THE JOURNAL OF BIOLOGICAL CHEMISTRY
55728 –55736, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Oligomerization of the ␥-Aminobutyric Acid Transporter-1 Is Driven
by an Interplay of Polar and Hydrophobic Interactions in
Transmembrane Helix II*
Received for publication, August 17, 2004, and in revised form, October 18, 2004
Published, JBC Papers in Press, October 20, 2004, DOI 10.1074/jbc.M409449200
Vladimir M. Korkhov‡, Hesso Farhan, Michael Freissmuth§, and Harald H. Sitte
From the Institute of Pharmacology, Medical University of Vienna, Waehringer Strasse 13a, Vienna A-1090, Austria
The available evidence indicates that members of the
neurotransmitter:sodium symporter family form consti-
tutive oligomers. Their second transmembrane helix
(TM2) contains a leucine heptad repeat proposed to be
involved in oligomerization. In artificial transmem-
brane segments, interhelical interactions are stabilized
by polar residues. We searched for these hydrogen bond
donors in TM2 by mutating the five polar residues in
TM2 of the ␥-aminobutyric acid transporter-1 (GAT1).
We tested the ability of the resulting mutants to oli-
gomerize by fluorescence microscopy, Foerster reso-
nance energy transfer, and ␤-lactamase fragment
complementation. Of all generated mutants, only Y86A-
(but not Y86F-), E101A-, E101Q-, and E101D-GAT1 were
judged by these criteria to be deficient in oligomeriza-
tion and were retained intracellularly. The observations
are consistent with a model where the leucine heptad
repeat in TM2 drives a homophilic association that is
stabilized by Tyr86 and Glu101; Tyr86 participates in hy-
drophobic stacking. Glu101 is in the a-position of the
leucine heptad repeat (where positions 1–7 are denoted
a– g, and each leucine is in the central d-position). Thus,
Glu101 is in the position predicted for the hydrogen bond
donor (i.e. sandwiched between Leu97 and Leu104, which
are one helical turn above and below Glu101). These key
residues, namely Tyr86 and Glu101, are conserved in re-
lated transporters from archaeae to humans; they are
therefore likely to support oligomeric assembly in trans-
porter orthologs and possibly other proteins with mul-
tiple transmembrane segments.
␥-Aminobutyric acid (GABA)1-mediated inhibitory synaptic
transmission is terminated by clearance of GABA from the
synaptic cleft by high affinity uptake proteins. The four known
GABA transporters (GAT1, BGT1 (betaine/GABA transporter),
* This work was supported by Austrian Science Fund Grants P15034
(to M. F.) and P17076 (to H. H. S.), by Austrian National Bank Grant
10507 (to H. F.), and by 5th Framework Programme of the European
Union Grant QLG3-CT-2001-00929, “Epileptosome.” The costs of pub-
lication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Recipient of a Bertha von Suttner-scholarship from the Austrian
Academic Exchange Service.
§ To whom correspondence should be addressed: Institute of Pharma-
cology, Vienna Medical University, Waehringer Strasse 13a, A-1090,
Vienna, Austria. Tel.: 43-1-4277-64171; Fax: 43-1-4277-9641; E-mail:
michael.freissmuth@meduniwien.ac.at.
1
The abbreviations used are: GABA, ␥-aminobutyric acid; GAT1, -2,
and -3, GABA transporter-1, -2, and -3, respectively; NSS, neurotrans-
mitter:sodium symporter; TM2, transmembrane domain II; FRET, flu-
orescence resonance energy transfer; DRAP, donor recovery after ac-
ceptor photobleaching; CFP, cyan fluorescent protein; YFP, yellow
fluorescent protein; ANOVA, analysis of variance.
55728
Vol. 279, No. 53, Issue of December 31, pp.
Printed in U.S.A.
GAT2, and GAT3) belong to the neurotransmitter:sodium sym-
porter (NSS) (1) family, which also includes carriers for dopam-
ine, serotonin, glycine, etc. (2). All members of the family share
sequence similarity, membrane topology, and common func-
tional features (e.g. ion channel-like properties, sodium, and
chloride) as transported cosubstrates and bidirectional trans-
port of substrates. The interest in transporters of this family is
motivated in part by their clinical relevance; they are targets
for drugs of abuse (e.g. cocaine, ecstasy, and metamphetamine)
and for therapeutic agents including antidepressants (amitryp-
tiline, desipramine, citalopram, and paroxetine) and anticon-
vulsants (tiagabine), etc. (reviewed in Ref. 3).
With the notable exception of the glycine transporter-1 (4),
all members of the neurotransmitter transporter family that
have so far been examined form oligomers (reviewed in Ref. 5).
This is also true for GAT1, for which oligomers have been
visualized in membranes of living cells by fluorescence reso-
nance energy transfer (FRET) microscopy (6). The TM2 of
GAT1 contains a leucine heptad repeat that seems to supports
oligomer formation (7). The leucine heptad repeat in TM2 is a
highly conserved feature throughout the NSS family. Consist-
ent with this finding, TM2-mediated dimer formation was also
suggested for dopamine transporter (8).
