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Molecular Endocrinology 18 (2): 339-349
Copyright © 2004 by The Endocrine Society

Carboxy Terminus of Glucose Transporter 3 Contains an Apical Membrane Targeting Domain

Kouichi Inukai, Annette M. Shewan, Wendy S. Pascoe, Shigehiro Katayama, David E. James and Yoshitomo Oka

Fourth Department of Internal Medicine (K.I., S.K.), Saitama Medical School, Saitama 350-0495, Japan; and Division of Molecular Metabolism and Diabetes (Y.O.), Department of Internal Medicine Tohoku University Graduate School of Medicine, Miyagi 980-8574, Japan; Institute for Molecular Bioscience (A.M.S., W.S.P., D.E.J.), University of Queensland, Brisbane 4072, Australia; and Garvan Institute of Medical Research (D.E.J.), St. Vincents Hospital, Sydney 2010, Australia

Address all correspondence and requests for reprints to: David E. James, Garvan Institute of Medical Research, St. Vincents Hospital, Sydney 2010, Australia. E-mail: d.james{at}garvan.org.au; or Yoshitomo Oka, Division of Molecular Metabolism and Diabetes, Department of Internal Medicine Tohoku University Graduate School of Medicine, Miyagi 980-8574, Japan. E-mail: oka{at}int3.med.tohoku.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473–484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE DELIVERY SYSTEM for the targeting of membrane proteins 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 (1, 2). A number of basolateral sorting signals described to date have been found to reside in the cytoplasmic domain of membrane proteins (3, 4). Most belong to two classes characterized by either a critical tyrosine-containing motif (YXXØ) (5) or a dileucine or leucine residue adjacent to another bulky hydrophobic amino acid (a.a.) (6, 7). These signals have been demonstrated to associate with adaptor protein 1 (8) and adaptor protein 2 (9, 10), which regulate clathrin assembly at the trans-Golgi network and the plasma membrane, respectively. These signals appear to mediate both efficient delivery to the basolateral membrane and endocytic recycling. Conversely, most of the apical signals that have been characterized to date are found in luminal or transmembrane domains. Although relatively little is known about apical sorting signals, both N-linked (11, 12) and O-linked glycosylation (13, 14) have been shown to play an important role.

Facilitative hexose transporters constitute a family of integral membrane proteins that mediate the transport of sugars across cellular membranes (15). These isoforms share a high level of a.a. sequence homology, and their predicted three-dimensional structure is conserved. Considerable evidence suggests that they contain 12 transmembrane domains with both the N and C termini located on the cytosolic side (15). Despite these similarities, major differences in intracellular trafficking have been noted between individual GLUTs. These differences are best demonstrated in polarized cell types in which different transporters have been localized to discrete surfaces. Glucose transporter 1 and 2 (GLUT1 and GLUT2) are principally found on the basolateral surface in epithelial cells whereas GLUT3 and GLUT5 are mainly targeted to the apical domain (16, 17, 18, 19). Similar results are obtained when these transporter isoforms are transfected into Madin-Darby canine kidney (MDCK) cells indicating that this is a universal feature of these proteins that can be recapitulated in a heterologous system (20). These results also demonstrate that MDCK cells provide a useful model for studying the vectorial membrane trafficking of facilitative hexose transporters. The asymmetric distribution of GLUTs in polarized cell types has physiological relevance. For example, with respect to the intestinal absorption of fructose, the apically targeted GLUT5 exhibits a high affinity for fructose [low Michaelis-Menten constant (Km)] (21), whereas the basolaterally targeted GLUT2 exhibits a high Vmax for fructose (22). Hence, these two facilitative transporters cooperate to achieve efficient absorption of fructose across the gut epithelium.

