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We previously identified Asp(340) in transmembrane segment 7 (TM7) as a key determinant of substrate affinity in Hxt7, a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by Hxt7, we performed cysteine-scanning mutagenesis of 21 residues in TM5 of a Cys-less form of Hxt7. Four residues were sensitive to Cys replacement, among which Gln(209) was found to be essential for high-affinity glucose transport activity. The 17 remaining sites were examined further for the accessibility of cysteine to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). Among the Cys mutants, T213C was the only one whose transport activity was completely inhibited by 0.5 mM pCMBS. Moreover, this mutant was protected from pCMBS inhibition by the substrate d-glucose and by 2-deoxy-D-glucose but not by L-glucose, indicating that Thr(213) is situated at or close to a substrate recognition site. The functional role of Thr(213) was further examined with its replacement with each of the other 19 amino acids in wild-type Hxt7. Such replacement generated seven functional transporters with various affinities for glucose. Only three mutants, those with Val, Cys, and Ser at position 213, exhibited high-affinity glucose transport activity. All of these residues possess a side chain length similar to that of Thr, indicating that side chain length at this position is a key determinant of substrate affinity. A working homology model of Hxt7 indicated that Gln(209) and Thr(213) face the central cavity and that Thr(213) is located within van der Waals distance of Asp(340) (TM7).

We previously identified Asn(331) in transmembrane segment 7 (TM7) as a key residue determining substrate affinity in Hxt2, a moderately high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by yeast glucose transporters, we have now studied Hxt7, whose affinity for glucose is the highest among the major hexose transporters. The functional role of Asp(340) in Hxt7, the residue corresponding to Asn(331) of Hxt2, was examined by replacing it with each of the other 19 amino acids. Such replacement of Asp(340) generated transporters with various affinities for glucose, with the affinity of the Cys(340) mutant surpassing that of the wild-type Hxt7. To examine the structural role of Asp(340) in the substrate translocation pathway, we performed cysteine-scanning mutagenesis of the 21 residues in TM7 of a functional Cys-less Hxt7 mutant in conjunction with exposure to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). The transport activity of the D340C mutant of Cys-less Hxt7, in which Asp(340) is replaced with Cys, was completely inhibited by pCMBS, indicating that Asp(340) is located in a water-accessible position. This D340C mutant showed a sensitivity to pCMBS that was approximately 70 times that of the wild-type Hxt7, and it was protected from pCMBS inhibition by the substrates d-glucose and 2-deoxy-d-glucose but not by l-glucose. These results indicate that Asp(340) is situated at or close to a substrate recognition site and is a key residue determining high-affinity glucose transport by Hxt7, supporting the notion that yeast glucose transporters share a common mechanism for substrate recognition.

Hxt2 and Hxt1 are, respectively, high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae. We have previously investigated which amino acid residues of Hxt2 are important for high affinity transport activity. Studies with all the possible combinations of 12 transmembrane segments (TMs) of Hxt2 and Hxt1 revealed that TMs 1, 5, 7, and 8 of Hxt2 are necessary for high affinity transport. Systematic shuffling of the 20 amino acid residues that differ between Hxt2 and Hxt1 in these TMs subsequently identified 5 residues as important for such activity: Leu(59) and Leu(61) (TM1), Leu(201) (TM5), Asn(331) (TM7), and Phe(366) (TM8). We have now studied the relative importance of these 5 residues by individually replacing them with each of the other 19 residues. Replacement of Asn(331) yielded transporters with various affinities, with those of the Ile(331), Val(331), and Cys(331) mutants being higher than that of the wild type. Replacement of the Hxt2 residues at the other four sites yielded transporters with affinities similar to that of the wild type but with various capacities. A working homology model of the chimeric transporters containing Asn(331) or its 19 replacement residues indicated that those residues at this site that yield high affinity transporters (Ile(331), Val(331), Cys(331)) face the central cavity and are within van der Waals distances of Phe(208) (TM5), Leu(357) (TM8), and Tyr(427) (TM10). Interactions via these residues of the four TMs, which compose a half of the central pore, may thus play a pivotal role in formation of a core structure for high affinity transport.

