Báo cáo khoa học: The intrinsic structure of glucose transporter isoforms Glut1 and Glut3 regulates their differential distribution to detergent-resistant membrane domains in nonpolarized mammalian cells potx

The intrinsic structure of glucose transporter isoforms
Glut1 and Glut3 regulates their differential distribution
to detergent-resistant membrane domains in nonpolarized
mammalian cells
Tomoko Sakyo1,2, Hiroaki Naraba1, Hirobumi Teraoka2 and Takayuki Kitagawa1,3
1 Pharmaceutical Research Center, Iwate Medical University, Morioka, Japan
2 Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
3 Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan

Keywords
detergent-resistant membrane; glucose
transporter 1; glucose transporter 3;
mammalian glucose transporter; sorting
signal
Correspondence
T. Kitagawa, Department of Cell Biology and
Molecular Pathology, Iwate Medical
University, School of Pharmacy, Iwate
028-3694, Japan
Fax: +81 19 698 1844
Tel: +81 19 651 5111 (Ext. 5150)
E-mail:
(Received 23 January 2007, revised 9 March
2007, accepted 30 March 2007)
doi:10.1111/j.1742-4658.2007.05814.x

The hexose transporter family, which mediates facilitated uptake in
mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site. We previously
demonstrated that glucose transporter 1 is organized into a raft-like detergent-resistant membrane domain but that glucose transporter 3 distributes
to fluid membrane domains in nonpolarized mammalian cells. In this study,
we further examined the structural basis responsible for the distribution by

using a series of chimeric constructs. Glucose transporter 1 and glucose
transporter 3 with a FLAG-tagged N-terminus were expressed in detergentresistant membranes and non-detergent-resistant membranes of CHO-K1
cells, respectively. Replacement of either the C-terminal or N-terminal cytosolic portion of FLAG-tagged glucose transporter 1 and glucose transporter 3 did not affect the membrane distribution. However, a critical sorting
signal may exist within the N-terminal half of the isoforms without affecting transport activity and its inhibition by cytochalasin B. Further shortening of these regions altered the critical distribution, suggesting that a large
proportion or several parts of the intrinsic structure, including the N-terminus of each isoform, are involved in the regulation.

The hexose transporter family, which mediates facilitated uptake in mammalian cells, consists of more than
10 members containing 12 membrane-spanning segments with a single N-glycosylation site [1,2] (Fig. 1A).
Among this family, glucose transporter (Glut) 1 is
widely expressed in a variety of cells and mediates much
of the basal, noninsulin-independent transport of
d-glucose with high affinity. Glut1’s function is thought
to be mainly regulated by expression through a variety of stimuli and agents, including serum, growth
factors, tumor viruses, and inhibitors of oxidative

phosphorylation [3–7]. This N-linked glycoprotein is
trafficked post-translationally to the cell surface [8].
We have investigated tumor-associated alterations in
Glut expression using human cell hybrids derived from
cervical carcinoma HeLa cells and normal fibroblasts
[9–11], whose tumorigenicity is controlled by a putative
tumor suppressor gene on chromosome 11 [12]. In these
studies, we found that the tumor-suppressed hybrid
cells express Glut1 alone, whereas tumorigenic cell
hybrids express both Glut1 and Glut3 as larger forms,
probably due to modifications of N-glycosylation

Abbreviations
CB, cytochalasin B; 2DG, 2-deoxy-D-glucose; DRM, detergent-resistant membrane; EGFP, enhanced green fluorescent protein; ECL,
enhanced chemiluminescence; FG1, FLAG-tagged Glut1; FG3, FLAG-tagged Glut3; GFP, green fluorescent protein; Glut, glucose transporter;

[3H]2DG, [3H]-2-deoxy-D-glucose.

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS

2843


Regulation of DRM sorting of Glut1

T. Sakyo et al.

[9–11]. However, differences in the membrane distribution and roles of these isoforms remain largely
unknown.
Glut1 and Glut3 share many similarities in structure
and function. About 65% of their amino acid
sequences are identical, but their C-terminal domains
and the extracellular loops are distinctive [1,13]
(Fig. 1B). Glut3 is expressed at the cell surface of various types of cell, including neuronal cells [13] and

many tumor cells [14,15]. These isoforms have a high
affinity for d-glucose when expressed at the cell surface
[16,17]. A striking difference between them is seen in
cellular localization. In polarized epithelial cells, such
as Caco-2 and MDCK cells, Glut1 is expressed on the
basolateral surface, whereas Glut3 is sorted to the apical surface [18–20]. It has been well established that
polarized cell membranes have structural characteristics
that are regulated by a unique sorting machinery to

A

B

Fig. 1. The topology of Glut1 and alignment
of the deduced amino acid sequences of
Glut1 and Glut3. (A) The predicted structure
of Glut1 [1]. The position of the single
endogenous site of N-linked glycosylation at
Asn is shown. CHO indicates N-linked oligosaccharide. An alignment of the deduced
amino acid sequences of rabbit Glut1 [42]
and human Glut3 [17] is shown in (B). The
amino acids are numbered to the left and
are written in the single-letter code. Identical amino acid sequences are indicated by
asterisks. The locations of predicted transmembrane domains are indicated by gray
boxes.

