane-spanning segment was confined to a stretch of 23 residues
between Gly-374 and Ser-399. Cysteine residues in three mutants in the
central part of the segment (M381C, V388C, and N391C) could be labeled
with the small and flexible reagent 2-aminoethyl methanethiosulfonate
hydrobromide only, suggesting accessibility via a narrow aqueous pore.
When the region was modeled as an Glutamate transporters in the mammalian central nervous system
remove the neurotransmitter glutamate from the synaptic cleft into
surrounding neurons and glial cells. Removal of glutamate prevents
neurotoxicity of high concentrations of glutamate and helps to end the
excitatory signal at some synapses (1-4). The proteins are secondary
transporters that couple glutamate transport against the concentration
gradient to the transport of protons, sodium ions, and potassium ions
across the membrane. The glutamate transporters belong to a large
family of transport proteins in which are also found bacterial
glutamate transporters including GltT of Bacillus
stearothermophilus (5). Computational analyses of the amino acid
sequences and hydropathy profiles of the glutamate transporters showed
that the proteins form a unique structural class of membrane proteins,
which is structurally not related to any other family of secondary
transporters (5, 6). Subsequent experimental studies confirmed that the
proteins contain unique structural features, like water-filled pores
and pore loops, which are not found in "regular" secondary
transporters (7-11).
The amino-terminal half of the transporters contains six
membrane-spanning -helix, all positions at which
cysteine mutations lead to inactive or severely impaired transporters
cluster on one face of this helix. The inactive mutants showed neither
proton motive force-driven uptake activity nor exchange activity nor
glutamate binding. The results indicate that transmembrane segment 8 forms an amphipathic
-helix. The hydrophilic face of the helix lines
an aqueous pore and contains many residues that are important for activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices, with the amino terminus of the proteins located in the cytoplasm. The membrane topology of the
carboxyl-terminal half of the transporters is still somewhat
controversial. However, in the most generally accepted model the six
amino-terminal
-helices are followed by a reentrant loop entering
the membrane from the cytoplasmic side, a seventh membrane-spanning
helix, a reentrant loop entering the membrane from the extracellular
side and, finally, an eighth membrane-spanning segment, leaving the
carboxyl terminus in the cytoplasm (Fig.
1). The eukaryotic and prokaryotic
glutamate transporters differ predominantly in the length of their
hydrophilic regions. Three hydrophilic stretches are considerably
longer in the eukaryotic proteins: the amino-terminal and
carboxyl-terminal extensions and the region between the third and
fourth membrane-spanning segment, which is glycosylated in the
eukaryotic members. Evidence is accumulating that the carboxyl-terminal
half of the transporters, which is particularly well conserved,
constitutes a major part of the translocation pathway and contains the
binding sites for the substrate and cotransported ions (7, 12-18). The
bacterial and eukaryotic glutamate transporters have different coupling ion specificity. The bacterial proteins catalyze the electrogenic symport of glutamate with two protons or a proton and a sodium ion. The
eukaryotic glutamate transporters catalyze the electrogenic symport of
glutamate with three sodium ions and one proton, whereas one potassium
ion is antiported.
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Fig. 1.
Model for the membrane topology of glutamate
transporters. Numbered rectangles indicate
membrane-spanning segments. The reentrant loops are numbered
6a and 7a.
The location of the most carboxyl-terminally located membrane-spanning
segment (segment 8), which enters the membrane from the extracellular
side and runs to the cytoplasmic side, is particularly ill defined.
Previous cysteine-scanning mutagenesis studies with two glutamate
transporters (human EAAT1 and rat EAAT2) have restricted the
membrane-spanning segment to a stretch of about 50 amino acids (10,
11). Hydropathy profile analysis is not very helpful in defining the
position of segment 8 more accurately because the region is relatively
hydrophilic and does not contain a stretch of ~20 hydrophobic
residues, which is characteristic for membrane-spanning -helices.