The molecular mechanisms by which GLUTs are differentially targeted in polarized cells remain to be clarified. Therefore, we attempted to characterize the structural determinants of GLUTs required for this differential targeting. We have expressed a panel of chimeric transporters utilizing various portions of GLUTs 1–5 in MDCK cells and assessed their differential distribution. Our data show that the carboxy-terminal tail of human (h)GLUT3 contains a dominant apical sorting signal that is capable of rerouting both GLUT1 and GLUT2 from the basolateral to the apical cell surface in MDCK cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression and Analysis of GLUT1/3 Chimeras in MDCK Cells
To clarify the molecular basis for the differential targeting of GLUTs in polarized epithelial cells, we undertook a chimeric strategy whereby different portions of a basolateral transporter and an apical transporter were spliced together and expressed in MDCK cells. Initially, we focused on hGLUT1 and hGLUT3, which are targeted to the basolateral and apical cell surfaces, respectively, in MDCK cells (20). MDCK cells express GLUT1 endogenously but not GLUT3 (20). Initially, we studied the targeting of recombinant hGLUT1 when overexpressed in MDCK cells. Stable cell lines were selected and screened for hGLUT1 expression using a monoclonal antibody that is specific for hGLUT1 (23). This antibody recognizes an epitope in the central loop of hGLUT1 and provides a useful tool for comparing relative expression levels between individual constructs and clones. To verify that this expression system did not result in marked overexpression, we analyzed the glucose transport activities of these clones. In wild-type cells, we observed glucose transport rates of 0.77 ± 0.16 nmol/mg·min and 0.04 ± 0.06 nmol/mg·min across the basolateral and apical membranes, respectively (n = 5, mean ± SD). The glucose transport rates across the basolateral membranes in GLUT1-expressing cell lines were increased at most by 1.5-fold as compared with that observed in wild-type cells. Moreover, we did not observe a significant change in apical transport in clones expressing GLUT1 at this expression level. On the other hand, in clones expressing GLUT3 over a broad range of expression levels, we observed a highly significant increase in transport across the apical membrane (0.56 ± 0.10 nmol/mg·min). Thus, these data provide a good indication that we have performed our studies using nonsaturating expression levels of recombinant transporters. As shown in Fig. 1CGo (left upper panel), at this level of expression the targeting of hGLUT1 was restricted to the basolateral surface as was the case for the endogenous protein. Also shown in Fig. 1CGo (right upper panel) is the distribution of hGLUT3 expressed in MDCK cells. Consistent with our previous findings (20), this protein was highly enriched at the apical cell surface. Whereas these transporters localize to either basolateral or apical membranes, intracellular labeling is also evident. Our focus in this study was the contribution of the cytosolic carboxy terminus to domain-specific cell surface localization and as such we did not characterize the intracellular vesicular compartments through which these chimeras traffic.



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  		Fig. 1. Immunofluorescence Localizations of GLUT1 and GLUT3 and Their Chimeric GLUTs

A, A diagrammatic representation of the constructs used in these studies is shown. B, Total membrane samples (15 µg) prepared from MDCK cells expressing the indicated proteins were subjected to SDS-PAGE and immunoblotting with an antibody raised against the C-terminal domain of GLUT3. NEG refers to a sample prepared in parallel from parental MDCK cells. Immunoblot data identical to those presented for parental membranes were obtained from nonexpressing but G418-resistant MDCK clones. C, MDCK cells expressing the indicated proteins were plated on glass coverslips, and immunofluorescence was performed as previously described (20 ). Exogenous transporters were detected using an antibody raised against a short peptide derived from the intracellular loop of hGLUT1 (GLUT1) and an antibody raised against the C-terminal domain of GLUT3 (GLUT3, GLUT1/3CT290, and GLUT1/3CT36). Confocal images were generated using a Zeiss Axiophot fluorescent microscope and a Bio-Rad MRC600 laser scanning head.

 
The above data provided the basis for our initial studies using hGLUT1/3 chimeras expressed in MDCK cells. We first designed two GLUT1/3 chimeras comprised of different portions of both proteins: hGLUT1/3CT290 contains the N-terminal half of hGLUT1 and the C-terminal half of GLUT3; and hGLUT1/3CT36 is comprised of hGLUT1 in which the carboxy-terminal tail (32 a.a.) has been replaced with that of hGLUT3 (36 a.a.). A scheme of these and other constructs is shown in Fig. 1AGo. As was the case for all of the constructs described in this study, we selected at least 12 different stable cell lines expressing the recombinant protein of interest and performed detailed analyses on at least three to four different clones for each construct covering a range of expression levels. The targeting of endogenous GLUT1, as well as inulin exclusion, was analyzed in each clone to verify polarity at the time of study (data not shown). Figure 1BGo shows a Western blot of cell lysates from cells expressing either full-length GLUT3 or the relevant chimeras using an antibody raised against the C-terminal domain of hGLUT3. This antibody did not detect a specific signal in nontransfected MDCK cells, but clearly detected the exogenous hGLUT3 epitope. The chimeras yielded proteins of the appropriate molecular size, i.e. similar to that observed for full-length GLUT3. Figure 1CGo shows the immunolocalization of the hGLUT1/3CT290 (left lower panel) and hGLUT1/3CT36 (right lower panel) chimeras in MDCK cells as compared with both GLUT1 and GLUT3. Both chimeras were concentrated on the apical domain, similar to the targeting observed for the full-length GLUT3 protein. We also observed labeling of intracellular structures (Fig. 1Go). This is consistent with our previous studies in which even GLUT1, which is highly concentrated on the basolateral surface (Fig. 1Go), was also found in intracellular vesicles in MDCK cells (20). These results suggest that the carboxy tails of these transporter proteins determine basolateral vs. apical delivery.