2844

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS


T. Sakyo et al.

play tissue-specific functions [21,22]. In platelets and
neuronal cells, Glut3 is also present in intracellular vesicles [3,23]. These results imply distinctive roles for
Glut1 and Glut3 in mammalian cells, although they
remain unclear. A recent study by Inukai et al. found
that a functional signal sorting Glut3 to the apical surface in MDCK cells lies in the C-terminal cytosolic tail
[24].
We have previously determined the differential membrane distribution of these Gluts in nonpolarized cells
such as HeLa cells and CHO-K1 cells, and found that

Glut1 distributes to a raft-like detergent-resistant membrane (DRM), whereas Glut3 localizes to a fluid membrane domain [25]. DRMs are recognized as specific
microdomains in the plasma membrane that are
enriched with cholesterol and sphingolipids to organize
an ordered lipid phase, including some proteins
such as caveolin, glycosylphosphatidylinositol-anchored
proteins, and tyrosine kinases. Mainly due to their
ordered lipid nature, these membrane domains are
relatively resistant to solubilization by nonionic detergents. The distribution of Glut1 within DRMs has
been reported in other cell types [26,27], suggesting
that it is mainly due to the intrinsic properties of
Gluts.
The molecular mechanisms by which Gluts are differentially recruited to membrane domains of nonpolarized cells remain to be clarified. Therefore, we
attempted to characterize the structural determinants
of Glut1 and Glut3 required for the distribution. We
have expressed a series of chimeric transporters utilizing various portions of Glut1 and Glut3 in CHO-K1
cells and assessed their DRM distribution. Our data
show that, despite apical sorting of Glut3, neither the
C-terminal nor the N-terminal cytosolic tail of Glut1
contains a sorting signal. This signal may exist within
the N-terminal half of the membrane-spanning segments of Glut1.

Results
Expression and DRM distribution of FLAG-tagged
Glut1 (FG1) and FLAG-tagged Glut3 (FG3)
Previously, we demonstrated that Glut1 is distributed
to raft-like DRMs and Glut3 is distributed to fluid
membranes in a nonpolarized mammalian cell line [25].
The differential distribution of these isoforms seems to
be independent of cell type or the amount of protein
expressed. To clarify the molecular basis for the control of the differential distribution of Glut1 and Glut3,

we adopted a chimeric strategy whereby different portions of Glut1 and Glut3 were spliced together and

Regulation of DRM sorting of Glut1

expressed in CHO-K1 cells, which express Glut1
endogenously but not Glut3. As a means of discriminating recombinant proteins from native Glut1, cDNA
constructs for either Glut1, Glut3 or the Glut1 ⁄ Glut3
chimera were prepared containing a FLAG epitope tag
or a green fluorescent protein (GFP)-encoded tag at
their N-termini, as described in Experimental procedures. These cDNA constructs were then ligated into
the expression vector pCMV and used for transient
expression by lipofection in CHO cells.
Initially, we examined whether FG1 and FG3 are
able to target DRMs and non-DRMs, respectively, in
CHO cells. To compare the solubility of the newly synthesized proteins in nonionic detergents, the cells,
which transiently expressed FG1 and FG3 proteins,
were treated with 0.5% Triton X-100 at 4 °C, following fractionation of the solubilized (S) and insoluble
(I) fractions. As described previously [25], immunoblotting of these samples indicated that caveolin-1,
which is a well-known marker as a detergent-insoluble
component [28], was present in the 0.5% Triton
X-100-insoluble fraction (Fig. 2A). In contrast, tubulin-a, which is another marker for solubilization, was
fully solubilized under this condition. Next, we examined the distribution of recombinant FG1 and FG3
proteins, using a monoclonal antibody that recognizes
a FLAG epitope (MDYKDDDDK). As expected,
FG3 was fully solubilized by 0.5% Triton X-100 at
4 °C. In contrast, FG1 remained in the insoluble fraction. These distributions were similar to those observed
with native Glut1 as well as overexpressed Glut1 and
Glut3 in CHO cells, which were detected with their
respective antibodies to C-terminal peptide (data not
shown). b-Actin, a component of the cytoskeleton,

distributed to both DRM and non-DRM fractions of
CHO cells under these conditions (Fig. 2A).
To directly determine the location of expressed
transporter proteins on the living cell surface, recombinant GFP-tagged Glut1 and GFP-tagged Glut3 were
also expressed in CHO cells. These GFP-tagged proteins were detected over the entire cell surface
(Fig. 2B), whereas GFP proteins were found in
the cytoplasm. The results indicate that the tagging of
the N-terminus with GFP did not impair the membrane trafficking of Gluts. We also observed that
GFP-tagged Glut1 was still distributed to rafts in
CHO-K1 cells (data not shown).
Glucose uptake by CHO cells transfected
with FG1 and FG3
To examine the influence of N-terminal FLAG tagging on glucose transport, the activity of CHO cells

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS

2845


Regulation of DRM sorting of Glut1

T. Sakyo et al.

was determined. Insertion of the FLAG epitope into
Glut1 or Glut3 at the N-terminus did not result in
significant alterations to the transport activity,
because CHO cells overexpressing these proteins
exhibited an increase in glucose transport activity
(Fig. 2C). Glucose transport in CHO cells expressing
FG1 was increased 4–6-fold as compared to that in

cells transfected with or without empty vector,
whereas cells transfected with FG3 showed only a
3–4-fold increase. This difference in the increase in
transport activity might be partly due to the level of
protein expression that was detected by western blot
analysis (Fig. 2C). The uptake of 2-deoxyglucose
(2DG) by CHO cells transfected with FG1, FG3 or
control vector was inhibited by about 90% by 10 lm
cytochalasin B (CB) (Fig. 2D), supporting a functional carrier-mediated process through these FLAGtagged proteins.
Neither the N-terminus nor the C-terminus
of Glut1 is needed for the DRM distribution
To determine the functional domains responsible for
the DRM distribution of Glut1, we generated a series
of Glut1 ⁄ Glut3 chimeric mutants by replacing the corresponding domains of Glut1 with the equivalent
regions of Glut3. As shown in Fig. 1B, the amino acid
regions that are most divergent between Glut1 and
Glut3 are the N-terminus and the C-terminus, and the
large intracellular loop between transmembrane
domains 6 and 7. As a unique sorting signal for the
recruitment of Glut3 to the apical membrane of polarized cells exists in the C-terminus [24], we first generated a set of FLAG-tagged Glut1 ⁄ Glut3 chimeras in
which the cytosolic N-terminus and C-terminus of
Glut1 were replaced by the corresponding amino acids
of Glut3 (Fig. 3A). These N31 and C13 chimeric proteins were transiently expressed in CHO cells, and
examined in terms of their detergent solubility. Both