The secondary structure of membrane-spanning segment 8 has been a
matter of debate, and it has been modeled both as an
-helix and a
-strand (10, 11, 16, 19). A
-strand structure was suggested
because of the hydrophilic nature of the region (11, 19), whereas an
-helical structure was suggested on the basis of computational
analysis of periodicity in the amino acid sequences of the glutamate
transporters (5, 16). No firm experimental evidence for either
structure has been presented.
Here, cysteine-scanning mutagenesis is applied to the region containing
membrane-spanning segment 8 in the glutamate transporter GltT of
B. stearothermophilus. The position of the membrane-spanning segment is confined to a stretch of 23 amino acids, which is likely to
adopt an -helical conformation. The segment lines an aqueous channel
and contains many residues that are of crucial importance for glutamate
binding and transport.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Growth Conditions
Escherichia coli strain ECOMUT2, which lacks the glutamate transporter GltP (20), was used to express GltT mutants and was grown in Luria Broth medium at 37 °C. Chloramphenicol and ampicillin were used at final concentrations of 30 µg/ml and 100 µg/ml, respectively. Expression of GltT mutants from pBAD24-derived plasmids (21) was induced by adding 0.15% L-arabinose at an A600 of 0.5, and cells were harvested 1.5 h after induction.
Recombinant DNA Techniques
General molecular biological techniques are described by Sambrook et al. (22). Enzymes for recombinant DNA work were obtained from Roche Molecular Biochemicals (Germany). The polymerase chain reaction overlap extension method (23) was used to introduce mutations in the gltT gene on a pBAD24-derived expression plasmid, which encodes GltT with an amino-terminal His tag consisting of six adjacent histidines (9). Oligonucleotides were obtained from Life Technologies, Inc. All polymerase chain reaction-amplified DNA fragments were sequenced by BMTC (Groningen, The Netherlands).
Preparation of Membrane Vesicles
Membrane vesicles with a rightside-out orientation were prepared from E. coli ECOMUT2 cells expressing GltT mutants by the osmotic lysis procedure, as described by Kaback (24). Randomly oriented membrane vesicles and membrane vesicles with an inside-out orientation were prepared with sonication and passage through a French pressure cell, respectively, as described previously (9). Membrane vesicles were stored in liquid nitrogen at a protein concentration of 10 mg/ml in 50 mM potassium phosphate, pH 7. The protein concentration was determined with the DC protein assay from Bio-Rad. Expression levels of His-tagged proteins were determined by running membrane vesicles (20 µg of protein) on SDS-polyacrylamide gels followed by transfer to poly(vinylidene difluoride) membranes (Roche Molecular Biochemicals) and detection with monoclonal antibodies directed against a 6 His tag (Dianova, Hamburg, Germany). Antibodies were visualized by using the Western light chemiluminescence detection kit (Tropix, Bedford, MA).
Purification and Reconstitution
GltT mutant proteins were purified and reconstituted into
proteoliposomes as described before (25) with the following minor modifications. The detergent
n-dodecyl--D-maltoside (Anatrace, Ohio) was
used instead of Triton X-100 to solubilize cytoplasmic membranes (0.4 mg/4 mg of membrane protein) and during purification of GltT (0.03%).
Triton X-100 was still used to destabilize preformed liposomes (25).
The lipid to protein ratio during reconstitution was 330 (w/w). The
protein concentration was determined with the DC protein assay from
Bio-Rad. Proteoliposomes were stored in liquid nitrogen an appropriate
buffer (see below).
Labeling of Membrane Vesicles and Proteoliposomes
Unless stated otherwise, membrane vesicles with a rightside-out orientation (10 mg of protein/ml) were labeled for 10 min at room temperature with 0.25 mM N-ethylmaleimide (NEM)1 or with the methanethiosulfonate reagents (2-(trimethylammonium)ethyl) methanethiosulfonate bromide (MTSET, 1 mM) and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 2.5 mM) (Anatrace, Ohio). Immediately after labeling, glutamate uptake experiments were performed with these membrane vesicles using artificial gradients.
