Transmembrane Segment 5 of the Glut1 Glucose Transporter Is
an Amphipathic Helix That Forms Part of the Sugar Permeation
Pathway*
Mike
Mueckler
and
Carol
Makepeace
From the Department of Cell Biology and Physiology, Washington
University School of Medicine, St. Louis, Missouri 63110
 |
ABSTRACT |
Transmembrane segment 5 of the Glut1 glucose
transporter has been proposed to form an amphipathic transmembrane
helix that lines the substrate translocation pathway (Mueckler, M.,
Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris,
H. R., Allard, W. J., Lienhard, G. E., and Lodish,
H. F. (1985) Science 229, 941-945). This hypothesis
was tested using cysteine-scanning mutagenesis in conjunction with the
membrane-impermeant, sulfhydryl-specific reagent,
p-chloromercuribenzenesulfonate (pCMBS). A series of 21 mutants was created from a fully functional, cysteine-less, parental
Glut1 molecule by changing each residue within putative transmembrane
segment 5 to cysteine. Each mutant was then expressed in
Xenopus oocytes and its steady-state protein level,
2-deoxyglucose uptake activity, and sensitivity to pCMBS were measured.
All 21 mutants exhibited measurable transport activity, although
several of the mutants exhibited reduced activity due to a
corresponding reduction in steady-state protein. Six of the amino acid
side chains within transmembrane segment 5 were clearly accessible to
pCMBS in the external medium, as determined by inhibition of transport
activity, and a 7th residue showed inhibition that lacked statistical
significance because of the extremely low transport activity of the
corresponding mutant. All 7 of these residues were clustered along one
face of a putative
-helix, proximal to the exoplasmic surface of the
plasma membrane. These results comprise the first experimental evidence
for the existence of an amphipathic transmembrane
-helix in a
glucose transporter molecule and strongly suggest that transmembrane
segment 5 of Glut1 forms part of the sugar permeation pathway.
 |
INTRODUCTION |
Facilitative transport of glucose into mammalian cells is mediated
by members of the Glut family of membrane glycoproteins (reviewed in
Refs. 1-3). Glut1, the prototype member of this family, may be the
most extensively studied of all mammalian membrane transporters.
Kinetic studies on human red blood cell Glut1 are mostly compatible
with a simple alternating conformation mechanism for sugar transport
(4), although anomalies have been observed that appear to contradict
this hypothesis (5). The human Glut1 polypeptide exhibits a
molecular mass of 54,117 and contains a single N-linked
oligosaccharide (6). Analysis of the amino acid sequence obtained by
translation of the cDNA sequence suggested the presence of 12 transmembrane segments (6), and this prediction has been experimentally
verified using glycosylation-scanning mutagenesis (7).
Five of the 12 putative transmembrane segments (segments 3, 5, 7, 8, and 11) are capable of forming amphipathic
-helices, which led to
the hypothesis that these amphipathic helices cluster together in the
membrane to form the walls of a water-filled pathway through which
glucose traverses the fatty acyl core of the lipid bilayer (6). It was
further suggested that hydroxyl- and amide-containing amino acid side
chains within these helices form the glucose binding pocket within
Glut1 via the formation of hydrogen bonds with sugar hydroxyl groups.
Several pieces of evidence are consistent with this model for the
structure of Glut1. First, glutamine-161 within helix 5 (8) and
glutamine-282 within helix 7 (9) both appear to participate in forming
the exofacial substrate binding site. Second, valine-165, which lies
near the center of helix 5 one helical turn distant from glutamine-161,
is accessible to aqueous sulfhydryl reagents and appears to be near the
exofacial substrate binding site based on mutagenesis and inhibitor
studies (10). Third, tryptophan-412 within helix 11 is essential for
transport activity (11). Fourth, hydrogen exchange studies indicate
that 30% of peptide hydrogen atoms are freely exposed to water in
purified reconstituted Glut1, consistent with the existence of a
water-accessible permeation pathway (12).
In this study we used cysteine-scanning mutagenesis in conjunction with
a sulfhydryl-specific chemical reagent to more directly address the
role of transmembrane segment 5 in forming the Glut1 sugar permeation
pathway. Our results suggest that transmembrane segment 5 is an
amphipathic
-helix with a polar water-accessible face that lines the
exofacial portion of the sugar permeation pathway.
 |
EXPERIMENTAL PROCEDURES |
Procedures for the site-directed mutagenesis and sequencing of
human Glut1 cDNA and the in vitro transcription and
purification of Glut1 mRNAs (13), isolation, microinjection, and
incubation of Xenopus oocytes (14), preparation of total
oocyte membranes (11), SDS-polyacrylamide gel electrophoresis and
immunoblotting with Glut1 C-terminal antibody (8), and 2-deoxyglucose
uptake measurements (15) have been described in detail previously.