By analogy with leucine zippers in soluble proteins, the
stretch of leucine residues in the TM2 of GAT1 lines one side of
a hypothetical ␣-helix. If this leucine heptad repeat is to serve
as an interface between two transporter molecules, there must
be additional forces that stabilize this arrangement. In aqueous
solution, the hydrophobic leucine (or isoleucine) side chains are
tightly packed to minimize the solvent exposed surface. It is,
however, difficult to conceive how the hydrophobic environ-
ment in the membrane provides the force to drive the associa-
tion of leucine residues. In fact, synthetic polyleucine segments
show little propensity for self-association in the membrane (9,
10). Nevertheless, in phospholamban and in the erythropoietin
receptor (11, 12), leucine and isoleucine-based packing interac-
tions do occur between transmembrane domains. Similarly,
artificial leucine-rich transmembrane segments can be forced
to self-associate, provided that the interaction is stabilized by a
polar hydrogen bond-donating residue (i.e. Ser, Thr, Gln, Glu,
and Asp in the a-position sandwiched between two leucine
residues in the d-positions of the heptad repeats) (9, 13). Based
on these findings, we surmised that a polar residue must be
essential to stabilize the homophilic interaction of TM2 seg-
ments in the GAT1 oligomer if the interaction is supported by
packing of the leucine heptad repeat. We therefore focused on
the polar or ionizable residues in TM2 of GAT1: Tyr86, Thr89,
Glu101, Cys102, and Ser103. Our experiments identified the two
residues Tyr86 and Glu101, both invariably present in the cor-
responding positions in all neurotransmitter:sodium symport-
ers, as key players in TM2-mediated intramembrane interac-
This paper is available on line at http://www.jbc.org
 TM2 Controls GABA Transporter-1 Oligomerization
TABLE I
Primers employed for the generation of mutated versions of GAT1
Primer Sequence
Y86A-fw 5⬘-cttcctaattccagctttcctg-3⬘
Y86A-rv 5⬘-gagcgtcaggaaagctggaat-3⬘
Y86F-fw 5⬘-cttcctaattccattcttcctg-3⬘
Y86F-rv 5⬘-gagcgtcaggaagaatggaat-3⬘
T89A-fw 5⬘-catatttcctggcgctcatc-3⬘
T89A-rv 5⬘-gcaaagatgagcgccaggaa-3⬘
T89F-fw 5⬘-tccatatttcctgttcctcatc-3⬘
T89F-rv 5⬘-ccgcaaagatgaggaacaggaa-3⬘
E101A-fw 5⬘-tctcttccttttggcgtgct-3⬘
E101A-rv 5⬘-ctagggagcacgccaaaagg-3⬘
E101D-fw 5⬘-tctcttccttttggactgctc-3⬘
E101D-rv 5⬘-ctagggagcagtccaaaagg-3⬘
E101Q-fw 5⬘-tctcttccttttgcagtgctc-3⬘
E101Q-RV 5⬘-ctagggagcactgcaaaagg-3⬘
C102A-fw 5⬘-ccttttggaggcctccctagg-3⬘
C102A-rv 5⬘-ctggcctagggaggcctcca-3⬘
S103A-fw 5⬘-cttttggagtgcgccctag-3⬘
S103A-rv 5⬘-actggcctagggcgcactc-3⬘
tion. However, in Tyr86 the hydrogen bond-donating hydroxyl
group is dispensable, and it is the aromatic ring (and hence a
hydrophobic interaction) that is (appears) essential for stabi-
lizing the oligomer. In contrast, both, the size of the side chain
and the hydroxyl group of Glu101 seem to be essential for
supporting oligomer formation. Glu101 is in the a-position of the
heptad repeat, sandwiched between Leu97 and Leu104, which,
in a helical arrangement, are one turn above and below Glu101.
Thus, Glu101 fulfills the criteria of the hydrogen bond donor
capable of stabilizing the interhelical contact.
EXPERIMENTAL PROCEDURES
cDNA Constructs and Mutagenesis—Plasmid DNA constructs YFP-
GAT1, CFP-GAT1, YFP-GAT1, YFP-GAT⌬37, CFP-GAT⌬37, and CFP-
GAT1-L97A were described elsewhere (7, 14). Mutated versions of
GAT1, carrying single amino acid substitutions (Y86A, Y86F, T89A,
T89F, G94A, E101A, E101D, E101Q, C102A, and S103A) were created
by PCR. For amplification of DNA fragments encompassing nucleotides
1–1032 of mutant GAT1 DNA sequences, we used the pairs of primers
listed in Table I.
The HindIII/AccI fragments excised from PCR products were cloned
into a HindIII/AccI-digested CFP-GAT1-L97A construct. At this step,
the L97A mutation in the former was eliminated, and the desired ones
were introduced. The derived transporters or truncated versions (amino
acids 1–110) were cloned in frame with F[1] and F[2] ␤-lactamase
fragment via AscI/NotI sites introduced by PCR. Sequences of all con-
structs were verified.
Cell Culture, Heterologous Expression, and Uptake Assays—Human
embryonic kidney 293 (HEK-293) cells were grown in Dulbecco’s mod-
ified Eagle’s medium containing L-alanyl-L-glutamine, 10% fetal bovine
serum, and 50 mg/liter gentamicin on 10-cm diameter cell culture
dishes at 37 °C in an atmosphere of 5% CO2, 95% air. One day before
transfection, cells were replated to obtain subconfluent cultures either
on glass coverslips (22 mm in diameter and placed into 6-well plates,
3 ⫻ 105 cells/well plate) or on 10-cm plates (1 ⫻ 106 cells/well plate) for
uptake experiments. Transient transfections were done using the
CaPO4 precipitation method (15) and whole-cell uptake assays as de-
scribed previously (6). In brief, 48 h after transfection with either
CFP-GAT1 or its mutated versions, HEK-293 cells were incubated for 3
min at room temperature in 0.1 ml of Krebs-Ringer-Hepes buffer (10
mM Hepes, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 20 mM
glucose, final pH 7.3) containing 15–20 nM [3H]GABA in the presence of
increasing concentrations of unlabeled GABA, with total GABA concen-
tration ranging from 1 to 60 ␮M (resulting in a specific activity ranging
from 1000 to 10 cpm/pmol). Nonspecific uptake was determined in the
presence of 10 ␮M tiagabine, with 3 min of preincubation. The uptake
buffer was aspirated, and the cells were washed with 1 ml of ice-cold
Krebs-Ringer-Hepes buffer and lysed with 0.5 ml of 1% SDS. The
amount of accumulated radioactivity was determined by liquid scintil-
lation counting. For vesicular uptake assays, membranes were pre-
pared from cells that expressed intracellularly retained GAT1 mutants
in isotonic sucrose; NaCl-driven uptake of [3H]GABA into these mem-
brane vesicles (50 –100 ␮g/assay) was measured by employing a filtra-
tion assay as described earlier (7).
55729
Fluorescence Microscopy—Fluorescence microscopy was performed
using a Zeiss Axiovert 200M inverted epifluorescence microscope
equipped with a CoolSNAP fx cooled CCD camera (Photometrics, Roper
Scientific, Tucson, AZ). The fluorescence filter sets were purchased from
Chroma (Chroma Technology Corp., Brattleboro, VT; CFP filter set:
excitation 436 nm, dichroic 455 nm, emission 480 nm; YFP filter set:
excitation 500 nm, dichroic 515 nm, emission 535 nm; FRET filter
set: excitation 436 nm, dichroic 455 nm, emission 535 nm). Coverslips
with attached cells were mounted in the microscope chamber and put on
the microscope stage. Images of cells with CFP- or YFP-tagged trans-
porters were acquired through corresponding filter channels. For donor
photobleaching, FRET cells expressing either CFP-tagged or CFP- and
YFP-tagged transporters were continuously illuminated with a 100-
watt mercury lamp and the CFP filter set for 1 min, a time interval
sufficient to bleach the donor to an extent of less than 20% (with
acquisition of one image every 3 s). Regions of interest were selected,
and fluorescence emission intensities were quantified. The resulting
fluorescence decay curves were fitted to the equation for a single expo-
nential decay approaching a constant value: fluorescence intensity ⫽
A0e⫺Kt ⫹ offset, where A0 denotes the starting value, offset is the final
fluorescence signal, and K is the decay constant. The photobleaching
lifetime constant ␶ is defined as 1/K. To measure donor recovery after
acceptor photobleaching (DRAP), we acquired a donor (CFP) image
before (Ib) and after (Ia) photobleaching using the YFP setting for 90 s
(excitation 500 nm, dichroic mirror 525 nm, and emission 535 nm).