Membrane vesicles with a random orientation (500 µl, 2 mg of protein/ml in 50 mM potassium phosphate, pH 7) were labeled with 0.25 mM fluorescein maleimide (Molecular Probes, Eugene, Oregon) for 10 min at room temperature. The reaction was stopped by adding a 10-fold excess of dithiothreitol, followed by small scale purification of His-tagged GltT mutants using Ni2+-nitrilotriacetic acid agarose (9). Purified proteins were run on SDS-polyacrylamide gels. Fluorescence of proteins labeled with fluorescein maleimide was visualized by UV excitation using a Gel-Doc system (Bio-Rad).
Proteoliposomes (30 µg of protein) were labeled with 0.25 mM NEM or 0.25 mM 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMdiS) at room temperature for 10 min in 50 mM potassium phosphate, pH 7. The reaction was stopped with a 10-fold excess of dithiothreitol. The labeled proteoliposomes were washed once with 50 mM potassium phosphate, pH 7, and solubilized in 300 µl of the same buffer containing 2% Triton X-100. 0.25 mM fluorescein maleimide was added, and the mixture was incubated for 10 min at room temperature. The reaction was stopped with a 10-fold excess of dithiothreitol. Proteins were precipitated with trichloroacetic acid and run on SDS-polyacrylamide gels. Fluorescence of proteins labeled with fluorescein maleimide was visualized using a Lumi-imager (Roche).
Glutamate Transport Assays
Membrane Vesicles-- Glutamate uptake in rightside-out membrane vesicles was measured by rapid filtration. Membrane vesicles were either energized using the potassium ascorbate/phenazine methosulfate electron donor system or by artificial gradients. In the former case, the membranes were diluted to a concentration of 0.6 mg/ml in 50 mM potassium phosphate, pH 7, and 10 mM potassium ascorbate. The uptake experiments were performed in 100 µl at 30 °C under a constant flow of water-saturated air. Phenazine methosulfate was added at a concentration of 100 µM, and the proton motive force was allowed to develop for 2 min, after which L-[14C]glutamate (Amersham Pharmacia Biotech) was added to a final concentration of 1.9 µM. The uptake was stopped by adding a 20-fold excess of ice-cold 0.1 M LiCl solution, followed by immediate filtration over cellulose nitrate filters (pore size, 0.45 µm). The filters were washed once with 2 ml of 0.1 M LiCl and assayed for radioactivity. When artificial gradients were used, a buffer system described previously was used (20). Membrane vesicles (10 mg protein/ml) were loaded with a buffer containing 25 mM potassium phosphate, pH 7, 100 mM potassium acetate, pH 7, and 1 µM valinomycin. Proton motive force-driven uptake was initiated by diluting the 4-µl vesicles into 330 µl of buffer containing 125 mM MES, adjusted to pH 6 with methylglucamine, 1 µM valinomycin, and 0.6 µM L-[14C]glutamate and incubated at 30 °C. The uptake was stopped as described above.
Proteoliposomes-- For counterflow experiments proteoliposomes were loaded with 25 mM potassium phosphate and 5 mM potassium glutamate, pH 7, by freeze thawing followed by extrusion through polycarbonate filters (pore size, 400 nm). Proteoliposomes were concentrated by centrifugation (250,000 × g, 20 min, 10 °C) and resuspended at a protein concentration of ~0.2 mg/ml. Counterflow was initiated by diluting 4 µl of proteoliposomes into 330 µl of 30 mM potassium phosphate, pH 7, prewarmed at 30 °C, and containing 1.2 µM L-[14C]glutamate. The uptake was stopped as described above. Glutamate uptake driven by artificial gradients in proteoliposomes was assayed using the same procedure as described for counterflow but with the buffer system for artificial gradient described above.
To measure efflux, proteoliposomes were loaded with
L-[14C]glutamate using the artificial
gradient method until ~10 pmol/µg of protein was taken up by the
proteoliposomes. 10 µM Carbonyl cyanide
p-trifluoromethoxyphenylhydrazone was added, and the efflux reaction was stopped at several time points by diluting aliquots of the
reaction mixture in ice-cold 0.1 M LiCl followed by rapid filtration as described above.