Hxt2 and Hxt1 are high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae, respectively, that differ at 75 amino acid positions in their 12 transmembrane segments (TMs). Comprehensive analysis of chimeras of these two proteins has previously revealed that TMs 1, 5, 7, and 8 of Hxt2 are required for high affinity glucose transport activity and that leucine 201 in TM5 is the most important in this regard of the 20 amino acid residues in these regions that differ between Hxt2 and Hxt1. To evaluate the importance of the remaining residues, we systematically shuffled the amino acids at these positions and screened the resulting proteins for high affinity and high capacity glucose transport activity. In addition to leucine 201 (TM5), four residues of Hxt2 (leucine 59 and leucine 61 in TM1, asparagine 331 in TM7, and phenylalanine 366 in TM8) were found to be important for such activity. Furthermore, phenylalanine 198 (TM5), alanine 363 (TM8), and either valine 316 (TM7) or alanine 368 (TM8) were found to be supportive of maximal activity. Construction of a homology model suggested that asparagine 331 interacts directly with the substrate and that the other identified residues may contribute to maintenance of protein conformation.

Chimeras of Hxt2 and Hxt1, high affinity and low affinity glucose transporters, respectively, of Saccharomyces cerevisiae, were previously constructed by random replacement of each of the 12 transmembrane segments (TMs) of Hxt2 with the corresponding region of Hxt1. Characterization of these chimeras revealed that at least TMs 1, 5, 7, and 8 of Hxt2 are required for high affinity transport activity. To determine which amino acid residues in these TMs are important for high affinity glucose transport, we systematically shuffled all of the 20 residues in these regions that differ between Hxt2 and Hxt1. Analysis of 60 independent mutant strains identified as expressing high affinity and high capacity glucose transport activity by selection on glucose-limited agar plates revealed that Leu-201 in TM5 of Hxt2 is most important for such activity and that either Cys-195 or Phe-198 is also required for maximal activity.

Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator superfamily. Hxt1 shares approximately 70% amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports D-glucose with an affinity about one-tenth of that of Hxt2. To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt1 by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters. Among > 20000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced. With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8. We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8. Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2. The K (m) and V (max) values for D-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2. These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2.

The Na(+)/glucose cotransporter gene SGLT1 was analyzed in a Japanese patient with congenital glucose-galactose malabsorption. Genomic DNA was used as a template for amplification by the polymerase chain reaction of each of the 15 exons of SGLT1. The amplification products were cloned and sequenced. About half of the exon 5 clones of the patient contained a C-->T transition, resulting in an Arg(135)-->Trp mutation, whereas the remaining clones contained the normal exon 5 sequence. In addition, whereas some exon 12 clones exhibited the normal sequence, others showed a CAgtaggtatcatc-->CAgacc mutation at the splice donor site of intron 12 that may result either in the skipping of exon 12 or in read-through of intron 12. Neither the Arg(135)-->Trp mutant nor either of the possible intron 12 mutant proteins exhibited Na(+)-dependent glucose transport activity when expressed in Xenopus oocytes. Immunocytochemical analysis indicated, however, that the Arg(135)-->Trp mutant was localized to the oocyte plasma membrane. DNA sequence analysis revealed that the missense mutation in exon 5 and the splice site mutation in intron 12 were inherited from the proband's father and mother, respectively. These results indicate that the patient is a compound heterozygote for this disease, and that the Arg(135)-->Trp mutant of SGLT1 undergoes normal trafficking to the plasma membrane but is non-functional.

This study deals with the cloning and characterization of monosaccharide transporter cDNAs in rice. OsMST1-3 (Oryza sativa monosaccharide transporters 1-3) have two sets of putative six transmembrane domains separated by a central long hydrophilic region. Heterologous expression of OsMST3 in the yeast Saccharomyces cerevisiae indicated that OsMST3 has transport activity for some monosaccharides in an energy-dependent H+ co-transport manner. Northern blot and in situ hybridization analyses showed that OsMST3 mRNA is detectable in leaf blades, leaf sheaths, calli and roots, especially the xylem as well as in sclerenchyma cells in the root. These results suggested that OsMST3 is involved in the accumulation of monosaccharides required for cell wall synthesis at the stage of cell thickening.