proteins were similarly retained in the insoluble fraction when the cells were treated with 0.5% Triton
X-100 at 4 °C, whereas the overproduced FG3 was
fully solubilized under these conditions (Fig. 3C). To
examine the relevance of N31 and C13 expression to
glucose transport, the activity of CHO cells was determined. As shown in Fig. 3B, the glucose transport

activity in the cells into which N31 was transfected
increased, as was the case in the FG1-transfected
CHO cells. The amount of 2DG taken up by C13transfected cells also increased, although it was lower
than that in N31-transfected cells. In any case, about
90% of this activity were inhibited by CB. Thus, substitution of the N-terminus (N31) or C-terminus (C13)
of Glut1 with that of Glut3 resulted in little or any
change in distribution to the DRM and transport
activity.
We also tested whether the substitution of both the
N-terminus and C-terminus of Glut1 with those of
Glut3, i.e. N31C3 (Fig. 4A), had an effect. The N31C3
protein expressed in CHO cells was as insoluble as
FG1, whereas FG3 was fully solubilized (Fig. 4B). In
any case, the distribution of endogenous Glut1 was
unaffected.
The N-terminal membrane-spanning regions
of Glut1 are needed for the DRM distribution
To further define the domains responsible for the
DRM distribution, additional FLAG-tagged chimeric
constructs, A and B, were generated (Fig. 5A). In A,
amino acids 2–12 and 272–492 of Glut1, which include
the N-terminal cytosolic tail and C-terminal six membrane-spanning regions but exclude a large intracellular loop, were replaced by the corresponding amino
acids of Glut3. Instead, in FLAG-tagged chimera B,
the C-terminal six membrane-spanning region amino
acids 272–451 correspond to Glut1, and the remainder
correspond to Glut3. The results are shown in Fig. 5.

Fig. 2. Effect of FG1 and FG3 in CHO cells. (A) Schematic composition of FG1 and FG3. Glut1 and Glut3 are in dark gray and pale gray, respectively. The location of the FLAG or GFP tag is indicated by hatched areas at the N-terminus. FG1 and FG3 were transfected with lipofectamine,
and their expression in CHO-K1 cells was determined as described in Experimental procedures. (B) CHO-K1 cells were also transfected with
GFP-tagged Glut1 and Glut3, and a control GFP vector. After 24 h, transfected cells were plated onto glass-based dishes and subjected to confocal fluorescence microscopy. The left panels show the GFP fluorescent images, and the right panels show the differential interference images

(upper, FG1; middle, FG3; lower, GFP). (C) Two days after transfection, cells were solubilized with 0.5% Triton X-100 at 4 °C. Total cell lysate (T)
was separated into soluble (S) and insoluble (I) fractions by centrifugation at 13 800 g for 30 min, and each sample (10 lg of protein per lane)
was subjected to SDS ⁄ PAGE and immunoblotting for FLAG, Glut1, Glut3, caveolin-1, and a-tubulin. The corresponding molecular masses are indicated in kDa. (D) After 48 h of transfection, the uptake of [3H]2DG was measured in the presence and absence of CB. The upper panel shows relative uptake values (% of control cells), and the lower panel indicates absolute uptake values normalized to the quantity of protein expressed in
CHO cells (nmoles per mg of protein per 10 min). Bars and brackets reflect the means ± SD of four determinations. DMSO, dimethylsulfoxide.

2846

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS


T. Sakyo et al.

Regulation of DRM sorting of Glut1

As these chimeric constructs contain both a FLAG
epitope at the N-terminus and a Glut3 epitope at the
C-terminus, the efficiency with which each of these

plasmids was expressed was comparable. Whereas the
total amounts detected with antibodies for FLAG and
Glut3 were similar, demonstrating a similar efficiency

B

A
FGl or GFP-G1
Intracellular
loop

GFP-G1

GFP or FLAG

N

C

FG3 or GFP-G3
GFP-G3
GFP or FLAG

C

N

GFP

C

D

CHO

Cell
Transfection

Fraction

Vector
T

S I

FG1

FG3

T S I T S

I

FLAG-M2

Glut3
Glut1
β-Actin
Caveolin-1

α-Tubulin

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS

2847


Regulation of DRM sorting of Glut1

A

T. Sakyo et al.

A

Intracellular
loop

FLAG

chimera C13
(G1 : 1 - 450)

