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

Glut1 and Glut3 regulates their differential distributionto detergent-resistant membrane domains in nonpolarized mammalian cells Tomoko Sakyo1,2, Hiroaki Naraba1, Hirobumi Teraoka2 and T

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

The hexose transporter family, which mediates

facilita-ted uptake in mammalian cells, consists of more than

10 members containing 12 membrane-spanning

seg-ments 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

vari-ety 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

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: tkitaga@iwate-med.ac.jp

(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 mem-brane-spanning segments with a single N-glycosylation site We previously demonstrated that glucose transporter 1 is organized into a raft-like deter-gent-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 detergent-resistant membranes and non-detergent-detergent-resistant membranes of CHO-K1 cells, respectively Replacement of either the C-terminal or N-terminal cyto-solic portion of FLAG-tagged glucose transporter 1 and glucose transpor-ter 3 did not affect the membrane distribution However, a critical sorting signal may exist within the N-terminal half of the isoforms without affect-ing transport activity and its inhibition by cytochalasin B Further shorten-ing of these regions altered the critical distribution, suggestshorten-ing that a large proportion or several parts of the intrinsic structure, including the N-termi-nus of each isoform, are involved in the regulation

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

[9–11] However, differences in the membrane

distribu-tion 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

var-ious 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 api-cal 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 oligo-saccharide 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 Identi-cal amino acid sequences are indicated by asterisks The locations of predicted trans-membrane domains are indicated by gray boxes.

play tissue-specific functions [21,22] In platelets and

neuronal cells, Glut3 is also present in intracellular

ves-icles [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

sur-face in MDCK cells lies in the C-terminal cytosolic tail

[24]

We have previously determined the differential

mem-brane 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

brane (DRM), whereas Glut3 localizes to a fluid

mem-brane 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

deter-gents 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

dif-ferentially recruited to membrane domains of

nonpo-larized 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

util-izing 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

seg-ments 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

con-trol of the differential distribution of Glut1 and Glut3,

we adopted a chimeric strategy whereby different

por-tions of Glut1 and Glut3 were spliced together and

expressed in CHO-K1 cells, which express Glut1 endogenously but not Glut3 As a means of discrimin-ating 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 proce-dures 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 syn-thesized proteins in nonionic detergents, the cells, which transiently expressed FG1 and FG3 proteins, were treated with 0.5% Triton X-100 at 4C, follow-ing fractionation of the solubilized (S) and insoluble (I) fractions As described previously [25], immuno-blotting 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, tubu-lin-a, which is another marker for solubilization, was fully solubilized under this condition Next, we exam-ined 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

4C In contrast, FG1 remained in the insoluble frac-tion 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, recom-binant GFP-tagged Glut1 and GFP-tagged Glut3 were also expressed in CHO cells These GFP-tagged pro-teins 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 mem-brane 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 tag-ging on glucose transport, the activity of CHO cells

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

func-tional carrier-mediated process through these

FLAG-tagged 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

cor-responding 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

polar-ized cells exists in the C-terminus [24], we first

gener-ated 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

pro-teins were transiently expressed in CHO cells, and

examined in terms of their detergent solubility Both

proteins were similarly retained in the insoluble frac-tion when the cells were treated with 0.5% Triton X-100 at 4C, 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 deter-mined 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 C13-transfected 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, sub-stitution 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 mem-brane-spanning regions but exclude a large intracellu-lar 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, respect-ively 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 confo-cal 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 indi-cated in kDa (D) After 48 h of transfection, the uptake of [ 3 H]2DG was measured in the presence and absence of CB The upper panel shows rel-ative 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.

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

GFP or FLAG

GFP or FLAG

Cell

Transfection

Fraction

CHO

Vector

FLAG-M2

Glut3

Glut1

β-Actin

α-Tubulin

Caveolin-1

GFP-G3 GFP-G1

GFP

FGl or GFP-G1

FG3 or GFP-G3

Intracellular loop

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,

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-trans-fected 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

FLAG

Intracellular loop

chimera N31

(G1 : 13 - 492)

chimera C13

(G1 : 1 - 450)

Cell

Transfection

Fraction

CHO

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

FLAG-M2

Glut3

Glut1

β-Actin

α-Tubulin

Caveolin-1

A

B

C

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-ter-minal 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

indi-cated by hatched areas at the N-terminus (B) CHO cells were

tran-siently 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

val-ues normalized to the quantity of protein expressed in CHO cells

are given in nmoles per mg of protein per 10 min One

representa-tive 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.

FLAG

Intracellular loop

A

Cell Transfection Fraction

CHO

T S I T S I T S I

FLAG-M2

Glut3

Glut1

β-Actin

B

chimera N31C3 (G1 : 13-450)

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 back-bone 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.