Treatment with p-Chloromercuribenzenesulfonate
(pCMBS)1--
Stage 5 Xenopus oocytes were injected with 50 ng of wild-type or
mutant Glut1 mRNA. Two days after injection, groups of ~20 oocytes were incubated for 15 min in the presence or absence of the
indicated concentrations of pCMBS in Barth's saline at 22 °C. The
100× concentrated reagent stock was prepared in 100% dimethyl sulfoxide, and control oocytes were treated with the appropriate concentration of vehicle alone. After a 15 min incubation period, the
oocytes were washed 4 times in Barth's saline and then used for the
determination of [3H]2-deoxyglucose uptake (50 µM, 30 min at 22 °C).
 |
RESULTS |
We previously constructed a mutant human Glut1 cDNA encoding a
cysteine-less (C-less) Glut1 polypeptide in which all 6 native cysteine
residues were changed to either serine or glycine residues (10). The
C-less transporter expressed in Xenopus oocytes exhibits transport activity nearly indistinguishable from wild-type Glut1. We
used C-less Glut1 cDNA to construct cysteine-scanning mutants for
transmembrane segment 5, which is predicted to form an amphipathic
-helix that lines the sugar permeation pathway (6). Each of the 21 residues within transmembrane segment 5 was individually changed to a
cysteine residue using oligonucleotide-mediated site-directed mutagenesis, producing a series of 21 mutant Glut1 molecules, each
containing only a single cysteine residue (see Table
I).
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Table I
Cysteine scanning mutagenesis of helix 5
cDNA encoding cysteine-less human Glut1 was subjected to
oligonucleotide-mediated site-directed mutagenesis creating a series of
21 mutant cDNAs in which each of the 21 residues within putative
transmembrane helix 5 was individually changed to cysteine. Residue
refers to the amino acid numbering for human Glut1 given in Ref. 6.
Amino acids are designated by the three-letter code.
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Mutant mRNAs were injected into Xenopus oocytes and the
expression and function of the corresponding mutant transporters were evaluated by immunoblotting oocyte membrane fractions and by conducting 2-deoxyglucose uptake assays. As demonstrated previously (13), the
immunoblots revealed the presence of two glycosylated forms of Glut1 in
oocyte membranes, a faster-migrating, core-glycosylated, high mannose
form, and a slower migrating, fully processed, complex N-glycosylated form (see Fig.
1). Roughly half of the cysteine-scanning mutants were expressed in oocyte membranes at levels similar to the
parental C-less construct, whereas the other half were expressed at
lower levels. V166C, V167C, and A171C were expressed at particularly low levels relative to the parental C-less transporter.

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Fig. 1.
Expression of helix 5-mutant transporter
proteins in Xenopus oocytes. Stage 5 Xenopus oocytes were injected with 50 ng of wild-type,
C-less, or mutant C-less mRNAs, and 2 days later oocytes were used
to prepare total membrane fractions for immunoblot analysis. Twenty
µg of total oocyte membrane protein were loaded per lane. Rabbit
antiserum A674 raised against the C-terminal 15 residues of human Glut1
was used at 1:500 dilution. The standard loaded on the gel in the
top and bottom panels was human erythrocyte
ghosts representing ~10 ng of Glut1 protein.
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Transport activity was detectable for all 21 mutants as determined by
uptake of [3H]2-deoxyglucose (Fig.
2). Consistent with our previously
published results (8), cysteine substitution at glutamine-161 greatly reduced transport activity, even when normalized to the amount of
steady-state protein. This residue has been shown to be involved in
exofacial substrate binding and in a conformational change during
transporter cycling (8). Cysteine substitutions at valine-166, glycine-167, alanine-171, and glutamine-172 also significantly inhibited transport activity, most likely because of the reduced steady-state levels of the mutant proteins relative to the parental C-less transporter (see Fig. 1). All of the other mutants exhibited transport activities that were at least 50% of the activity of the
parental C-less transporter. Cysteine substitution at leucine-164 enhanced transport activity by ~46%, perhaps because of the
increased steady-state level of the mature glycosylated form of this
mutant relative to that of the parental C-less construct (see Fig. 1). We previously demonstrated that only the fully glycosylated form of
Glut1 is present in the plasma membrane (13).

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Fig. 2.
2-Deoxyglucose uptake activity of helix 5 mutants. [3H]2-deoxyglucose uptake (50 µM, 30 min at 22 °C) was measured 2 days after
injection of mRNAs. Results represent the mean ± S.E. of four
to twelve independent experiments, each experiment using 15-20 oocytes
per experimental group. The background uptake values of water-injected
controls were subtracted.
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To determine which transmembrane residues are accessible to the
external aqueous solvent and therefore may comprise part of the sugar
permeation pathway, transport activity was measured for each of the 21 mutants after incubation in the presence of the membrane-impermeant
sulfhydryl-specific reagent, pCMBS, and compared with the activities
measured in the presence of vehicle alone (see Fig.