N

C
1

chimera N31
(G1 : 13 - 492)

chimera N31C3 (G1 : 13-450)
FLAG

N

450

C

N
13

C
450

13

492

Intracellular
loop

B
B

CHO

Cell

FG1

Transfection
Fraction

T

S I

FG3

N31C3

T S I

T S I

FLAG-M2
C

CHO

Cell
Transfection

FG1

Fraction

T S I

FG3

C13

N31

Glut3

T S I T S I T S I

FLAG-M2

Glut3

Glut1

Glut1

β-Actin

β-Actin
Caveolin-1
α-Tubulin

Fig. 3. Effect of replacing the N-terminal or C-terminal tail of Glut1
and Glut3 on DRM distribution. (A) Schematic composition of
FLAG–Glut1 ⁄ Glut3 chimeras. Each construct contains the FLAG
sequence DYKDDDDK inserted immediately after the methionine
start codon. In chimera C13, the last 42 amino acids of the C-terminal tail of Glut1 are replaced with the corresponding 48 amino
acids of Glut3. By contrast, chimera N31 contains the first 12
amino acids of the N-terminal tail of Glut1 and, subsequently,
amino acids 11–496 of Glut3. The location of the FLAG-tag is indicated by hatched areas at the N-terminus. (B) CHO cells were transiently transfected with the C13 or N31 chimera, as described in
Fig. 2. (B) After 48 h of transfection, the uptake of [3H]2DG was
measured in the presence and absence of CB. Absolute uptake values normalized to the quantity of protein expressed in CHO cells
are given in nmoles per mg of protein per 10 min. One representative datum of several independent determinations is shown here.
DMSO, dimethylsulfoxide. (C) Transiently transfected cells were
solubilized by 0.5% Triton X-100 at 4 °C and fractionation and
immunoblotting for Glut1 and Glut3 were performed as described
in Fig. 2.

in the expression of these constructs, their distribution
to the DRM fraction was distinctive. As was seen

with FG1 and endogenous Glut1, chimeric protein A
was distributed to DRMs (Fig. 5C). In contrast,
2848

Fig. 4. Effect of N-terminal and C-terminal substitution of Glut1 on
DRM distribution. (A) The FLAG-tagged chimera N31C3 contains
the N-terminal and C-terminal amino acids of Glut3 and the backbone of Glut1. (B) CHO-K1 cells were transiently transfected with
FG1, FG3 or N31C3, as described above. Cells were solubilized by
0.5% Triton X-100 at 4 °C, and fractionation and immunoblotting
for Glut1, Glut3 and FLAG were performed as described in Fig. 2.

chimeric protein B was distributed only to the soluble
fraction, as seen with FG3. Increased glucose uptake
was evident in the cells that overproduced chimera A
in a CB-sensitive manner. However, a small increase in
glucose uptake was evident with the chimera B-transfected cells.
Role of a large intracellular loop of Glut1
in the DRM distribution
The role of a large intracellular loop of Glut1 in the
DRM distribution was further examined, as this domain
was included in the most effective chimeric construct A
(Fig. 5), and is one of the major characteristics of these
isoforms [25]. However, chimeric construct D, in which
this intracellular loop was replaced with Glut3, was not
distributed to DRMs (Fig. 6A). The shortening of this
loop, i.e. construct C, reduced the ability to distribute

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS

T. Sakyo et al.

Regulation of DRM sorting of Glut1

A

A

Intracellular
loop

Intracellular
loop

FLAG

chimera C

chimera A
(G1 : 13-271)

13

chimera D

chimera B
272

(G1 : 272-450)

N

(G1 : 13-206)

271

(G1 : 207-271)

450

B

B

C
206

13

N

C
207

CHO

Cell
Transfection

Fraction

271

FG1

FG3 chimera C chimera D

T S I T S I T S I T S I

FLAG-M2

Glut3

C

CHO

Cell

Glut1

FG1

Transfection
Fraction

T

S I

FG3

chimera A chimera B

T S I T S I

T S

I

FLAG-M2

Glut3

β-Actin

Fig. 6. Role of a large intracellular loop of Glut1 in DRM distribution. (A) Chimera C contains amino acids 13–206 of Glut1 corresponding to the N-terminal half of the membrane-spanning
segments and the backbone of Glut3. In chimera D, a large intracellular loop of Glut3 (amino acids 204–269) is replaced with the corresponding amino acids 207–271 of Glut1. (B) CHO-K1 cells were
transiently transfected with these chimeras, and the distribution
was determined after 48 h of transfection.

Glut1

Discussion
β-Actin
Fig. 5. Effects of the N-terminal half of the membrane-spanning
segments and a large intracellular loop of Glut1A. Chimera A contains amino acids 1–10 of Glut3, amino acids 13–271 of Glut1, and
amino acids 291–496 of Glut3. Chimera B also contains amino
acids 1–290 of Glut3, amino acids 272–450 of Glut1, and amino
acids 449–496 of Glut3. The chimeras also have a FLAG epitope in

the N-terminus. (B) CHO cells were transiently transfected with the
indicated FLAG-tagged chimeric construct, and the uptake of
[3H]2DG was measured in the presence and absence of CB after
48 h of transfection. Absolute uptake values normalized to the
quantity of protein expressed in CHO cells are presented in nmoles
per mg of protein per 10 min. The data are representative of three
different experiments performed in duplicate. DMSO, dimethylsulfoxide. (C) The transfected CHO-K1 cells were solubilized by 0.5%
Triton X-100 at 4 °C, and fractionation and immunoblotting for Glut1
and Glut3 were performed as described in Fig. 2.

DRMs, indicating some role for this loop region. Further replacement within the N-terminal membrane-spanning regions of Glut1, included in chimera C, gave
uncertain results.