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RESULTS |
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Construction, Expression, and Activity of Single Cysteine
Mutants--
The cysteineless version of GltT, which was shown
previously to have glutamate uptake activity similar to that of the
wild type (9), was used as background for the construction of a set of
21 single cysteine mutants in the region of membrane-spanning segment 8 (Fig. 1). Each residue in the amino acid stretch that is highly
amphipathic when modeled an -helix (residues 374-392) (5) was
mutated to cysteine, as well as selected residues at the
carboxyl-terminal end of the stretch. Out of the 21 mutants 19 could be
expressed in E. coli and were present in the cytoplasmic membrane as judged from Western blots using antibodies raised against
the amino-terminal His tag which is present in all mutants (Fig.
2A). Only two mutants, D376C
and G390C, were not found in the membrane of E. coli (not
shown). The glutamate transport activity of the mutants that were
expressed was measured in membrane vesicles with a rightside-out
orientation. At most of the positions cysteine mutations were well
tolerated by the transporter resulting in glutamate transport
activities between 40 and 125% of the cysteineless mutant (Fig.
2B). In four mutants (D380C, R383C, T384C, and N387C) glutamate transport was completely abolished. In two other mutants, R377C and M381C, glutamate uptake was measurable but severely impaired
(5 and 17% of the activity of the cysteineless mutant, respectively)
(Fig. 2B). In none of these mutants was the impaired or
abolished activity caused by a reduced level of expression (Fig.
2A).
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Labeling of Single Cysteine Mutants with Fluorescein Maleimide-- The accessibility of the cysteine residues in the GltT mutants for the large maleimide compound fluorescein maleimide (molecular weight 427) was determined. Randomly oriented membrane vesicles were prepared from cells expressing the various GltT mutants and labeled with fluorescein maleimide. Subsequently, the vesicles were solubilized, and His-tagged GltT mutants were purified, followed by SDS-polyacrylamide gel electrophoresis. Fluorescence of GltT mutants labeled with fluorescein maleimide was detected by UV excitation.
The cysteineless mutant was used as a negative control and was not
modified by fluorescein maleimide (not shown). Only four single
cysteine mutants (G374C, I375C, S399C, and Q404C) were labeled with
fluorescein maleimide (Fig. 3). The
cysteine residues in the four mutants are located at the extreme amino
and carboxyl termini of the mutated stretch. The accessibility of these
residues for fluorescein maleimide indicates a location in the protein structure, which is well exposed to the aqueous environment and is
consistent with a position in a loop structure. The other single cysteine mutants could not be labeled with fluorescein maleimide, as
exemplified by mutants R377C, A382C, and S392C (Fig. 3). The cysteine
residues in these mutants are either buried in the protein structure or
exposed to the lipid environment.
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Although mutants G374C, I375C, and S399C were readily labeled with fluorescein maleimide, the accessibility of the cysteine residues in these mutants is not completely unrestricted: biotin maleimide (molecular weight 524), which was used previously in membrane topology studies of GltT (9), did not label these mutants (not shown). Only the single cysteine in mutant Q404C was readily labeled with biotin maleimide in membrane vesicles with an inside-out orientation, and labeling was prevented by preincubation with the charged maleimide AMdiS, suggesting a cytoplasmic location (9). In agreement, Q404C was not labeled with biotin maleimide in whole cells (not shown).
Effect of NEM, MTSEA, and MTSET on Transport Activity--
The
effect of cysteine modification on glutamate transport activity was
determined in membrane vesicles with a rightside-out orientation
prepared from E. coli cells expressing the single cysteine
mutants. Three small modifying reagents were used: NEM and MTSEA, which
are membrane-permeable, and MTSET, which is charged and reportedly
membrane-impermeable (26, 27). Treatment with NEM did not affect
glutamate uptake of the cysteineless mutant when the vesicles were
energized with the artificial electron donor system ascorbate/phenazine
methosulfate (9). In contrast, the methanethiosulfonate reagents
severely reduced glutamate uptake activity by the cysteineless mutant
using the same energizing method. Apparently, the reagents interfere
with the generation of a proton motive force by inhibition of one of
the components of the electron transport chain. Therefore, the proton
motive force was generated using a combination of acetic acid and
K+ diffusion gradients (20). Glutamate transport catalyzed
by the cysteineless mutant driven by the diffusion gradients was not
significantly affected by the methanethiosulfonate reagents or NEM
(Fig. 4A).