DRAP was quantified by FRET efficiency (E) according to the following
equation: E ⫽ (Ia ⫺ Ib)/Ia (16) and corrected for cross-bleaching of CFP
through the YFP filter set (⬃9 –10%). Fluorescence images were ac-
quired and analyzed using the MetaMorph and MetaFluor of MetaSer-
ies software package (release 4.6; Universal Imaging Corp., Downing-
town, PA). For statistical comparisons of time constants of fluorescence
efficiencies, ANOVA followed by Dunnett’s or Tukey’s test was used.
␤-Lactamase Protein Fragment Complementation Assay—For the
␤-lactamase protein fragment complementation assay, we used plas-
mids pcDNA3.1-Zeo/F[1]m182T and pcDNA3.1-Zeo/F[2] to express of
proteins fused to ␤-lactamase fragments F[1] and F[2] (17). Constructs
of wild type and mutant GAT1 were fused by PCR cloning. Cells were
transfected with the indicated combinations of plasmids using the cal-
cium phosphate precipitation method. After 24 h, cells were detached
mechanically, lysed by a freeze-thaw cycle, and homogenized in phos-
phate-buffered saline. Membranes were sedimented by centrifugation
at 50,000 ⫻ g for 20 min, and pellets were resuspended in phosphate-
buffered saline. The membrane suspension (20-␮l aliquot containing
50 –100 ␮g of membrane protein) was incubated in the presence of 100
␮M nitrocefin in phosphate-buffered saline in a total volume of 200 ␮l;
the incubation times were as indicated for the indicated periods at
37 °C. Absorption at 492 nm was measured using an automated plate
reader. The comparisons were performed between the activities meas-
ured within linear range, before onset of substrate (nitrocefin) deple-
tion. The results were analyzed by one-way ANOVA followed by Dun-
nett’s test.
Molecular Graphics—The Swiss-PDBViewer was used to generate
the molecular model of TM2 dimer. The sequence of GAT1 TM2
(AFLIPYFLTLIFAGVPLFLLECSL) was fitted onto the ␣-carbon back-
bone of the two GCN-4 (2ZTA) leucine zipper helices. The improbable
side chain conformations were excluded by selection of allowed conform-
ers following energy minimization of the whole structure by Swiss-
PDBViewer implementation of GROMOS96. The derived model was
rendered by POV-Ray 3.0.
RESULTS
Site-directed Mutagenesis—The hydrophobic environment of
the membrane does not provide any driving force for the as-
sembly or stabilization of a leucine zipper (9, 10). Thus, the
amino acid residues of TM2 were examined for their ability to
form hydrogen bonds and selected by sequence comparison
between the members of the NSS family. For lack of structural
information, we considered the TM2 region as the sequence
between Ala81 and Leu104 (Fig. 1A); the leucine heptad repeat
comprises residues Leu83–Leu104. We identified residues tyro-
sine 86 (Tyr86), threonine 89 (Thr89), glutamate 101 (Glu101),
cysteine 102 (Cys102), and serine 103 (Ser103) as the candidates
for site-directed mutagenesis (marked by the arrows in Fig.
1A). Side chains of each of these residues may participate in
inter- or intrahelical interactions between transmembrane do-
55730 TM2 Controls GABA Transporter-1 Oligomerization

FIG. 1. Sequence conservation of TM2 within NSS family, [3H]GABA uptake, and fluorescence microscopy of the corresponding
mutated GAT1 proteins in living cells. A, amino acid residues 81–104 conform to the putative TM2 of GAT1. The homologues from archaeal,
bacterial, and mammalian sources share high sequence similarity with rat GAT1 TM2. The arrows indicate the residues mutated herein. B, cells
(1–2 ⫻ 105/well) expressing wild type GAT1 and the indicated GAT1 mutants were incubated with 5 ␮M [3H]GABA (specific activity ⬃160
cpm/pmol) for 3 min, and uptake was determined as outlined under “Experimental Procedures.” Data are means from two independent experiments
that were carried out in triplicate and done in parallel on cells expressing the wild type transporter and each indicated mutant. The uptake of the
wild type was 50 – 80 pmol䡠min⫺1䡠10⫺6 cells and was set to 100%. C, HEK-293 cells were transfected with equal amounts of vectors encoding for
GAT1 (WT) or its mutated versions (substitution indicated in each image). The next day, images (⫻ 63, oil immersion) were taken using the
acceptor (WT) or donor filter set. The images are representative of 4 – 6 independent transfections.

mains of polytopic proteins. Tyr86 and Glu101 are conserved in
all members of the family including bacterial and archaeal
transporters (three examples shown in Fig. 1A). This suggests
similar or related functional roles in different transporters.
Ser103 is present in close relatives of GAT1, namely glycine and
proline transporters. The equivalent positions in TM2 of more
distantly related transporters are predominantly occupied by
alanine. Thr89 and Cys102 are present exclusively in GAT1.
Thus, we searched for the hydrogen donor that may support
oligomer formation by generating the following point-mutated
versions of GAT1 (see also Fig. 1A); the Tyr86 residue was
mutated to alanine (the smallest side chain, incapable of form-
ing hydrogen bonds) and phenylalanine (lacking only the hy-
droxyl group when compared with tyrosine). Thr89 was substi-
tuted by alanine and phenylalanine (present in analogous
positions of dopamine transporter, norepinephrin transporter,
GAT2, GAT3, etc.). Glu101 was changed to alanine and aspar-
tate (a charged residue one -CH2- group shorter than gluta-
mate) and glutamine (devoid of charge but same size as gluta-
mate). Cys102 and Ser103 were substituted by alanine residues.