The plasma membrane of mammalian cells is composed of functionally distinct membrane domains and
their components. This requires ordered gene expression as well as intricate post-translational sorting
machinery that delivers proteins and lipids to the correct membrane domains during cell growth [29]. The
best characterized system comprises polarized epithelial
cells, and the sorting machinery for membrane proteins
that are recruited to different cell surfaces in polarized
cells has been a subject of considerable interest. Many
studies have concentrated on identifying the determinants of basolateral and apical sorting signals at the
molecular level [20,24,30]. Heterogeneous membrane
domains also exist in nonpolarized cells [8].
These microdomains, called ‘lipid rafts’ or ‘DRMs’,
because of their physicochemical nature, are enriched
with ordered lipids such as cholesterol, glycolipids, and
sphingolipids, which are present in cell membranes [31].
Several proteins are preferentially distributed to these
microdomains, including glycosylphosphatidylinositolanchored protein, the Src-family tyrosine kinases,

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS

2849


Regulation of DRM sorting of Glut1

T. Sakyo et al.

heterotrimeric G proteins, and phospholipid-binding
protein [21]. Rafts constitute the scaffolding for signal
transduction and several pathogens [22].
We previously demonstrated the differential distribution of Glut isoforms Glut1 and Glut3 in the plasma
membrane of nonpolarized HeLa cells and CHO-K1
cells. Glut1 is distributed to raft-like DRMs, whereas
Glut3 is predominantly found in fluid lipid domains
[25]. The distribution of Glut1 to DRMs in nonpolarized Clone 9 cells [27] and 3T3-L1 cells [26] has also
been reported. However, the mechanism by which
Glut1 but not Glut3 is recruited to DRMs is unknown.
It is well established that the C-terminus of Glut isoforms has various important roles in subcellular protein trafficking [24,32,33] and glucose uptake [34]. In
polarized epithelial cells, such as MDCK and Caco-2
cells, it has been shown that Gluts are, respectively,
sorted to either the apical or basolateral surface. Glut1
is principally found on the basolateral cell surface,
whereas Glut3 is mainly recruited to the apical domain
[18]. Inukai et al. have shown that Glut1 contains a
basolateral sorting signal in its intracellular loop
region [30], whereas the C-terminal tail of Glut3 contains a targeting motif directing the trafficking of basolateral-sorting Glut1 to the apical cell surface in
MDCK cells [24]. Recently, two proteins binding to
the C-terminus of Glut1 have been reported. One is

the Glut1 transporter-binding protein Glut1CBP,
which controls normal Glut1 trafficking in polarized
epitherial cells, helping to regulate the level of Glut1 in
the plasma membrane [32,33]. The other is stomatin, a
type 2 membrane protein that interacts with the C-terminus of Glut1 in DRMs of Clone 9 cells [26,35]. We
therefore speculated that the C-terminus of Glut1 has
some role in the DRM distribution. However, in the
present study, we observed that replacement of neither
the C-terminal nor the N-terminal amino acids of
Glut1 domains with the corresponding amino acids
of Glut3 had any significant effect on the distribution
of Glut1 in CHO-K1 cells (Fig. 3A). The expression
levels of these chimeric proteins and enhanced glucose
uptake were not greatly affected as compared to those
in the vector-transfected control cells. The results suggested that a Glut1 ⁄ Glut3 chimera that has the C-terminus of Glut3 can be trafficked to the cell surface
and distributed to DRMs like Glut1. Analysis of chimeric constructs A and B demonstrated that a large
region from the N-terminal half of TM1 to the large
cytoplasmic loop of Glut1 is necessary for the DRM
distribution (Fig. 5C). Further shortening of this
region or replacement of the large cytoplasmic loop of
Glut1 with the appropriate region of Glut3 clearly
affected the distribution to DRMs (Fig. 6B), and a
2850

chimeric analysis within these regions provided indefinite results. Thus, our data suggest that the regulatory
elements for the DRM distribution of Glut1 in nonpolarized cells are different from those for the apical ⁄ basolateral sorting signals of Glut1 and Glut3 in
polarized epitherial cells. Rather, the DRM distribution may require a specific tertiary structure to be oriented in liquid-ordered lipid phases.
Some recent reports have discussed the biological
significance of the DRM distribution of Glut1, suggesting that the redistribution of Glut1 among different microdomains of the plasma membrane in
nonpolarized cells may have a role in the stressinduced activation of glucose transport [27,32,36]. The

results imply that the distribution to the DRM of
Glut1 in nonpolarized cells is closely related to several
regulatory systems for glucose transport, which might
be distinct from those in polarized cells. Further studies are needed to clarify the molecular mechanism by
which Glut1 is distributed to DRM domains, and its
physiologic roles under various conditions.

Experimental procedures
Antibodies and reagents
The rabbit polyclonal antibody against C-terminal peptides
of human Glut1 was purchased from Millipore (Billerica,
MA, USA). The rabbit polyclonal antibody to Glut3 was
purchased from Medical & Biological Laboratories
(Nagoya, Japan). The mouse monoclonal antibodies to
b-actin, a-tubulin and FLAG M2 were from Sigma
(St Louis, MO, USA), and the mouse monoclonal antibodies to human caveolin-1 were from BD Biosciences (Bedford, MA, USA). An enhanced chemiluminescence (ECL)
kit and [3H]2-deoxy-d-glucose ([3H]2DG) (1 lCi mL)1)
were obtained from GE Healthcare (Chalfont St Giles,
UK). CB was provided by Sigma and Calbiochem (La
Jolla, CA, USA).

Plasmids
The wild-type Glut1–enhanced green fluorescent protein
(EGFP) and wild-type Glut3–EGFP cDNA constructs were
prepared by subcloning the full-length rabbit Glut1 or
human Glut3 cDNA into the Bgl2-Xho1 site of the vector
pEGFP-C2 (Clontech, Mountain View, CA, USA) to generate N-terminal EGFP fusion proteins. FG1, FG3 and
FLAG-tagged Glut1 ⁄ Glut3 chimeric cDNAs were produced according to previously described methods [39]. A
pUC ⁄ Glut1 or pSRa ⁄ Glut3 vector [11] was used as a
template for PCR. The mammalian expression vector

pCMV-Script was kindly provided by O. Kuge (School of

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS


T. Sakyo et al.

Sciences, Kyushu University). cDNAs encoding FG1, FG3
and chimeric Gluts were ligated into the Sal1–Not1 site of
pCMV. All of the FLAG-tagged constructs have the
sequence MDYKDDDDK inserted after the first methionine. Inserts were fully sequenced, and were observed to have
no unexpected mutations. The six chimeras had the compositions shown in Fig. 1.