DRMs, indicating some role for this loop region

Fur-ther replacement within the N-terminal

membrane-span-ning regions of Glut1, included in chimera C, gave

uncertain results

Discussion

The plasma membrane of mammalian cells is com-posed of functionally distinct membrane domains and their components This requires ordered gene expres-sion as well as intricate post-translational sorting machinery that delivers proteins and lipids to the cor-rect 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 determi-nants 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 glycosylphosphatidylinositol-anchored protein, the Src-family tyrosine kinases,

FLAG

450 272

Intracellular loop

A

B

C

chimera A

(G1 : 13-271)

chimera B

(G1 : 272-450)

Cell Transfection

Fraction

CHO FG1 FG3 chimera A chimera B

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

FLAG-M2

Glut3

Glut1

β-Actin

Fig 5 Effects of the N-terminal half of the membrane-spanning

segments and a large intracellular loop of Glut1A Chimera A

con-tains 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

[ 3 H]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,

dimethylsul-foxide (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.

207 271

Intracellular loop

Cell Transfection Fraction

CHO FG1 FG3 chimera C

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

FLAG-M2

Glut3

Glut1

β-Actin

chimera D

B

A

chimera C

(G1 : 13-206)

chimera D

(G1 : 207-271)

Fig 6 Role of a large intracellular loop of Glut1 in DRM distribu-tion (A) Chimera C contains amino acids 13–206 of Glut1 corres-ponding to the N-terminal half of the membrane-spanning segments and the backbone of Glut3 In chimera D, a large intracel-lular loop of Glut3 (amino acids 204–269) is replaced with the cor-responding 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.

heterotrimeric G proteins, and phospholipid-binding

protein [21] Rafts constitute the scaffolding for signal

transduction and several pathogens [22]

We previously demonstrated the differential

distribu-tion 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

nonpola-rized 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

iso-forms has various important roles in subcellular

pro-tein 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

con-tains a targeting motif directing the trafficking of

baso-lateral-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-ter-minus 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

sug-gested that a Glut1⁄ Glut3 chimera that has the

C-ter-minus of Glut3 can be trafficked to the cell surface

and distributed to DRMs like Glut1 Analysis of

chi-meric 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

chimeric analysis within these regions provided indefin-ite results Thus, our data suggest that the regulatory elements for the DRM distribution of Glut1 in nonpo-larized cells are different from those for the api-cal⁄ basolateral sorting signals of Glut1 and Glut3 in polarized epitherial cells Rather, the DRM distribu-tion may require a specific tertiary structure to be ori-ented in liquid-ordered lipid phases

Some recent reports have discussed the biological significance of the DRM distribution of Glut1, sug-gesting that the redistribution of Glut1 among dif-ferent microdomains of the plasma membrane in nonpolarized cells may have a role in the stress-induced 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 stud-ies 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 antibod-ies to human caveolin-1 were from BD Biosciences (Bed-ford, 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 gener-ate N-terminal EGFP fusion proteins FG1, FG3 and FLAG-tagged Glut1⁄ Glut3 chimeric cDNAs were pro-duced 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

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

methion-ine Inserts were fully sequenced, and were observed to have

no unexpected mutations The six chimeras had the

composi-tions 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 UÆmL)1) and

streptomycin (100 lgÆmL)1) under humidified 5%

CO2⁄ 95% air at 37 C, as described previously [40] These

cells were free from mycoplasma contamination

Lipofecta-mine reagent and Opti-MEM were purchased from

Invitro-gen 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

supple-mental Opti-MEM in 100 mm dishes Cells were incubated

in the presence of the lipofectamine⁄ DNA mixture for 5 h

at 37C, 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 37C Living cells were

visualized by confocal microscopy (LSM 510, Carl Zeiss

Microimaging, Jena, Germany) Digital images were

proc-essed 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

buf-fer 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

phenylmethanesulfonyl fluoride for 30 min at 4C, as des-cribed previously [25] An aliquot of the treated cells was preserved as the total fraction (T) The remainder was cen-trifuged at 13 000 g for 30 min at 4C using a TMA-4 rotor, MRX-150, and the supernatant was used as the sol-uble 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 4C 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 phenyl-methanesulfonyl fluoride (pH 7.5) The protein concentra-tion was determined using bicinchoninic reagent (Pierce, Rockford, IL, USA) with BSA as a standard Protein sam-ples (10 lg) were subjected to 10% SDS⁄ PAGE, and trans-ferred 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 chi-meric forms of Glut1 and Glut3, as described in ‘Cell cul-ture and transfection’ Duplicate culcul-ture 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 dimethylsulfox-ide containing CB at a final concentration of 10 lm, as pre-viously 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% tri-chloroacetic acid for more than 20 min at 4C, and cellular radioactivity was determined by liquid scintillation count-ing For protein extraction, cells were washed with 2 mL of NaCl⁄ Pi and incubated with 0.5 m NaOH for 30 min at

37C 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 sugges-tions regarding the construction of FLAG-tagged chimeric constructs We also thank Toshie Gamou for technical assistance in chimeric construction, and

Yumi Ikeda for assistance in the transfection and

immunoblotting This study was supported in part by

the Human Science Foundation of Japan (T

Kita-gawa) and the Japan Science Society, Sasagawa

Foun-dation (T Sakyo)

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