Transport of GABA by GAT1 Mutants—Cellular uptake as-
says showed that the mutated transporters were capable of
transporting GABA with a high rate and that transport was
blocked by the competitive inhibitor tiagabine. By definition,
cellular uptake demonstrates that the transporters are ex-
pressed on the cell surface. When assayed at a low fixed con-
centration of [3H]GABA, the transport velocities of the mutants
Y86F, C102A, and S103A were comparable with that of the
fluorescently tagged wild type transporter (Fig. 1B). Accord-
ingly, in saturation experiments, these mutants had Km values
that were indistinguishable from those of wild type GAT1 (Ta-
ble II). In contrast, substitutions of Thr89 by alanine and by
phenylalanine progressively reduced the affinity for substrate
(see Fig. 2A and Table II). The Km reflects the overall process of
substrate transport (i.e. binding of substrate and the cosub-
strates Na⫹ and Cl⫺, their translocation, and intracellular
release). Because there is no suitable radioligand for GAT1
binding assays, it is not possible to determine substrate bind-
ing affinity directly. We probed the binding pocket of GAT1
indirectly by determining the IC50 of tiagabine for GAT1 at a
very low substrate concentration. Under these conditions, dif-
ferences in substrate affinity are irrelevant, and the IC50 ap-
proaches the Ki value of the inhibitor. As can be seen from Fig.
2B, mutation of Thr89 did not impair binding of the inhibitor
regardless of whether threonine was substituted by alanine or
by the bulky phenylalanine (see also Table II). These findings
argue against a role of Thr89 in initial inhibitor recognition and
binding but imply a role in translocation of the substrate.
Y86A-GAT1 transported substrate with a significantly lower
rate when compared with wild type GAT1 (second bar from left
in Fig. 1B). In experiments performed with E101A, E101D, and
E101Q-GAT1, the transport of [3H]GABA could barely be de-
 TM2 Controls GABA Transporter-1 Oligomerization
TABLE II
Km values for uptake of 关3H兴GABA uptake by wild type (WT) and
mutated versions of GAT1 and IC50 values for tiagabine-induced
inhibition thereof
Km Km IC50 for
TM2 mutation (whole cell)a (vesicular uptake)b
tiagabine
␮M ␮M nM
WT GAT1 4.2 ⫾ 1.0 4.1 ⫾ 0.6c 408 ⫾ 26
Y86A 9.2 ⫾ 3.0
Y86F 2.0 ⫾ 0.4
T89A 17.6 ⫾ 2.3 233 ⫾ 63
T89F 57.8 ⫾ 15.0 244 ⫾ 91
E101A NMd
E101D 1.6 ⫾ 0.5
E101Q NM
C102A 3.0 ⫾ 0.5
S103A 4.7 ⫾ 0.9
a
Km values were calculated from whole cell uptake experiments
(performed as shown in Fig. 1A); the differences between wild type
GAT1, T89A-GAT1, and T89F are statistically significant judged by
one-way ANOVA followed by Tukey’s post hoc test (n ⫽ 4 – 6).
b
Km values were calculated from uptake assays in vesicular mem-
brane preparations (n ⫽ 4).
c
Km was determined for intracellularly retained GAT1-⌬37.
d
NM, not measurable.
tected irrespective of the transfection method employed (cal-
cium phosphate precipitation in Fig. 1B). This observation
indicated that the mutated transporters were either not prop-
erly folded or failed to get inserted into the plasma membrane.
We verified that all mutants were expressed by visualizing the
transporters in living cells by means of fluorescence micros-
copy. Consistent with [3H]GABA uptake measurements, CFP-
tagged GAT containing Y86F, T89A, T89F, C102A, and S103A
substitutions were expressed at the surface of heterologously
transfected HEK-293 cells (Fig. 1C). By contrast with wild type
CFP-GAT1 (and the other mutants), there was little, if any,
fluorescence recorded over the plasma membrane with Y86A-
GAT1, E101A-GAT1, E101D-GAT1, and E101Q-GAT1; these
mutants were trapped within the cell.
Transport of GABA by Intracellularly Retained GAT1 Mu-
tants—The mutations Y86A, E101A, and E101D caused ineffi-
cient targeting of GAT1 to the plasma membrane of HEK-293
cells. This may have resulted from misfolding of these mutated
transporters. To assess the conformational integrity of the mu-
tants, we prepared membrane vesicles from appropriately
transfected cells and measured vesicular [3H]GABA uptake. As
an internal control, we used GAT1-⌬37, which lacks the last 37
amino acids. This truncated version is retained within the cell,
because it lacks the docking sites for the coatomer protein II
components, but it has a wild type transmembrane core and
hence transports at normal rates (14). The transporters were
tagged with a fluorescent protein; this allowed us to correct for
the variability in expression inherent in transient transfections
by normalizing the transport velocity for recorded fluorescence.
It is evident from Fig. 3A that Y86A-GAT1 transported
[3H]GABA with a Km and a Vmax value that were reasonably
similar to that of the wild type transporter. Likewise, E101D-
GAT1 translocated [3H]GABA with an apparent affinity that
was within the range seen with GAT1-⌬37 (see Table II). In
contrast, we failed to detect any measurable [3H]GABA influx
with vesicles containing the other two Glu101 mutants (i.e.
E101A-GAT1 and E101Q-GAT1). Thus, while we cannot rule
out the possibility that the latter two mutants are misfolded, it
is clear that Y86A-GAT1 and E101D-GAT1 adopt a conforma-
tion that binds and translocates substrate with an affinity that
is only modestly different from that of the wild type trans-
porter. In other words, retention of Y86A-GAT1 and of E101D-
GAT1 is unlikely to be due to misfolding.