Expression of a Low-Density Lipoprotein Receptor (LDL-R)/GLUT1 Chimera in MDCK Cells
It was shown previously that the cytoplasmic tail of the LDL-R possesses two tyrosine-dependent basolateral targeting signals (24, 25). Deletion of these residues resulted in rerouting of the LDL-R to the apical cell surface (24). To determine whether the GLUT1 carboxy tail possesses a basolateral sorting signal, we studied the trafficking of an LDL-R chimera containing the carboxy tail of GLUT1 (LDL-R/G1CT24), in MDCK cells. Several clones expressing the full-length LDL-R and the chimeras were obtained and grown on transwell filters. Trafficking of these proteins was analyzed using a cell surface biotinylation assay (Fig. 2Go). In agreement with previous studies (24), the wild-type LDL-R was almost entirely targeted to the basolateral membrane, whereas more than 85% of mutated LDL-R (CT37Y-A18) was targeted to the apical membrane. When 24 a.a. from the C terminus of GLUT1 were grafted onto CT37Y-A18, a small portion of the apical proteins relocalized to the basolateral membrane, but the majority remained at the apical membrane (Fig. 2CGo). These findings suggest that the C-terminal tail of GLUT1 does not contain an autonomous basolateral sorting signal. This does not exclude the possibility of a basolateral sorting signal elsewhere in the GLUT1 molecule. In fact, this seems likely given that it is a multispanning membrane protein with at least three major cytosolic domains. It is also conceivable that sorting domains in these more complex molecules are comprised of discontinuous elements found in discrete domains that interact in vivo.



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  		Fig. 2. Cell Surface Distributions of Wild and Mutated LDL-Rs Using Biotinylation Assay

A, A diagrammatic representation of the constructs used in these studies is shown. B, MDCK cells expressing various chimeric proteins were plated onto transwell filters. Apical (A) or basal (B) cell surfaces were incubated in 0.5 mg/ml EZ-link Sulfo-NHS-biotin (Pierce Chemical Co.) for 15 min on ice. Filters were quenched and transferred into lysis buffer, and the solubilized material was cleared by centrifugation. An aliquot of cleared supernatant served as the total expression sample, and the remainder was incubated with streptavidin-agarose to recover biotinylated proteins. Immunoprecipitated proteins were subjected to SDS-PAGE and subsequent Western blot analysis using a polyclonal LDL-R antibody, LB1, with detection performed using goat antirabbit-horseradish peroxidase antibody and enhanced chemiluminescence. C, Values are given as the percent of total cell surface receptors. Bars represent the mean ± SD of three independent experiments.

 
Trafficking of a GLUT1/5 Chimera in MDCK Cells
It was shown previously that GLUT5 is targeted to the apical cell surface in polarized epithelial cells and that apical targeting is regulated via information contained within the central portion of the protein (26). Thus, it is highly unlikely that the C-terminal tail of GLUT5 contains targeting information relevant to trafficking in polarized epithelial cells. Therefore, we reasoned that a GLUT1 chimera containing the C terminus of GLUT5 should provide a useful tool for the present studies. If this chimera targets to the apical cell surface, this will provide definitive proof that the GLUT1 tail contains a basolateral sorting motif. This chimera was expressed in MDCK cells and localized by immunofluorescence microscopy. As shown in Fig. 3Go, whereas full-length GLUT5 was targeted to the apical surface, the GLUT1/5 chimera was found almost exclusively on the basolateral membrane. In combination with the studies described above, these data strongly indicate that the C-terminal tail of GLUT1 does not contain a basolateral targeting motif.