Cell culture and transfection
CHO-K1 cells were cultured in F-12 medium (Invitrogen,
Carlsbad, CA, USA) containing 10% fetal bovine serum
(MBL, Nagoya, Japan), penicillin (100 mL)1) and
streptomycin (100 lgỈmL)1) under humidified 5%
CO2 ⁄ 95% air at 37 °C, as described previously [40]. These
cells were free from mycoplasma contamination. Lipofectamine reagent and Opti-MEM were purchased from Invitrogen. One day before transfection, CHO-K1 cells were
trypsinized and seeded onto 100 mm plastic culture dishes
at 2 · 106 cells per dish. On the following day, transfection
procedures were performed using 30 lL of lipofectamine
diluted in 70 lL of Opti-MEM (Gibco-BRL) and 6 lg of
Glut ⁄ pCMV plus 1 lg of GFP diluted in 100 lL of supplemental Opti-MEM in 100 mm dishes. Cells were incubated
in the presence of the lipofectamine ⁄ DNA mixture for 5 h
at 37 °C, in 5% CO2, and then incubated overnight in F-12
medium in the presence of 10% fetal bovine serum. At 48 h
post-transfection, cells were used for immunoblotting and
immunofluorescence analysis as described. For 2DG uptake

assays, the cells were seeded in 3 cm dishes at 2 · 105 cells
per dish.

Immunofluorescence analysis
CHO-K1 cells were transfected with GFP-tagged Glut1 and
Glut3, as described under ‘Cell culture and transfection’. At
24 h post-transfection, cells were seeded in a glass-based
dish and incubated overnight at 37 °C. Living cells were
visualized by confocal microscopy (LSM 510, Carl Zeiss
Microimaging, Jena, Germany). Digital images were processed with photoshop (Adobe, San Jose, CA, USA).

Detergent solubilization and immunoblotting
The cells growing in two 10 cm dishes (CHO-K1 cells,
about 1 · 107 cells per dish) were washed once with cold
NaCl ⁄ Pi and scraped. They were then centrifuged for
10 min at 180 g using a swinging bucket rotor RS-240,
2100 (Kubota, Tokyo, Japan), and washed with Hepes buffer. After clarification by centrifugation at 280 g using a
TMA-4 rotor, MRX-150 (TOMY, Tokyo, Japan) for
5 min, pellets were treated with 0.1–0.5 mL of Hepes
buffer containing 0.5% Triton X-100 (Sigma), 10 mm
sodium Hepes, 150 mm NaCl, 5 mm EDTA and 0.5 mm

Regulation of DRM sorting of Glut1

phenylmethanesulfonyl fluoride for 30 min at 4 °C, as described previously [25]. An aliquot of the treated cells was
preserved as the total fraction (T). The remainder was centrifuged at 13 000 g for 30 min at 4 °C using a TMA-4
rotor, MRX-150, and the supernatant was used as the soluble fraction (S). The pellet was washed in 1 mL of cold
Hepes buffer without Triton X-100, and was centrifuged
at 13 800 g for 10 min at 4 °C using a TMA-4 rotor,
MRX-150. The pellet (insoluble fraction, I) was solubilized

with 0.1–0.5 mL of lysis buffer, containing 10 mm
Tris ⁄ HCl, 150 mm NaCl, 1% Triton X-100, 0.5% Nonidet
P-40, 1 mm EDTA, 1 mm EGTA, and 0.5 mm phenylmethanesulfonyl fluoride (pH 7.5). The protein concentration was determined using bicinchoninic reagent (Pierce,
Rockford, IL, USA) with BSA as a standard. Protein samples (10 lg) were subjected to 10% SDS ⁄ PAGE, and transferred to Immobilon-P membranes (Millipore), which were
incubated in NaCl ⁄ Tris ⁄ Tween (500 mm NaCl, 20 mm
Tris ⁄ HCl, pH 7.5, plus 0.1% Tween-20) containing 5%
skimmed milk (Sanko Junyaku, Tokyo, Japan), followed by
rabbit polyclonal antibody or mouse monoclonal antibody
(1 : 1000–2000). The membranes were further incubated
with horseradish peroxidase-conjugated anti-(rabbit IgG) or
anti-(mouse IgG) serum (Amersham Pharmacia Biotech),
and visualized with the ECL detection kit.

[3H]2DG uptake assays
CHO cells were transiently transfected with 6 lg of chimeric forms of Glut1 and Glut3, as described in ‘Cell culture and transfection’. Duplicate culture plates were washed
with NaCl ⁄ Pi, and then incubated for 10 min in glucose
uptake medium consisting of 1 mL of glucose-free DMEM
(Sigma) containing 2.5 lCi of [3H]2DG, 5 mm 2DG and
10 lL of either dimethylsulfoxide alone or dimethylsulfoxide containing CB at a final concentration of 10 lm, as previously described [11,41]. Uptake was terminated by
removal of the medium followed by three rapid washes with
2 mL of NaCl ⁄ Pi. Then, cells were incubated with 5% trichloroacetic acid for more than 20 min at 4 °C, and cellular
radioactivity was determined by liquid scintillation counting. For protein extraction, cells were washed with 2 mL of
NaCl ⁄ Pi and incubated with 0.5 m NaOH for 30 min at
37 °C. The protein concentration was determined using
bicinchoninic acid reagent with BSA as a standard.