55731
Endoplasmic Reticulum Export of the Wild Type Transporter
in the Presence of GAT1 Mutants—Previous work by us and
others has led to the hypothesis that the export of the NSS
members GAT1 (7, 14), serotonin transporter (18), and dopa-
mine transporter (8, 19) is contingent on oligomerization. This
model also explains the dominant negative phenotype (i.e. fa-
milial postural hypotension) that results from a point mutation
in one allele of the norepinephrine transporter (20). A compar-
ison of Fig. 4, A and B, illustrates this phenomenon; cells were
transfected with CFP-tagged GAT1-⌬37 (visualized in Fig. 4A)
and YFP-tagged wild type GAT1 (visualized in Fig. 4B). In the
cell that expressed GAT1-⌬37 (visualized in the left corner of
Fig. 4A), the wild type transporter was trapped in the endo-
plasmic reticulum (Fig. 4B). In contrast, in the cell that only
expressed YFP-tagged GAT1, the transporter was found at the
plasma membrane (top right corner of Fig. 4B). As mentioned
earlier, GAT1-⌬37 is trapped in the endoplasmic reticulum,
because it lacks the docking site for the coatomer protein II
component Sec24 (14). Thus, this approach can serve as a
means to detect the capacity of mutant transporters to associ-
ate with wild type GAT1. It is evident that all Glu101-substi-
tuted GAT1 variants (Fig. 4, E, G, and I) did not impair cell
surface targeting of the wild type transporter to any apprecia-
ble extent (Fig. 4, F, H, and J). In cells that expressed high
levels of Y86A-GAT1 (cell on the left in Fig. 4C), there was some
intracellular retention of wild type GAT1 (Fig. 4D); wild type
GAT1 was, however, visualized predominantly at the cell sur-
face in cells that expressed Y86A-GAT1 at moderate levels
(exemplified by the cell on the right in Fig. 4, C and D). We
interpret these observations as indicative of a reduced associ-
ation of the Y86A mutant and the Glu101-substituted versions
with the wild type transporter.
Impaired Oligomerization of Intracellular GAT1 Mutants—
The fact that the Y86A and Glu101 mutants were retained within
the cell and failed to retain the wild type transporter in the
endoplasmic reticulum provided circumstantial evidence for a
defect in their ability to form oligomers. To directly visualize the
oligomeric status of individual transporters in living cells, we
performed FRET microscopy. FRET microscopy relies on the
energy transfer that occurs if donor and acceptor fluorophore are
in close vicinity; in the pair CFP (donor) and YFP (acceptor), this
Foerster distance is in the range of 50 Å (Fig. 5A) (21) and thus
requires the formation of an oligomer. Here we used approaches
that rely either on the protection of the donor against bleaching
in the presence of acceptor (donor photobleaching FRET) or on
the increase in donor emission after acceptor bleaching (donor
recovery after acceptor photobleaching). Donor photobleaching
FRET did not reveal any difference in the lifetime (␶) of the
fluorescent decay upon bleaching the mutant transporters ex-
pressed in the plasma membrane (Fig. 5, B and C). Thus, the
mutations of residues Thr89, Cys102, and Ser103 as well as sub-
stitution of Tyr86 to phenylalanine did not impede the interaction
with wild type GAT1. In other words, the hydrophilic side chains
of these residues are unlikely to participate in forming the oligo-
meric interface.
We have also verified this conclusion by using DRAP-FRET
microsocopy, an independent approach that relies on bleaching
of the acceptor rather than the donor. The data set depicted in
Fig. 5D shows a representative experiment for GAT1-⌬37 (top
row) and for GAT1–Y86A (bottom row), where the fluorescence
intensity is shown in pseudocolors. We used GAT1-⌬37 to
gauge the oligomeric status of those mutants that are retained
within the cell; because GAT1-⌬37 is trapped in the endoplas-
mic reticulum, it serves as a good control and obviates a com-
parison over different membrane compartments. The increase
in fluorescence that can be seen for GAT1-⌬37 (cf. CFP images
55732 TM2 Controls GABA Transporter-1 Oligomerization

FIG. 2. Uptake of [3H]GABA by cells expressing wild type and mutant GAT1. A, cells (1–2 ⫻ 105/well) expressing wild type GAT1 (f),
T89A (Œ), or T89F (
) were incubated with the indicated total concentrations of GABA for 3 min, and uptake was determined as outlined under
“Experimental Procedures.” B, cells (as in A) were incubated with 1 ␮M [3H]GABA (specific activity 800 cpm/pmol) and increasing concentrations
of tiagabine; data represent means ⫾ S.E. in at least three independent experiments performed in duplicate. Transport velocity in the absence of
inhibitor amounted to ⬃20 pmol/min/106 cells (⬃8000 –10,000 cpm) and was set to 1.0 to normalize for interassay variation. The solid lines were
drawn by subjecting the data to a nonlinear, least-squares curve fit using the equation for a rectangular hyperbola.

FIG. 3. Uptake of [3H]GABA in vesicles incorporating retained GAT1 mutants. A, the vesicles (20 –50 ␮g of protein/assay) were prepared
from cells expressing CFP-GAT1 (f) and Y86A-GAT1 (Œ) and were incubated with the indicated concentrations of [3H]GABA at 20 °C for 3 min
as described under “Experimental Procedures.” The solid lines were drawn by subjecting the data to a nonlinear, least-squares curve fit using the
equation for a rectangular hyperbola. B, membrane vesicles were prepared from HEK-293 cells, expressing wild type CFP-GAT1, Y86A-GAT1,
E101A-GAT1, and E101D-GAT1. The vesicle preparations (40 ␮g of protein/assay) were incubated with a single concentration of [3H]GABA (⬃500
nM) in a final volume of 50 ␮l. Nonspecific uptake/binding determined in the presence of 100 ␮M tiagabine was subtracted.

FIG. 4. Intracellular retention of in-

tact GAT1 by intracellular mutants.
Images were captured from HEK-293
cells co-expressing YFP-GAT1 and the in-
dicated CFP-tagged mutants by fluores-
cence microscopy as described in the leg-
end to Fig. 1C. Images were captured
through the CFP filter set for cells co-
expressing CFP-tagged GAT1-⌬37, Y86A,
E101A, E101D, and E101Q mutants, re-
spectively (A, C, E, G, and I), and then
through the YFP filter set to visualize the
YFP-tagged wild type GAT1 (B, D, F, H,
and J).

before and after bleaching in the top row) is not readily detect-
able for the Y86A mutant (bottom row). Fig. 5E summarizes the
results from DRAP-FRET microscopy (Fig. 5, D and E), which
allows for quantitative comparisons of energy transfer efficien-
cies; it is evident that the FRET efficiency is comparable for
wild type GAT1 and Y86F (white columns) and that they are
similar in magnitude to the C-terminally truncated GAT1-⌬37.