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  		Fig. 3. Immunofluorescence Localizations of GLUT5 and GLUT1/5 Chimeric GLUTs

A, Scheme of the constructs generated for this analysis. B, MDCK cells expressing the indicated proteins were plated onto flamed, glass coverslips and incubated for 5 d to enable tight juction formation and establishment of polarity. Cells were fixed and the domain-specific localizations of exogenous GLUT5 and the GLUT1/5 chimera were assessed by immunofluorescent microscopy using an antibody raised against the C-terminal domain of GLUT5. The GLUT1/5 chimera consists of residues 1–450 of hGLUT1 joined in frame to residues 459–502 of GLUT5.

 
Expression and Analysis of GLUT2/3 Chimeras in MDCK Cells
To further investigate the apical sorting signal in the C terminus of GLUT3, we undertook an analysis of a panel of chimeras based on hGLUT2 and hGLUT3. Like GLUT1, GLUT2 is targeted to the basolateral cell surface in MDCK cells (20). Initially, we analyzed the targeting of two chimeras, which contained reciprocal portions of GLUT2 and 3. The first, designated GLUT2/3, comprised hGLUT2 up to the end of the last transmembrane domain followed by the cytosolic C-terminal tail of hGLUT3. The second chimera, hGLUT3/2, comprised hGLUT3 up to and including the last transmembrane domain followed by the cytosolic tail of hGLUT2 (Fig. 4AGo). Both chimeras produced protein products of the correct molecular size when transfected into MDCK cells (data not shown). Confocal immunofluorescence microscopy was used to analyze the targeting of both chimeras in MDCK cells. As shown in Fig. 4BGo, GLUT2/3 (left lower panel) was targeted to the apical membrane and GLUT3/2 (right lower panel) to the basolateral membrane. These results support the conclusion that the cytoplasmic carboxy tail of GLUT3 possesses an autonomous apical targeting motif.



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  		Fig. 4. Immunofluorescence Localizations of GLUT2, GLUT3, and Their Chimeric GLUTs

A, The GLUT2, GLUT3, and the two chimeras generated are represented in this line drawing. The chimeras consist of either GLUT2 or 3 up to and including the 12th transmembrane domain of each GLUT with the opposite C-terminal domain fused in frame, generating GLUT2/3 and GLUT3/2. B, At least 4 d before the immunofluorescence study depicted here, MDCK cells expressing the various proteins, as indicated, were plated onto coverslips. The proteins of interest were detected utilizing either an antibody raised against the C-terminal domain of GLUT2 (GLUT2 and GLUT3/2) or an antibody raised against the C-terminal domain of GLUT3 (GLUT3, GLUT2/3). Confocal images were collected and representative images are shown.

 
Expression and Analysis of GLUT4/3 Chimeras in MDCK Cells
We have previously shown that a chimera composed of the full-length GLUT4 protein and the last 12 a.a. of hGLUT3 appended to the C terminus of GLUT4, when expressed in adipocytes, behaves indistinguishably from GLUT4 (27). This chimera, termed GLUT4/3tag, was expressed in MDCK cells and found to be targeted to intracellular membranes (Fig. 5CGo, left panel) in a manner similar to that previously described for the wild-type GLUT4 protein (20). The intracellular sequestration of GLUT4 is controlled, in part, by an aromatic a.a.-based signal (FQQI) in the cytosolic N terminus of the protein (28). Phenylalanine at position 5 was mutated to alanine in this GLUT4/3tag chimera and expressed in MDCK cells. Localization studies indicated that some GLUT4/3tagF5A was targeted to the basolateral cell surface, whereas others stayed in an intracellular compartment (Fig. 5CGo, middle panel). Conversely, the chimera GLUT4/3CT24F5A composed of GLUT4 and the cytosolic C-terminal tail of GLUT3 and possessing the Phe5 to Ala mutation was targeted apically (Fig. 5CGo, right panel). Collectively, these data show that the GLUT3 tail possesses an apical targeting signal that does not include the C-terminal 12 a.a.