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One of the four mutants that were labeled with fluorescein maleimide (Fig. 3), G374C, was inactivated by both the membrane-permeable and membrane-impermeable modifying reagents, consistent with accessibility from the outside (Fig. 4A). In contrast, mutant S399C was inactivated by the membrane-permeable reagents MTSEA and NEM but not by the membrane-impermeable MTSET. This behavior is consistent with a cytoplasmic accessibility. The other mutants that were labeled with fluorescein maleimide, I375C and Q404C, were not inactivated by NEM (Fig. 4A). Similarly the MTS reagents did not inactivate mutant Q404C, whereas the activity of mutant I375C was only slightly affected by the reagents. Apparently, modification of the single cysteines in these mutants is well tolerated.
In the set of single cysteine mutants that were not labeled with fluorescein maleimide, three mutants, M381C, V388C, and N391C, could be inactivated by the membrane-permeable reagent MTSEA but not by the membrane-impermeable reagent MTSET and also not by NEM (Fig. 4A). Apparently, accessibility of the cysteine residues is very much restricted, allowing the small and flexible MTSEA reagent, but not NEM, which has a more constrained structure, to reach the residues. Because the membrane-impermeable analogue MTSET did not inhibit the three mutants, the cysteines in the mutants appear to be accessible through a narrow aqueous pore from the cytoplasmic side of the membrane.
Finally, the activity of most of the mutants that were not labeled with
fluorescein maleimide was not affected by any of the modifying
reagents. These mutants either tolerated modification or could not be
modified at all because they were not accessible. Based on the results
described here it is not possible to distinguish between the two
possibilities. When the region between Gly-374 and Ser-392 is modeled
as an -helix, all inactive, severely impaired, and nonexpressed
mutants cluster on one face of the helix (black circles in
Fig. 5B), whereas the active
mutants cluster on the other face (bars in Fig.
5A and open circles in 5B). The
mutants that were inactivated by the cysteine-modifying reagents also cluster on one face of the helix. This face partially overlaps with the
face of the helix containing the inactive mutants (gray bars
and gray circles in Fig. 5, A and B,
respectively).
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Kinetics of MTSET Labeling-- Mutant G374C that is accessible from the periplasmic side of the membrane was inactivated with the membrane-impermeable reagent MTSET with a half-time of 1-2 min (Fig. 4B). Mutant S399C that is located at the cytoplasmic side of the membrane was not significantly inactivated in 10 min. However, longer incubations did result in partial inactivation of the mutant. This is most likely caused by a slow permeability of the reagent followed by access from the cytoplasmic side of the membrane. Similar observations were made for mutants M381C, V388C, and N391C (shown for V388C and N391C in Fig. 4B), suggesting a similar accessibility from the cytoplasmic side of the membrane following the slow permeation of the reagent.
Orientation of GltT in Proteoliposomes--
A more detailed
kinetic characterization of the single cysteine mutants was done in
proteoliposomes. A procedure to purify GltT and to reconstitute the
protein into proteoliposomes was described before (25). To interpret
correctly results obtained with single cysteine mutants reconstituted
in proteoliposomes (below), the orientation of GltT in the liposomal
membrane was determined. Two single cysteine mutants, S129C and S292C,
which were characterized before and which contain cysteines with well defined extra- and intracellular locations, respectively (9), were
purified and reconstituted into proteoliposomes. The
membrane-impermeable maleimide AMdiS was used to label cysteines that
are accessible from the outside of the proteoliposomes, whereas NEM was
used to label cysteines on both sides of the membrane. Subsequently, unreacted cysteines were labeled with fluorescein maleimide, and the
extent of fluorescein labeling was measured. In proteoliposomes, mutant
S129C, which has an extracellular cysteine, was fully accessible for
both AMdiS and NEM, indicating that the orientation of the protein in
the proteoliposomes is rightside-out (Fig.