In contrast, the FRET efficiency is reduced by about 50% for
the mutants Y86A-GAT1 and E101A/D/Q-GAT1 regardless of
whether they are allowed to interact with GAT1-⌬37 or with
themselves (Fig. 5E). Consistent with these DRAP measure-
ments, donor photobleaching experiments showed reduced pho-
tobleaching lifetimes with the representative pairs of the in-
tracellularly retained mutants: Y86A versus GAT1 and E101D
versus GAT1 (black columns in Fig. 5C). Taken together, these
results indicate that the phenyl ring of Tyr86 and the side chain
of Glu101 are crucial for formation of the GAT1 oligomer. Two
different FRET-based methods that monitor protein-protein
interaction lead to the same conclusions. Furthermore, FRET
microscopy provides evidence for a homophilic TM2-driven oli-
gomerization. The mutated transporters fused to CFP and YFP
comprise the FRET pairs with FRET efficiencies roughly equal
to those of pairs with the acceptor molecule being GAT1-⌬37.
Mutation of a single residue (Tyr86 or Glu101) in a TM2 of one
of the interacting molecules was sufficient to impair this inter-
action. Mutation of the respective residue in both interacting
subunits did not increase the difference in FRET efficiency,
compared with the GAT1-⌬37 control. This suggests that the
mutated residues form contacts in a mutually dependent fash-
ion, constituting a homophilic (TM2-TM2) interaction domain.
 TM2 Controls GABA Transporter-1 Oligomerization 55733

FIG. 5. Donor photobleaching and DRAP FRET microscopy of intracellular GAT1 mutants. The indicated pairs of CFP- or YFP-labeled
GAT1 mutant constructs were coexpressed in HEK-293 cells at a 1:1 ratio of plasmids encoding donor and acceptor. Donor photobleaching (B, C)
and DRAP (D, E) FRET microscopy experiments were performed 24 –28 h after transfection. A, scheme illustrating the principle of FRET. B,
photobleaching of the donor (CFP) was achieved by illuminating the cell at the center of the visual field with a mercury arc lamp (Zeiss HBO;
100-watt intensity) for 60 s. Images were captured every 3 s, digitized, and stored. Decay of intensity (in the regions of interest) was quantified
in successive images to generate the decay curves and calculate photobleaching lifetimes, ␶. C, a summary of ␶-values determined as shown in B
for the indicated mutants. The ␶ values for all mutants that were expressed at the cell surface (white bars) were not significantly different from
those of the wild type GAT1; for mutants that were retained within the cell (represented by black bars), there was a significant difference between
the ability of self-association of the control protein GAT1-⌬37 and its association with the Y86A and the E101 mutant (*, p ⬍ 0.05; **, p ⬍ 0.01;
significance judged by one-way ANOVA followed by Dunnett’s test; values represent mean ⫾ S.E. from 15–22 individual curves). D, to measure
DRAP, a donor (CFP) image was acquired before (left image) and after photobleaching (middle image) using the YFP setting for 90 s (excitation
500 nm, dichroic mirror 525 nm, and emission 535 nm); shown are pseudocolor images for the control protein (GAT1-⌬37) and a representative,
intracellularly retained mutant, Y86A-GAT1; the YFP image prior to bleaching is also shown to document the presence of acceptor within the same
cell. E, summary of FRET efficiencies obtained from DRAP experiments carried out as in C. The FRET efficiency (mean ⫾ S.E.; n ⫽ 11–29,
calculated as described under “Experimental Procedures”) determined for GAT1-⌬37 versus GAT1-⌬37 was significantly different from that
determined for all other intracellularly retained mutants (represented by black bars; p ⬍ 0.05; one-way ANOVA followed by Dunnett’s test) but
comparable with the ransporters expressed at the cell surface (indicated by white bars) (i.e. self-association of wild type GAT1 (abbreviated as CrG
versus YrG) and Y86F versus wild type GAT1 (abbreviated as Y86F versus YrG).

A decrease in FRET efficiencies may arise from an increase
in the distance between the fluorophores, a change in the
relative orientation of the fluorophore dipoles, or a decline in
the steady state number of oligomeric complexes. The data in
Fig. 4 also suggest that the mutations reduced the level of
oligomeric complexes. We obtained independent evidence for
this interpretation by employing a third approach, namely a
␤-lactamase fragment complementation assay (17). This
method of proving interaction between two proteins relies on
reconstitution of enzyme activity (cleavage of nitrocefin) upon
interaction of two proteins that are tagged by two split parts of
the enzyme ␤-lactamase (Fig. 6A). We fused GAT1 and its
mutated versions in frame with two complementary fragments
(fragment F[1] and fragment F[2]) of TEM ␤-lactamase. Mem-
branes were prepared from HEK-293 cells that co-expressed
these constructs, and the reconstitution of enzymatic activity
was assessed colorimetrically. As predicted, the rates of nitro-
cefin cleavage in membranes containing GAT1-F[1] and GAT1-
F[2] were comparable with those of membranes from cells
expressing GAT1-F[1] and Y86F-GAT1-F[2] (squares and
downward pointing triangles in Fig. 6B). However, Y86A-
GAT1-F[2] and the corresponding fusion protein containing the
mutations E101A, E101D, and E101Q reconstituted ␤-lacta-
mase activity with GAT1-F[1] to a lower extent (Fig. 6, B and
C). This finding is consistent with the results of FRET micros-
copy (Fig. 5) and of the retention assay (Fig. 4); taken together,
the observations show that the mutations Y86A and E101A/
D/Q impair the ability of the transporter to form an oligomer.
55734 TM2 Controls GABA Transporter-1 Oligomerization
FIG. 6. ␤-Lactamase fragment complementation. A, the scheme
depicts the principle of the ␤-lactamase protein complementation assay;
the two fragments of ␤-lactamase (labeled F[1] and F[2]) refold upon
interaction of transporter molecules and cleave substrate-nitrocefin (tri-
angle labeled with N). B, representative experiment of ␤-lactamase
complementation by full-length wild type GAT1-F[1] co-expressed with
full-length wild type or mutant GAT1-F[2]; HEK-293 cells were co-
transfected with plasmids encoding the indicated combinations of con-
structs fused to either fragment of ␤-lactamase (F[1] or F[2]). The enzy-
matic activity was measured in duplicate as described under
“Experimental Procedures.” The inset shows the initial 6 min that were
used to calculate the rate of enzymatic activity from the linear portion. C,
summary of three independent experiments, performed in duplicate as in
B. The activity determined with wild type GAT1-containing constructs
was the reference (equal to 100%). D, the ␤-lactamase activities of coex-
pressed TM1-TM2-F[1]/TM1-TM2-F[2] pairs were determined in a man-
ner analogous to that described for B. Data are means ⫾ S.E. from three
independent experiments carried out in duplicate. The asterisks (p ⬍ 0.05)
and double asterisks (p ⬍ 0.01) indicate statistical significance of differ-
ence from control (one-way ANOVA followed by Dunnett’s test).