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  		Fig. 5. Immunofluorescence Localizations of GLUT4 and GLUT4/3 Chimeric GLUTs

A, The cDNAs generated for this study and used to transfect MDCK cells are depicted here. As shown, all molecules bear the GLUT3 epitope tag at the extreme carboxy terminus with the Phe5 of GLUT4 mutated to Ala in both GLUT4/3tagF5A and GLUT4/3CT24F5A. B, Western blot analysis of total membrane samples prepared from MDCK cells. Recombinant GLUTs were detected with an anti-GLUT3 antibody. C, MDCK cells expressing GLUT4/3tag, GLUT4/3 tagF5A, or GLUT4/3CT24F5A were plated at confluence and allowed to polarize over 5 d. The cellular localization of each of these proteins was assessed by confocal immunofluorescence microscopy using an antibody raised against the GLUT3 carboxy-terminal 12 residues. Representative images are presented.

 
Further Analysis of GLUT1/3 Chimeras in MDCK Cells
To further define the composition of the apical targeting signal, we examined the targeting of a panel of GLUT1/3 chimeras comprising different portions of the C terminus of GLUT3. Consistent with the data described above (Fig. 5Go), a chimera in which the last seven a.a. of GLUT1 were replaced with the last 13 a.a. of hGLUT3 (GLUT1/3CT13) was targeted to the basolateral cell surface (Fig. 6CGo, middle lower panel). Intriguingly, addition of a further six a.a. from hGLUT3 (GLUT1/3CT19) resulted in a significant increase in apical targeting although some basolateral targeting was still evident (Fig. 6CGo, left lower panel). However, addition of a further five a.a. from GLUT3 to generate the chimera GLUT1/3CT24 resulted in predominantly apical targeting with little evidence of basolateral localization (Fig. 6CGo, right upper panel). To verify these data, we performed vectorial biotinylation as described above and found that GLUT1/3CT19 was targeted predominantly to the apical side whereas GLUT1/3CT13 was targeted primarily to the basolateral side (Fig. 6DGo), consistent with our immunofluorescence studies.



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  		Fig. 6. Immunofluorescence Localizations of GLUT1, GLUT3, and Their Chimeric GLUTs

A, Stable MDCK cell lines were generated by transfection with cDNA, as depicted, and selection in G418. GLUT1/3CT24 consists of a.a. 1–473 of GLUT1 and a.a. 473–496 of GLUT3. Similarly, GLUT1/3CT19 and GLUT1/3CT13 were constructed by fusing a.a. 1–478 (CT19) or a.a. 1–485 (CT13) of GLUT1 to a.a. 478–496 (CT19) or a.a. 484–496 (CT13) of GLUT3. B, Membrane samples were prepared and subjected to SDS-PAGE and Western blot analysis to assess expression levels of GLUTs in MDCK cells. Membranes were incubated with an antibody raised against the C-terminal domain of GLUT3. C, MDCK cells were transfected (with cDNA as indicated in panel A) and clones were analyzed by immunofluorescence microscopy to ascertain the domain-specific localization of the recombinant transporters. Cells were fixed, permeabilized, and immunolabeled using an antibody raised against a short peptide derived from the intracellular loop of hGLUT1 (GLUT1) and an antibody raised against the C-terminal domain of GLUT3 (GLUT3, GLUT1/3CT24, GLUT1/3CT19, and GLUT1/3CT13). Confocal images were generated using a Zeiss Axiophot fluorescent microscope and a Bio-Rad MRC600 laser scanning head. D, Cell surface localizations of overexpressed hGLUT1, hGLUT1/3CT19, and hGLUT1/3CT13 were assessed using a domain-specific biotinylation assay. Cells were grown at confluence for 4–5 d on Transwell filters. Apical and basolateral cell surface proteins were biotinylated by addition of 0.5 mg/ml EZ-Link-Sulfo-NHS-Biotin (Pierce Chemical Co.) for 15 min. Biotinylated proteins were recovered and subjected to SDS-PAGE followed by Western blotting with antibodies specific for hGLUT1. Chemiluminescent bands were quantified using a Bio-Rad 600 densitometer, and signals detected were corrected to account for loading differences.