3). The activity of 7 cysteine-scanning
mutants was inhibited by at least 40% after incubation with pCMBS,
indicating that the corresponding 7 cysteine-substituted amino acid
side chains reacted with the pCMBS and therefore must be accessible from the external aqueous solvent. Interestingly, none of the 5 side
chains predicted to lie closest to the cytoplasmic surface of the
membrane appeared to be accessible to the external reagent. Note that
the reduction in relative transport activity after pCMBS treatment did
not reach statistical significance for the glutamine-161 mutant because
of the extremely low absolute transport activity for this particular
mutant (see Fig. 2). However, we have previously demonstrated an
important role for glutamine-161 in transport activity consistent with
this residue being exposed to the sugar permeation pathway (8).

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Fig. 3.
Effect of pCMBS on transport activity of
helix 5 mutants. Three days after injection of mRNAs, groups
of 15-20 oocytes were incubated in the presence or absence of 0.5 mM pCMBS in Barth's saline at 22 °C for 15 min. Oocytes
were washed four times in Barth's saline and then subjected to
2-deoxyglucose uptake measurements under the conditions described in
the legend to Fig. 2. Results are expressed as mean ± S.E. of
four to twelve independent experiments, each experiment using 15-20
oocytes per experimental group. Each uptake value was normalized to the
uptake observed in the presence of vehicle (100% dimethyl sulfoxide)
alone for each mutant, so that the results of independent experiments
could be directly compared and averaged. The normalization was
necessary because the absolute uptake values can vary considerably with
different batches of oocytes. The background uptake values of
water-injected controls were subtracted before normalization. C-less
represents the parental cysteine-less Glut1 construct; *,
p < 0.01 for 2-deoxyglucose uptake in the presence
versus absence of pCMBS.
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DISCUSSION |
Helical wheel analysis of the results of the pCMBS
inhibition experiments revealed that the 7 residues accessible to pCMBS from the external aqueous solvent are clustered together on one face of
a putative
-helix formed by transmembrane segment 5 (see Fig.
4). Interestingly, this helical face
encompasses glutamine-161 and valine-165, both of which were
hypothesized to be exposed within the aqueous sugar permeation pathway
based on two separate series of experiments (8, 10). The opposite face
of helix 5, which is presumably in contact with the fatty acyl core of the lipid bilayer, is composed primarily of highly hydrophobic amino
acid side chains. These data comprise the first experimental evidence
for the existence of an amphipathic transmembrane helix in a glucose
transporter and also support our original hypothesis that transmembrane
segment 5 comprises part of the sugar permeation pathway (6).

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Fig. 4.
Helical wheel representation of helix 5. Transmembrane helix 5 of Glut1 as viewed from the exoplasmic surface of
the plasma membrane. Amino acids are represented by the single letter
code. Arrows point to residues that are accessible to pCMBS
from the external solvent.
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Our results with Glut1 are reminiscent of those reported for
cysteine-scanning mutagenesis experiments conducted on the
Escherichia coli lac permease (reviewed in Ref. 16) and the
glucose-6-P antiporter (17), both of which belong to the same 12 transmembrane helix superfamily of membrane transporters as Glut1.
Helix 7 of the glucose-6-P antiporter and several helices within the
lac permease have been shown to possess solvent-accessible faces that appear to line a portion of their respective substrate permeation pathways. Our results extend these findings to a mammalian facilitative transporter, and the results when taken together imply that a similar
structural paradigm may exist for cotransporters, antiporters, and
facilitative transporters in organisms ranging from E. coli to the human.
Yan and Maloney (17) demonstrated that residues within helix 7 of the
glucose-6-P antiporter that are accessible to the external solvent are
clustered along the water-accessible face proximal to the exoplasmic
surface of the plasma membrane, and that the 6 residues along the
water-accessible face most proximal to the cytoplasmic surface of the
plasma membrane are not accessible from the external solvent. Our
results with helix 5 of Glut1 are completely consistent with these
findings, in that none of the 5 residues predicted to lie closest to
the cytoplasmic surface of the membrane displayed sensitivity to pCMBS
in the external solvent. These results are consistent with a simple
alternating conformation or carrier model for the mechanism of
transport whereby the outer binding site is largely inaccessible to
cytoplasmic substrate and the inner binding site is largely
inaccessible to substrate in the external medium. At least in the case
of the glucose-6-P antiporter, there also appears to exist a central transmembrane region that is alternately accessible from either aqueous
domain as the transporter moves between its two basic configurations.
As pointed out by Yan and Maloney (17), this general model for membrane
transport was proposed decades ago based purely on kinetic analyses,
and it is only now that molecular biological approaches are providing
more direct experimental support for this fundamental hypothesis.
 |
FOOTNOTES |
*
This work was supported in part by a Grant from the National
Institutes of Health (DK 43695) and by the Diabetes Research and
Training Center at Washington University School of Medicine.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 Cell Biology
and Physiology, Washington University School of Medicine, 660 S. Euclid
Ave., St. Louis, MO 63110. Tel.: 314-362-4160; Fax: 314-362-7463;
E-mail: mike{at}cellbio.wustl.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
pCMBS, p-chloromercuribenzenesulfonate;
C-less, Glut1 molecule in
which all 6 native cysteine residues were changed to either glycine or
serine.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.