6). Consistent with this result, the
single cysteine in mutant S292C, which has an intracellular location,
was accessible for NEM but not for the membrane-impermeable AMdiS. The
rightside-out orientation of GltT in proteoliposomes allows a fair
comparison of the studies with membrane vesicles and
proteoliposomes.
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Glutamate Transport Activity in Proteoliposomes--
Transporter
mutants that do not show activity when transport is driven by the
proton motive force may show activity when assayed for a partial
transport reaction like exchange (see, e.g. Refs. 13 and 28). In such
cases the mutant transporter may be unable to complete the catalytic
cycle because the reorientation of the unloaded carrier is impaired, a
step that is omitted in the exchange mode, whereas the reorientation of
the substrate-loaded carrier is unaffected. The exchange reaction
catalyzed by GltT was measured using a previously described counterflow
assay in proteoliposomes (25). Mutants R377C, D380C, R383C, T384C, and
N387C, which were inactive or showed severely impaired glutamate
transport activity (Fig. 2), as well as the cysteineless mutant and
mutants G374C and N391C, were successfully purified and reconstituted
into proteoliposomes using the protocol developed for the wild-type
protein (Fig. 7A). Proton
motive force-driven glutamate uptake of the mutant proteins in
proteoliposomes was comparable with the activity in membrane vesicles
(Fig. 7B, solid bars). G374C and N391C showed
approximately 50% of the activity of the cysteineless mutant,
transport by R377C was severely impaired, but measurable and the other
transporters were inactive.
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When counterflow was measured the results did not differ significantly from proton motive force-driven transport for most mutants (Fig. 7B, open bars), indicating that mutants R377C, D380C, R383C, T384C, and N387C are not only impaired in the complete reaction cycle but also in the partial exchange reaction. Surprisingly, counterflow by mutant N391C was severely reduced compared with proton motive force-driven transport. Because efflux catalyzed by mutant N391C is also severely reduced (Fig. 7C), the impaired counterflow activity of the mutant was not caused an inability to maintain the necessary gradient of glutamate across the membrane. Therefore, the reduced counterflow activity of mutant N291C may be the result of a reduced affinity for cytoplasmic glutamate, an impaired reorientation of the substrate loaded carrier, or a stringent dependence on the presence of the proton motive force even in the exchange mode.
Glutamate Binding--
A qualitative method was developed to
measure glutamate binding in GltT mutants R377C, D380C, R383C, T384C,
and N387C, which were inactive or severely impaired in proton motive
force-driven glutamate uptake. The previously described single cysteine
mutant S269C in the reentrant loop between membrane-spanning helix 6 and 7 (Fig. 1) could be labeled with biotin maleimide in whole cells,
and labeling was prevented by the presence of L-glutamate (Fig. 8) (9). In contrast, none of the
inactive mutants described here (R377C, D380C, R383C, T384C, and N387C)
could be labeled with biotin maleimide, either in whole cells or in
membrane vesicles (not shown). Double mutants were constructed in which
mutation S269C was combined with each of the five mutations that
impaired glutamate transport. The double mutants were expressed in
E. coli and could be labeled in whole cells with biotin
maleimide, just like the single mutant S269C (Fig. 8). In the double
mutants the cysteine at position 269 serves as indicator for glutamate
binding. Labeling of the cysteine in double mutant R377C/S269C could be prevented by the presence of glutamate, indicating normal glutamate binding, even though mutant R377C displayed strongly reduced glutamate transport activity (Figs. 2 and 7). 5 mM
L-glutamate did not prevent the labeling with biotin
maleimide of the double mutants D380C/S269C, R383C/S269C, T384C/S269C,
and N387C/S269C, indicating that in these mutants glutamate binding is
completely abolished.