 
As shown in Fig. 6CGo, expressed GLUT1 occasionally exhibits apparent apical staining in addition to the basolateral staining in immunohistochemical analysis. However, this does reflect, not mixed labeling to different surface domains, but rather both basolateral and intracellular labeling. When the GLUTs reside intracellularly, the outlines of cell nuclei can be seen clearly in x-y images. In contrast, when they localize in apical membranes, the outlines can barely be visualized. When GLUT1s are expressed (Fig. 6CGo, left upper panel), the staining pattern appears to be mixed, similar to those of GLUT1/5 (Fig. 3BGo) and GLUT3/2 (Fig. 4BGo). However, this expressed GLUT1 was proved to be located exclusively in the basolateral membrane by biotinylation experiments (Fig. 6DGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we obtained evidence that the cytosolic carboxy-terminal tail of hGLUT3 contains a targeting motif capable of directing the trafficking of GLUTs 1, 2, and 4 to the apical cell surface in MDCK cells. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence for the presence of an apical targeting signal involving residues 473–484 (DRSGKDGVMEMN) in the carboxy tail. The finding of special interest is that chimeras containing the last 12 or 13 a.a. of GLUT3 did not exhibit significant apical targeting, whereas a chimera containing a further seven a.a. from GLUT3 (GLUT1/3CT19) exhibited significant apical targeting. Thus, fine mapping of the C-terminal tail of hGLUT3 has narrowed the apical sorting information to a small region encompassing residues DGVMEMN, which corresponds to residues PAGVELN in rodent GLUT3. It is noteworthy that this chimera (GLUT1/3CT19) was partially targeted to the basolateral membrane whereas GLUT1/3CT24, which contained an additional five a.a. from GLUT3, was targeted almost indistinguishably from wild-type GLUT3. To our knowledge, no similar targeting motifs have been reported in other apical membrane proteins, and we have been unable to identify a similar motif in other apical membrane proteins. Most noteworthy is the presence of two conserved hydrophobic residues surrounding an acidic residue in both the rodent (VEL) and human (MEM) sequences. Future studies will be aimed at identifying the roles of these residues.

Based upon our initial studies with GLUT1/3 chimeras (Fig. 1Go), we hypothesized that the GLUT1 tail contained a basolateral signal that was disrupted in this chimera, resulting in default trafficking to the apical cell surface. However, detailed studies showed that this was not the case and argued in favor of an apical signal in GLUT3. Most notably, the GLUT3 tail facilitated apical targeting when grafted onto GLUT1, 2, or 4, all of which normally recycle via the basolateral cell surface. Thus far, two separate classes of basolateral sorting signals have been identified. These are characterized by either an essential tyrosine residue, or a dileucine motif (5, 6, 7). Similar kinds of motifs could not be found in the C termini of either GLUT1 or GLUT2 (Fig. 7Go). The LGA residues are conserved between GLUT1 and GLUT2 in the C terminus. However, mutating all of these residues did not disrupt the basolateral targeting of GLUT1 (data not shown). We also prepared a GLUT1 mutant missing the last three a.a. because this domain has been shown to act as a binding site for PDZ domain-containing proteins (29, 30). However, the distribution of this truncated GLUT1 was exactly the same as that of wild-type GLUT1 (data not shown). Thus, using this type of approach, we were unable to identify a basolateral targeting domain in the tail of GLUT1. Consistent with this conclusion, the GLUT1 tail did not significantly alter the apical targeting of a tail minus LDL-R, a construct that has been successfully used to map basolateral sorting signals in cytosolic tails (Fig. 2Go). Moreover, the cytosolic tail of GLUT5, which does not contain a basolateral sorting signal, did not disrupt the targeting of GLUT1 (Fig. 3Go). Taken together, these studies provide compelling evidence that the C-terminal tail of GLUT3 contains an apical sorting signal. This signal is obviously very dominant, being able to overcome basolateral sorting signals in GLUTs 1, 2, and 4.



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  		Fig. 7. Comparison of the Cytoplasmic Tails of Human and Rodent GLUTs

The putative a.a. sequence, which is suggested to have an apical sorting signal, is underlined.

 
It was reported previously that some apical targeting motifs function by selectively partitioning into lipid raft domains in the Golgi (31). Intriguingly, it was reported recently that, in nonpolarized epithelial cells, whereas a substantial portion of GLUT1 was found in detergent-resistant membrane domains, this was not the case for GLUT3 (32). Hence, these data support the notion that GLUT3 is sorted via a non-lipid raft-mediated domain; this is consistent with the present data indicating such a domain to be located in the cytosol, perhaps allowing the transporter to interact with cytosolic targeting machinery. Several other reports have recently found narrowly defined sequences or short peptides in the C-terminal cytoplasmic tails of membrane proteins to have a role in apical targeting (33, 34, 35, 36, 37). Among them, multiple autonomous signals for apical targeting in the same protein have been reported (33, 34). These signals include PDZ-interacting domains (35) and ß-turn structures (36) as well as less well-defined signals. Taken together, these observations suggest the apical sorting mechanism defined by cytosolic sorting signals to be less well defined than luminal or transmembrane signals, and further work is required to determine whether there is some type of homology among these motifs.