Acknowledgements
We are grateful to Dr K. Ishidate for encouragement
and advice. We thank Dr O. Kuge for helpful suggestions regarding the construction of FLAG-tagged
chimeric constructs. We also thank Toshie Gamou

for technical assistance in chimeric construction, and

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS

2851


Regulation of DRM sorting of Glut1

T. Sakyo et al.

Yumi Ikeda for assistance in the transfection and
immunoblotting. This study was supported in part by
the Human Science Foundation of Japan (T. Kitagawa) and the Japan Science Society, Sasagawa Foundation (T. Sakyo).

13

References
1 Bell GI, Burant CF, Takeda J & Gould GW (1993)
Structure and function of mammalian facilitative sugar
transporters. J Biol Chem 268, 19161–19164.
2 Joost HG & Thorens B (2001) The extended GLUTfamily of sugar ⁄ polyol transport facilitators: nomenclature, sequence characteristics, and potential function of
its novel members. Mol Membr Biol 18, 247–256.
3 Hiraki Y, Rosen OM & Birnbaum MJ (1988) Growth
factors rapidly induce expression of the glucose transporter gene. J Biol Chem 263, 13655–13662.
4 Flier JS, Mueckler MM, Usher P & Lodish HF (1987)
Elevated levels of glucose transport and transporter
mRNA are induced by ras or src oncogenes. Science
235, 1492–1495.
5 Kitagawa T, Tanaka M & Akamatsu Y (1989) Regulation of glucose transport activity and expression of glucose transporter mRNA by serum, growth factors and

phorbol ester in quiescent mouse fibroblasts. Biochim
Biophys Acta 980, 100–108.
6 Kitagawa T, Masumi A & Akamatsu Y (1991) Transforming growth factor-b1 stimulates glucose uptake and
the expression of glucose transporter mRNA in quiescent mouse 3T3 cells. J Biol Chem 266, 18066–18071.
7 Younes M, Lechago LV, Somoano JR, Mosharaf M &
Lechago J (1996) Wide expression of the human erythrocyte glucose transporter Glut1 in human cancers.
Cancer Res 56, 1164–1167.
8 Keller P & Simons K (1997) Post-Golgi biosynthetic
trafficking. J Cell Sci 110, 3001–3009.
9 Noto Y, Iwazaki A, Nagao J, Sumiyama Y, Redpath
JL, Stanbridge EJ & Kitagawa T (1997) Altered N-glycosylation of glucose transporter-1 associated with
radiation-induced tumorigenesis of human cell hybrids.
Biochem Biophys Res Commun 240, 395–398.
10 Kitagawa T, Tsuruhara Y, Hayashi M, Endo T &
Stanbridge EJ (1995) A tumor-associated glycosylation
change in the glucose transporter GLUT1 controlled by
tumor suppressor function in human cell hybrids. J Cell
Sci 108, 3735–3743.
11 Suzuki T, Iwazaki A, Katagiri H, Oka Y, Redpath JL,
Stanbridge EJ & Kitagawa T (1999) Enhanced expression of glucose transporter GLUT3 in tumorigenic
HeLa cell hybrids associated with tumor suppressor dysfunction. Eur J Biochem 262, 534–540.
12 Saxon PJ, Srivatsan ES & Stanbridge EJ (1986) Introduction of human chromosome 11 via microcell transfer

2852

14

15

16

17

18

19

20

21
22
23

24

25

controls tumorigenic expression of HeLa cells. EMBO J
5, 3461–3466.
Kayano T, Fukumoto H, Eddy RL, Fan YS, Byers
MG, Shows TB & Bell GI (1988) Evidence for a family
of human glucose transporter-like proteins. Sequence
and gene localization of a protein expressed in fetal
skeletal muscle and other tissues. J Biol Chem 263,
15245–15248.
Nagamatsu S, Sawa H, Wakizaka A & Hoshino T (1993)
Expression of facilitative glucose transporter isoforms in
human brain tumors. J Neurochem 61, 2048–2053.
Younes M, Brown RW, Stephenson M, Gondo M &
Cagle PT (1997) Overexpression of Glut1 and Glut3 in

stage I nonsmall cell lung carcinoma is associated with
poor survival. Cancer 80, 1046–1051.
Gould GW, Thomas HM, Jess TJ & Bell GI (1991)
Expression of human glucose transporters in Xenopus
oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms. Biochemistry 30, 5139–5145.
Asano T, Katagiri H, Takata K, Tsukuda K, Lin JL,
Ishihara H, Inukai K, Hirano H, Yazaki Y & Oka Y
(1992) Characterization of GLUT3 protein expressed in
Chinese hamster ovary cells. Biochem J 288, 189–193.
Harris DS, Slot JW, Geuze HJ & James DE (1992)
Polarized distribution of glucose transporter isoforms in
Caco-2 cells. Proc Natl Acad Sci USA 89, 7556–7560.
Heijnen HFG, Oorschot V, Sixma JJ, Slot JW & James
DE (1997) Thrombin stimulates glucose transport in
human platelets via the translocation of the glucose
transporter GLUT-3 from alpha-granules to the cell surface. J Cell Biol 138, 323–330.
Pascoe WS, Inukai K, Oka Y, Slot JW & James DE
(1996) Differential targeting of facilitative glucose transporters in polarized epithelial cells. Am J Physiol 271,
547–554.
Rajendran L & Simons K (2005) Lipid rafts and membrane dynamics. J Cell Sci 118, 1099–1102.
Simons K & Toomre D (2000) Lipid rafts and signal
transduction. Nat Rev Mol Cell Biol 1, 31–39.
Thoidis G, Kupriyanova T, Cunningham JM, Chen P,
Cadel S, Foulon T, Cohen P, Fine RE & Kandror KV
(1999) Glucose transporter Glut3 is targeted to secretory
vesicles in neurons and PC12 cells. J Biol Chem 274,
14062–14066.
Inukai K, Shewan AM, Pascoe WS, Katayama S, James
DE & Oka Y (2004) Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain.
Mol Endocrinol 18, 339–349.