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DISCUSSION |
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Cysteine-scanning mutagenesis has been used to study the membrane topology of several members of the family of glutamate transporters (7-11). The proteins have a highly unusual structure, consisting of eight membrane-spanning segments and two reentrant loops (Fig. 1). There is still some controversy about the exact membrane topology of part of the proteins and, in particular, on the exact position and secondary structure of the most carboxyl-terminal, inward-going, membrane-spanning segment (segment 8). In this report, membrane-spanning segment 8 was studied by cysteine-scanning mutagenesis in the glutamate transporter GltT of the bacterium B. stearothermophilus. 21 single cysteine mutants were constructed in a stretch of 30 residues from Gly-374 to E-404, and the membrane-spanning segment could be confined to this stretch by the following criteria.
One mutant (G374C) reacted with both charged and uncharged cysteine-modifying reagents in membrane vesicles with a rightside-out orientation, consistent with a location in an aqueous environment at the extracellular side of the membrane. Furthermore, the bulky molecule fluorescein maleimide labeled the cysteine as well as the cysteine at the adjacent position in mutant I375C, indicative of a spacious access route and a location in a loop structure outside of the membrane. The result is consistent with cysteine-scanning mutagenesis studies in the human glutamate transporter EAAT1, where a cysteine at the corresponding position (A470C) was modeled at the extracellular side of the membrane (11).
In contrast, mutant S399C, the more carboxyl-terminal mutant Q404C, and the previously described mutant Q412C had labeling characteristics of residues located in a loop structure in the aqueous environment at the cytoplasmic side of the membrane. The cysteines in these mutants were accessible for the bulky molecule fluorescein maleimide. Furthermore, mutant S399C was labeled with membrane permeable cysteine-modifying reagents only in membrane vesicles with a rightside-out orientation. Cysteines at positions between Ile-375 and Ser-399 were not labeled with fluorescein maleimide. Therefore, the location of the eighth membrane-spanning segment seems to be confined to a stretch of 23 residues between Ile-375 and Ser-399, which is significantly shorter compared with previous studies that showed at least 50 residues between residues at the extra- and intracellular side of the membrane (10, 11).
The secondary structure of membrane-spanning segment 8 has been a
matter of debate, and it has been modeled as both as an -helix and a
-strand (10, 11, 16, 19). Because a hydrophobic stretch of
approximately 20 residues, which is indicative of a membrane-spanning
-helix, is not found at the position of segment 8, the presence of a
membrane-spanning
-strand was suggested (11, 19). On the other hand,
the stretch of amino acid residues that we experimentally showed to be
membrane-spanning has been modeled as an
-helix before, based on
computational analysis of the periodicity in the amino acid sequences
of glutamate transporters (5, 16). When modeled as an
-helix the
region between Gly-374 and Ser-392 is highly amphipathic (Fig.
5A) with all well conserved charged and polar residues
clustering on one face of the helix and all hydrophobic residues, which
are not conserved, clustering on the opposite face (16). Periodicity
consistent with an
-helical conformation is also observed in the
properties of the cysteine mutants described here. All positions at
which cysteine mutations are tolerated cluster on the hydrophobic,
nonconserved face of the helix, whereas residues that cannot be mutated
to cysteine without loss of activity cluster on the opposite face of
the helix (Fig. 5, A and B). Furthermore,
cysteine mutants that can be inactivated by cysteine-modifying reagents
also cluster on the same face of the helix. The results strongly
suggest that membrane-spanning segment 8 is
-helical. An
-helical
conformation of the segment would solve the problem of accommodating
the charged and polar residues in the hydrophobic core of the membrane.
If the hydrophilic face of the helix were buried within the protein
core it would be effectively shielded from the hydrophobic lipid
bilayer by the hydrophobic face.