GLUT3 has been shown to undergo specialized targeting in other cell types. It is targeted to the limited membrane of secretory granules in platelets (38) and to the tail of sperm cells (39). Moreover, there is evidence that GLUT3 is targeted to axons in neurons (our unpublished data) and also to neuronal vesicles (40). It will be intriguing to determine whether the cytosolic C terminus is involved in each of these unique trafficking processes, which would imply a common mechanism. Consistent with this, it was previously suggested that similar rules govern the targeting of membrane proteins in epithelial cells and neurons (41).

The identification of this apical targeting signal in the GLUT3 tail may have significant utility in studying the structure/function of sugar transporters in mammalian cells. A major complication of studying transport kinetics of GLUTs after their expression in mammalian cells is that there is always a very high background due to the presence of endogenous transporters. We previously found that glucose transport across the apical cell surface in MDCK cells is negligible because most of the uptake occurs via the basolateral surface (20). By transplanting the apical signal from GLUT3 onto other transporters it should be feasible to redirect them to the apical surface, as shown here, which should afford ideal conditions for studying transport kinetics in a more appropriate environment.

In conclusion, we have analyzed the asymmetric distribution of facilitative GLUTs by expressing chimeric transporters utilizing various portions of GLUTs (GLUT1–GLUT5) in MDCK cells. Our present data strongly suggest that the apical sorting signal resides in the C-terminal tail of GLUT3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
cDNA Constructs
Chimeric cDNAs were produced according to previously described methods (42), which allowed us to swap different domains from different transporter isoforms at any desired junction. A hGLUT1 cDNA (23), a hGLUT2 cDNA (42), a hGLUT3 cDNA (27), a hGLUT4 cDNA (28), and a rat GLUT5 cDNA (43) were used as PCR templates. Cytomegalovirus-based expression plasmid pCB6 was generously supplied by Dr. Mellman (4). Fragments prepared by PCR were fully sequenced and observed to have no unexpected mutations. cDNAs encoding wild-type and chimeric GLUTs were ligated into pCB6 vectors.

Cell Culture and Transfection
MDCK cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C, in 5% CO2. Lipofectamine reagent, Opti-MEM I, and G-418 (geneticin) were purchased from GIBCO Life Technologies (Eggenstein, Germany). One day before transfection, MDCK cells were trypsinized and seeded onto a 60-mm plastic culture dish at 6 x 105cells per dish. The following day, transfection procedures were performed using 30 µl lipofectamine diluted in 300 µl Opti-MEM I (supplemented with 20 mM L-glutamine) and 6 µg GLUT/pCB6 diluted in 300 µl of supplemental Opti-MEM-I/60-mm dish. Cells were incubated in the presence of the lipofectamine-DNA mix for 5 h at 37 C, in 5% CO2, and then incubated overnight in DMEM-10% fetal calf serum-L-glutamine. Forty-eight hours after transfection, each transfected 60-mm dish was split into three 150-mm dishes and incubated with G-418 (0.8 mg/ml). Colonies resistant to G-418 were isolated after 10–14 d and screened for protein expression by Western blotting.

Western Blotting
To screen for positive expression of the chimera of interest, membrane protein samples (20 µg protein) were subjected to SDS-PAGE employing a 10% resolving gel. Proteins were transferred to a nitrocellulose membrane. Membranes were incubated for 1 h at 37 C with the appropriate primary antibody. After three 10-min washes in PBS-0.1% Tween 20, the membranes were incubated at room temperature for 1 h with a horseradish peroxidase-labeled donkey antirabbit secondary antibody (Amersham Life Science, Little Chalfont, Buckinghamshire, UK) diluted 1:10,000 in PBS-0.2% BSA. After three further washes, labeled proteins were visualized using the enhanced chemiluminescence detection method (Amersham Life Science).

Preparation of Total Membrane Fractions
Total membrane fractions were prepared from MDCK cells after homogenization in 20 mM HEPES, pH 7.4, 1 mM EDTA, 255 mM sucrose buffer containing protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 250 µM phenylmethylsulfonylfluoride) and centrifugation at 50,000 rpm in a Beckman TLA100–3 rotor (Beckman Coulter, Inc., Fullerton, CA) for 60 min. The membrane pellet was resuspended in 20 mM HEPES, pH 7.4, 1 mM EDTA, 255 mM sucrose buffer and stored at -80 C before use.