␤-Lactamase Fragment Complementation by Isolated Trans-
membrane Domains—It is evident that the point mutations in
TM2 (at positions Tyr86 and of Glu101) neither fully eliminated
resonance energy transfer (Fig. 5E) nor completely abolished
␤-lactamase complementation (Fig. 6B). These observations
suggest that there are additional contact sites in the oligomeric
interface (e.g. TM4 (22) and TM11/12 (18)). To study the ho-
mophilic interaction between TM2 segments, we generated
constructs that comprised TM1, TM2, and either the ␤-lacta-
mase fragment F[1] or F[2] (schematic rendering in Fig. 6E).
TM1, which functions as a (noncleavable) signal sequence, was
included to ascertain the correct orientation of the polypeptide.
These short GAT1-derived constructs carrying mutations
Y86A/F and a representative of the glutamate 101 (E101D)
were co-expressed as F[1]- and as F[2]-tagged versions. The
activities of these pairs of fusion constructs were compared
with those of the wild type pair: Y86F was again indistinguish-
able from wild type, whereas Y86A and E101D had lower
activities (Fig. 6D). A comparison of Fig. 6, C and D, shows that
the differences in association of Y86A and E101D mutants was
more pronounced when presented in the context of the short
TM1-TM2 construct.
DISCUSSION
For many membrane proteins, a correct quaternary assem-
bly is a prerequisite for efficient exit from the endoplasmic
reticulum and subsequent delivery to the plasma membrane.
Prominent examples are GABAB receptors and ATP-sensitive
K⫹ channels (23, 24). The mechanisms that drive the oligo-
meric assembly of proteins with multiple transmembrane seg-
ments are, in many instances, poorly understood. Neurotrans-
mitter transporters are of particular interest, because they
contain multiple motifs known to support the association of
transmembrane segments: a glycophorin-like motif in TM6
(19), a leucine heptad repeat in TM2 (7), and additional inter-
action motifs in TM4 (22) and TM11–12 (18).
In the present work, we focused on TM2 of GAT1 and
searched specifically for the polar residue that may be required
to drive the association of leucine heptad repeats in the mem-
brane (9). Our observations exclude Tyr86, Thr89, Cys102, and
Ser103 as hydrogen bond donors required for stabilization of the
oligomer. In contrast, Glu101 meets several essential criteria. (i)
Glu101 occupies the a-position in the leucine heptad repeat (i.e.
it is sandwiched between Leu97 and Leu104, which are one
helical turn above and below Glu101 and thus occupy the d-
position in their respective heptad repeat (where positions 1–7
are denoted a– g) (9); (ii) replacement of Glu101 by mutation
greatly reduced the ability of the resulting mutants to support
oligomerization: cell surface delivery, resonance energy trans-
fer, and ␤-lactamase complementation; and (iii) the loss of
oligomerization cannot be attributed to a major misfolding.
Although E101A and E101Q failed to translocate substrate, the
activity of E101D-GAT1 was readily measurable and its affinity
for substrate was within the range observed with the wild type
transporter. This unequivocally shows that E101D-GAT1 adopts
an active conformation. We therefore rule out the possibility that
the E101D mutant is retained in the endoplasmic reticulum due
to misfolding at the level of tertiary structure. Loss of function in
E101A and E101Q mutants may not be directly addressed. How-
ever, in norepinephrin transporter (25), these mutations result in
a transporter with at least partial activity. Taken together, these
observations suggest that a major folding problem is unlikely to
be present in GAT1-E101D.
Substitution of Tyr86 also affected oligomerization of GAT1.
The bulk of the Y86A mutant was trapped in the intracellular
compartment and showed reduced interaction with itself or the
wild type transporter. Because of its hydroxyl group, tyrosine is
a polar residue; nevertheless, it is the hydrophobic moment of
Tyr86 that matters for TM2-mediated transporter association.
Rather than donating a hydroxyl for a hydrogen bond, Tyr86 is
likely to engage in a hydrophobic interaction via the aromatic
 TM2 Controls GABA Transporter-1 Oligomerization
FIG. 7. Molecular modeling of the dimeric interface formed by
the TM2 of GAT1. A, proposed model of the TM2 dimer of GAT1, as
viewed in the plane of the membrane. The stretch Ala81 to Leu104 of
GAT1 were fitted onto the ␣-carbon backbone of the two GCN-4 (2ZTA)
leucine zipper helices as outlined under “Experimental Procedures.”
The features responsible for TM2 dimer formation are shown in color:
leucine heptad repeat (green), Tyr86 (yellow), Glu101 (red). Residues
Thr89 and Ser103 (both in yellow) are shown to emphasize their irrele-
vance for oligomerization. B and C, the enlarged Glu101-containing
portion of TM2 dimer. If shielded from access of water from the cytosol
(indicated by the space-filling representation in B), Glu101 on one helix
forms a strong hydrogen bond with Glu101 on the other helix. Substitu-
tion of Leu104 to alanine abolishes hydrogen bonding by making Glu101
water-accessible and ionized (indicated by the gap in C).
ring (e.g. ␲-␲ stacking), which supports transporter oligomer-
ization and subsequent plasma membrane targeting. The phe-
nol ring of tyrosine and the phenyl-side chain of phenylalanine
have a propensity to interact with the planar hydrophobic
moieties in a stacked or T-shaped conformation (26). These
interactions between phenyls were shown to drive self-associ-
ation of a transmembrane segment of IsK ion channel (27).
Thus, Tyr86-mediated stacking is a plausible mechanism. In
case of parallel stacking of aromatic rings, the hydroxyl-group
obviously must be satisfied by intramolecular hydrogen bond-
ing (i.e. an interaction within the same transporter moiety) or
by an interaction with a lipid head group.
In order to visualize how the transmembrane dimer interface
may look, we threaded the sequences of two GAT1 TM2 do-
mains on the ␣-carbon backbone of the GCN4 leucine zipper
(28). The geometry of the obtained structure was optimized to
exclude improbable side chain conformations. Due to the pres-
ence of two prolines and one glycine residue, which may distort
the ␣-helical structure of the transmembrane segment (29), the
derived molecular model is but a rough estimation of the TM2
dimer structure. According to our model, the association of two
TM2 helices may be supported in part via a net of extensive van
der Waals contacts between the corresponding hydrophobic
side chains. In this case, the dimer interface would involve the
leucines of the heptad repeat and Tyr86 (shown in green and
yellow, respectively, in Fig. 7A). These residues would stabilize
the packing of the TM segments against each other. However,
we cannot rule out that Tyr86, in particular, may mediate
packing against residues on other helices of the full-length
transporter. Most importantly, however, the dimer formation
appears to be dependent on the glutamates on adjacent TM2
domains of two transporters (shown in red in Fig. 7, A and B).