Interestingly, cysteine residues in three mutants (M381C, V388C, and N391C), which are distributed along the long axis of membrane-spanning helix 8 at the hydrophilic face, were accessible for the cysteine-modifying reagent MTSEA (Fig. 4). This indicates that there must be an aqueous path through the protein which provides an access route to the residues. The water-filled pore is not very spacious because only small and flexible cysteine-modifying reagents can enter it. The narrow aqueous path along membrane-spanning helix 8 contrasts with the very spacious water-filled pore, which was found around the reentrant loop between helices 6 and 79. Therefore, it is unlikely that the two water-accessible pores are parts of one single larger pore. Cysteine-scanning mutagenesis in two related glutamate transporters, human EAAT1 and rat EAAT2, showed that helix 7 also contains water-accessible residues of which the accessibility is restricted to small reagents (7, 11, 12). The similar accessibility behavior of helices 7 and 8 suggests that the two helices may be close together in space.
Since the first mutagenesis experiments of glutamate transporters it is
known that certain residues in membrane-spanning helix 8 are of crucial
importance for function (11, 28-30). Particularly well characterized
is the arginine residue at the corresponding position of Arg-383 in
GltT, which is conserved in all glutamate transporters of the family
(5). Mutations at this position in the human glutamate transporters
EAAT1 and EAAT3 as well as the glutamate transporters EAAT1 and EAAT2
from rat completely abolished glutamate transport (11, 28-30).
Interestingly, the arginine mutant in EAAT3 (R447C) was still able to
transport cysteine, which is a substrate of EAAT3 but not of the other
glutamate transporters. It was concluded that Arg-447 is involved in
the binding of the -carboxylate group of glutamate (28). The
observation that mutant R383C in GltT was completely inactive and could
not bind glutamate is consistent with the findings in EAAT3.
In addition, the results presented here show that many other residues at the hydrophilic face of membrane-spanning helix 8 at positions ranging from the extracellular to the cytoplasmic side of the membrane are of crucial importance for the transporter's function. Starting at the amino-terminal end of the helix, mutant R377C was severely impaired in glutamate transport but could still bind glutamate. Subsequently, three mutants, D380C, T384C, and N387C, behaved similarly to mutant R383C: These mutants do not transport glutamate in either the proton motive force-driven uptake mode or the exchange mode and do not bind glutamate. Finally, three mutants, M381C, V388C, and N391C, with cysteines at the hydrophilic face of the helix, could be inactivated by cysteine-modifying reagents. In the latter mutant glutamate exchange and efflux activities were severely impaired, whereas proton motive force-driven glutamate uptake was only mildly affected. Possibly, Asn-391 is involved in binding of cytoplasmic glutamate, which would be consistent with its location at the cytoplasmic end of membrane-spanning segment 8.
In addition to membrane-spanning segment 8 several other regions in the
glutamate transporters are important for binding of glutamate, binding
of cotransported cations, and translocation. These regions include the
reentrant loop between helices 6 and 78, 9, helix 77, 13, 15, and the
reentrant loop between helices 7 and 814. Further work is required to
find the spatial relation between these regions. In such studies the
multimeric structure of the transporters will also have to be
considered. Recently, it was reported that the human glutamate
transporter EAAT3 is present as a pentamer in oocyte membranes (31). It
is not known whether a pentameric structure is common among glutamate
transporters and, importantly, whether the monomer subunits
functionally interact. Such information will be required to build a
model for the substrate binding site and the translocation path.
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FOOTNOTES |
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* This work was supported by the Ministry of Economic Affairs, the Ministry of Education, Culture, and Science, and the Ministry of Agriculture, Nature Management, and Fishery in the framework of an industrial relevant research program of the Netherlands Association of Biotechnology Centers in The Netherlands.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology,
University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. Tel.: 31-50-363-2155; Fax: 31-50-363-2154; E-mail: j.s.lolkema@biol.rug.nl.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M011064200
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ABBREVIATIONS |
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The abbreviations used are: NEM, N-ethylmaleimide; MTSET, (2-(trimethylammonium)ethyl) methanethiosulfonate bromide; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; AMdiS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid..
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