Protein Assay
The protein concentration of total membrane fractions was determined using the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions.

Immunofluorescence Microscopy
Cells were plated at near-confluent density on glass coverslips and fixed 5 d later in 2% paraformaldehyde-PBS for 1 h at room temperature. Polarization was indicated by the presence of domes or blisters in the cultures. Coverslips were washed three times in PBS, and then quenched for 15 min in 0.2% Triton X-100. After an additional three washes in PBS, coverslips were blocked for 30 min in 2% horse serum-PBS and then washed twice in PBS. Primary antibodies were diluted in 0.1% horse serum-PBS, and incubations were carried out at 4 C overnight. Fluorescein isothiocyanate-conjugated antirabbit Ig secondary antibody diluted in 0.1% horse serum-PBS was applied after three 5-min washes in PBS. After a 1-h incubation, at room temperature, coverslips were washed three times in PBS for 5 min each and then mounted in 1% propyl gallate-50% glycerol-PBS. Confocal images were generated with a Zeiss Axiophot microscope (Carl Zeiss, Thornwood, NY) and a Bio-Rad MRC600 confocal laser scanning head (Bio-Rad Laboratories, Inc., Hercules, CA). At least one G-418-resistant clone, which was negative by immunoblotting, served as a negative control for immunofluorescence microscopy.

Cell Surface Biotinylation
Localization of the LDL-R constructs and GLUT1/3 chimeras was assessed utilizing a domain-specific biotinylation assay. Briefly, MDCK cells expressing various chimeric proteins were plated onto transwell filters (Corning, Inc., Corning, NY) and grown for at least 4 d at 37 C and 5% CO2. To assess the integrity of the monolayer, growth media containing [14C]inulin (0.1 mCi/ml at 1:2000 dilution) were added to the apical chamber and incubated for 1 h. After this incubation, aliquots of medium from both the apical and basal chambers were counted (Coulter Counter, BD, Beckman Coulter, Inc.). Transwells with less than 0.5% transport were used in experiments. Filters were washed three times with ice-cold PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 (PBS+). Apical or basal cell surfaces were incubated with 0.5 mg/ml EZ-link Sulfo-NHS-biotin (Pierce Chemical Co.) for 15 min on ice. This process was repeated, and the free biotin reagent was then quenched by washing with PBS+ containing 50 mM glycine. Filters were transferred into lysis buffer (1% TX-100, 20 mM HEPES, 1 mM EDTA, 100 mM NaCl, aprotinin 10 µg/ml, leupeptin 10 µg/ml, and phenylmethylsulfonylfluoride 250 µM) for 30 min on ice and vortexed, and solubilized material was cleared by centrifugation at 14,000 rpm at 4 C for 15 min. An aliquot of cleared supernatant served as the total expression sample, and the remainder was incubated with streptavidin-agarose (Sigma Chemical Co., St. Louis, MO) to recover biotinylated proteins. Immunoprecipitated proteins were subjected to SDS-PAGE and subsequent Western blot analysis using either a polyclonal LDL-R antibody, LB1 (gift from Dr. P. Kroon, Department of Biochemistry, University of Queensland), or antibodies specific to hGLUT1 (gift from Dr. G. Lienhard, Department of Biochemistry, Dartmouth Medical School, Hanover, NH). Detection was performed using goat antirabbit-horseradish peroxidase antibody (Amersham Life Science) and enhanced chemiluminescence (SuperSignal, Pierce Chemical Co.).


   FOOTNOTES
 
K.I. and A.M.S. contributed equally to this work and should both be considered first authors.

This work was supported by the Research Council of Australia, of which D.E.J. is a Senior Principal Research Fellow of the National Health and Medical Research Council of Australia. This work was also supported by Grant-in Aid for Scientific Research no. 13470226 (to Y.O.) and Creative Basic Research Grant no. 10NP0201 (to Y.O.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Abbreviations: a.a., Amino acid(s); GLUT, glucose transporter; LDL-R, low-density lipoprotein receptor; MDCK, Madin-Darby canine kidney.

Received for publication March 18, 2003. Accepted for publication October 30, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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