55735
The side chain of Glu101 is a likely hydrogen bonding entity
that may provide the driving force for TM2 dimerization in the
hydrophobic environment of the membrane. In accordance with
our hypothesis, the protonated E101 may donate its hydrogen
for a hydrogen bond with a heteroatom of the Glu101 of a
juxtaposed helix. The acidic residues in TM spans have been
shown to cause strong hydrogen bond-based association within
the lipid bilayer of ErbB2 receptor and cystic fibrosis trans-
membrane conductance regulator Cl⫺ channel (30, 31). Glu101
is flanked along its helical side by two leucine residues, Leu97
and Leu104, which may protect Glu101 from the aqueous cyto-
solic milieu (Fig. 7B). Mutation of Leu97 to alanine destabilizes
the dimer, whereas mutation of both Leu97 and Leu104 com-
pletely abolishes TM2-TM2 dimerization (7). This may be ex-
plained by a loss of hydrophobic contacts between leucine res-
idues, which allows for access of water and thus results in
deprotonation of the ␦-carboxyl group of Glu101 (Fig. 7C). Our
interpretation is that the hydrogen bonding of Glu101 occurs in
the context of hydrophobic side chains of its neighbors and is
strictly directional; mutations to Asp or Gln are detrimental to
hydrogen bond formation. The E101Q mutant may not form the
equivalent hydrogen bond. Likewise, the transporter with
E101D substitution is unable to form the hydrogen bond and
may be more easily accessible to water from the cytosol, leading
to deprotonation and subsequent mutual helix repulsion. In
serotonin transporter, the substitution of the homologous
Glu136 by cysteine renders this residue sensitive to modifica-
tion by a hydrophilic sulfhydryl-reactive compound (32). How-
ever, this observation may be compatible with our model if
replacement by cysteine creates a water-accessible cavity; as
mentioned earlier, in the hydrophobic environment of the lipid
bilayer, the stable association of leucine heptad repeats re-
quires a hydrogen bond donor in the a-position, which is sand-
wiched between two leucines that are one helical turn below
and above in the d-position of the repeat (9, 13). If the side
chain of cysteine is too short to stabilize the leucine zipper, the
driving force that keeps the leucine side chains aligned may be
reduced, and the cysteine at the bottom of TM2 is likely to
become accessible to water.
These conjectures are well supported by a statistical assess-
ment of Adamian and Liang (33); their survey scored the prob-
abilities for residues in TM segments to interact with each
other within the membrane. In agreement with our results,
Adamian and Liang reported that a Glu-Glu pair is frequently
found in contact regions within the membrane, whereas Asp-
Asp and Gln-Gln pairs are very unlikely to occur (33). Thus, the
interaction between two Glu residues are expected to support a
strong intramembrane association of two transporter mole-
cules. Recent work suggests that the sequence context strongly
modulates transmembrane hydrogen bonding interactions (34).
Accordingly, the specificity of hydrogen bonding between GAT1
TM2 helices could be provided by intrinsic properties of the
Glu101 side chain and by the surrounding residues, which
would form a hydrophobic cage.
Our model is strongly supported by the evidence obtained
from the ␤-lactamase complementation experiments performed
with full-length transporters and fragments thereof. The asso-
ciation patterns are similar, and, in fact, the effects of the
mutations are more pronounced when assessed in the isolated
TM1-TM2 segments. This approach demonstrates that the pu-
tative transmembrane domain interface involving Tyr86 and
Glu101 is present in full-length GAT1 as well as in its severed
TM1-TM2 segment. Thus, the available evidence supports a
model where the interface depicted in Fig. 7A may play a role
in helical packing; this is apparently true even when the con-
straints are relieved that are projected on TM1-TM2 in the con-
55736 TM2 Controls GABA Transporter-1 Oligomerization
text of an intact transporter and despite the concomitant increase
in the conformational flexibility of isolated TM segments.
It is worth pointing out that a cysteine scanning mutagenesis
of TM2 also identified the homologous residues (i.e. Tyr121 and
Glu136) in serotonin transporter as essential for cell surface
expression and for transporter activity (32). Previously, the
residue Glu101 of GAT1 was proposed to act as the counterion
for binding of the cosubstrate Na⫹ (35). This interpretation
fails to account for the reduction in cellular [3H]GABA uptake
that Keshet et al. (35) observed with the mutant E101D-GAT1
if we assume that aspartate can still provide a counterion for
Na⫹ binding. However, the reduction in cellular uptake and in
transporter-associated currents is readily explained by the im-
paired cell surface expression of the Glu101 mutants that we
documented in the present study. Accordingly, we consider our
model more plausible, where the carboxyl group of Glu101 is
shielded from the aqueous phase and is thus unavailable for
binding of Na⫹. Our results rather emphasize the central role
of this polar residue Glu101 in driving GAT1 oligomerization by
intramembrane contacts via adjacent TM2 helices. In our opin-
ion, this association is supported by a meshwork of hydrophobic
residues (leucine heptad repeat and Tyr86) dispersed along the
helical interface. It is worth pointing out that the residues
involved are conserved among the homologues of GAT1 from
archaeae to humans (Fig. 1A). In addition, the leucine zipper-
like motifs are present in other transport proteins (e.g. GLUT4,
Sglt1, etc.). Thus, the mode of transmembrane association that
we propose for GABA transporter molecules may also govern
the oligomerization of other members of the transporter family.
Finally, our work proves that the concepts developed for arti-
ficial single transmembrane segments (9, 10, 34) are applicable
to proteins with a more complex architecture (i.e. several trans-
membrane ␣-helices). Related combinations of hydrophobic and
polar interactions are likely to support oligomer formation in
other proteins with multiple transmembrane spans.
Acknowledgments—We are grateful to Stephen Michnick for the
plasmids encoding ␤-lactamase fragments. We thank Frank Eisenhaber
for helpful suggestions regarding molecular modeling and Christoforos
Charalambous for critical reading of the manuscript.
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