Sakyo T & Kitagawa T (2002) Differential localization
of glucose transporter isoforms in non-polarized
cells: distribution of GLUT1 but not GLUT3 to
detergent-resistant membrane domain. Biochim Biophys
Acta 1567, 165–175.

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS


T. Sakyo et al.

26 Kumar A, Xiao YP, Laipis PJ, Fletcher BS & Frost SC
(2004) Glucose deprivation enhances targeting of
GLUT1 to lipid rafts in 3T3-L1 adopocytes. Am J
Physiol Endocrinol Metab 286, 568–576.
27 Rubin D & Ismail-Beigi F (2003) Distribution of Glut1
in detergent-resistant membranes (DRM) and nonDRM domains: effect of treatment with azide. Am J
Physiol Cell Physiol 285, 377–383.
28 Travis AJ, Merdiushev T, Vargas LA, Jones BH,
Purdon MA, Nipper RW, Galatioto J, Moss SB,
Hunnicutt GR & Kopf GS (2001) Expression and localization of caveolin-1, and the presence of membrane
rafts, in mouse and guinea pig spermatozoa. Dev Biol
240, 599–610.
29 Schuck S & Simons K (2004) Polarized sorting in
epithelial cells: raft clustering and the biogenesis of the
apical membrane. J Cell Sci 117, 5955–5964.
30 Inukai K, Takata K, Asano T, Katagiri H, Ishihara H,
Nakazaki M, Fukushima Y, Yazaki Y, Kikuchi M &
Oka Y (1997) Targeting of GLUT1–GLUT5 chimeric
proteins in the polarized cell line Caco-2. Mol Endocrinol 11, 442–449.

31 Simons K & Ikonen E (1997) Functional rafts in cell
membranes. Nature 387, 569–572.
32 Bunn RC, Jensen MA & Reed BC (1999) Protein
interactions with the glucose transporter binding
protein GLUT1CBP that provide a link between
GLUT1 and the cytoskeleton. Mol Biol Cell 10,
819–832.
33 Reed BC, Cefalu C, Bellaire BH, Cardelli JA, Louis T,
Salamon J, Bloecher MA & Bunn RC (2005)
GLUT1CBP (TIP2 ⁄ GIPC1) interactions with GLUT1
and myosin VI: evidence supporting an adapter function
for GLUT1CBP. Mol Biol Cell 16, 4183–4201.
34 Oka Y, Asano T, Shibasaki Y, Lin JL, Tsukuda K,
Katagiri H, Akanuma Y & Takaku F (1990) C-terminal
truncated glucose transporter is locked into an inwardfacing form without transport activity. Nature 345,
550–553.

Regulation of DRM sorting of Glut1

35 Zhang JZ, Hayashi HE, Yousuke Prohaska R &
Ismail-Beigi F (1999) Association of stomatin (band
7.2b) with Glut1 transporter. Arch Biochem Biophys
372, 173–178.
36 Barnes K, Ingram JC, Bennett MDM, Stewart GW &
Baldwin SA (2004) Methyl-beta-cyclodextrin stimulates
glucose uptake in Clone 9 cells: a possible role for lipid
rafts. Biochem J 378, 343–351.
37 Barnes K, Ingram JC, Porras OH, Barros LF, Hudson
ER, Lee GD, Fryer Foufelle F, Carling D, Hardie DG &
Baldwin SA (2002) Activation of GLUT1 by metabolic

and osmotic stress: potential involvement of AMPactivated protein kinase (AMPK). J Cell Sci 115,
2433–2442.
38 Baldwin SA, Barros LF, Griffiths M, Ingram J,
Robbins EC, Streets AJ & Saklatvala J (1997) Regulation of GLUT 1 in response to cellular stress. Biochem
Soc Trans 25, 954–958.
39 Katagiri H, Asano T, Ishihara H, Tsukuda K, Lin JL,
Inukai K, Kikuchi M, Yazaki Y & Oka Y (1992) Replacement of intracellular C-terminal domain of GLUT1
glucose transporter with that of GLUT2 increases Vmax
and Km of transport activity. J Biol Chem 267, 22550–
22555.
40 Kitagawa T & Akamatsu Y (1981) Control of passive
permeability of Chinese hamster ovary cells by external
and intracellular ATP. Biochim Biophys Acta 649,
76–82.
41 Oka Y, Asano T, Shibasaki Y, Kasuga M, Kanazawa
Y & Takaku F (1988) Studies with antipeptide antibody
suggest the presence of at least two types of glucose
transporter in rat brain and adipocyte. J Biol Chem 263,
13432–13439.
42 Asano T, Shibasaki Y, Kasuga M, Kanazawa Y,
Takaku F, Akanuma Y & Oka Y (1988) Cloning of a
rabbit brain glucose transporter cDNA and alteration
of glucose transporter mRNA during tissue
development. Biochem Biophys Res Commun 154,
1204–1211.

FEBS Journal 274 (2007) 2843–2853 ª 2007 The Authors Journal compilation ª 2007 FEBS

2853