Three critical aromatic sites have been identified in the yeast galactose transporter Gal2: Tyr(352) at the extracellular boundary of putative transmembrane segment (TM) 7, Tyr(446) in the middle of TM10 and Phe(504) in the middle of TM12. The relationship between these sites was investigated by random mutagenesis of each combination of two of the three residues. Galactose transport-positive clones selected by plate assays encoded Tyr(446) and specific combinations of aromatic residues at sites 352 and 504. Double-site mutants containing aromatic residues at these latter two positions showed either essentially full galactose transport activity (Phe(352)Trp(504) and Trp(352)Trp(504)) or no significant activity (Phe(352)Tyr(504) and Trp(352)Tyr(504)), whereas single-site mutants showed markedly reduced activity. These results are indicative of a specific interaction between sites 352 and 504 of Gal2.

Tyr(446) in putative transmembrane segment 10 (TM10) of the yeast galactose transporter Gal2 has previously been identified as essential for galactose recognition. In the present study, alignment of the amino acid sequences of 63 sugar transporters or related proteins revealed 14 aromatic sites, including Tyr(446) of Gal2, that are conserved in >75% of these proteins. The importance of the remaining 13 conserved aromatic amino acids was examined individually by random mutagenesis using degenerate primers. Galactose transport-positive clones were identified by plate selection and subjected to DNA sequencing. For those transport-positive clones corresponding to Tyr(352), and Phe(504) mutants, all the amino acid substitutions comprised aromatic residues. The importance of the aromatic residues at these sites was further investigated by replacing them individually with each of the other 19 amino acids and measuring the galactose transport activity of the resulting mutants. Among both Tyr(352) and Phe(504) mutants, the other aromatic amino acids supported galactose transport; no other amino acids conferred high affinity transport activity. Thus, at least three aromatic sites are critical for galactose transport: one at the extracellular boundary of putative TM7 (Tyr(352)), one in the middle of putative TM10 (Tyr(446)), and one in the middle of putative TM12 (Phe(504)).

The comprehensive study of chimeras between the Gal2 galactose transporter and the Hxt2 glucose transporter of Saccharomyces cerevisiae has shown that Tyr446 is essential and Trp455 is important for galactose recognition by Gal2. Consistent with this finding, replacement of the corresponding Phe431 and Tyr440 residues of Hxt2 with Tyr and Trp, respectively, allowed Hxt2 to transport galactose, suggesting that the two amino acid residues in putative transmembrane segment 10 play a definite role in galactose recognition (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721-16724). Replacement of Trp455 of Gal2 with any of the other 19 amino acids was shown to reduce galactose transport activity to between 0 and <20% of that of wild-type Gal2. The role of Phe431 in Hxt2 was similarly studied. Other than Phe, only Tyr at position 431 was able to support glucose transport activity, at the reduced level of <20%. In contrast, replacement of Tyr440 of Hxt2 with other amino acids revealed that most replacements, with the exception of Pro and charged amino acids, supported glucose transport activity. The importance of residue 431 in sugar recognition was more pronounced in a modified Hxt2 in which Tyr440 was replaced with Trp. Glucose transport was supported only by the aromatic amino acids Phe, Tyr, and Trp at position 431, and galactose transport was supported only by Tyr. These results suggest that an aromatic amino acid located in the middle of transmembrane segment 10 (Tyr446 in Gal2 and Phe431 in Hxt2) plays a critical role in substrate recognition in the yeast sugar transporter family to which Gal2 and Hxt2 belong.

The molecular mechanism of substrate recognition in membrane transport is not well understood. Two amino acid residues, Tyr446 and Trp455 in transmembrane segment 10 (TM10), have been shown to be important for galactose recognition by the yeast Gal2 transporter; Tyr446 was found to be essential in that its replacement by any of the other 19 amino acids abolished transport activity (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721-16724). The Glut1 glucose transporter of animal cells belongs to the same Glut transporter family as does Gal2 and thus might be expected to show a similar mechanism of substrate recognition. The role of the two amino acids, Phe379 and Trp388, in rat Glut1 corresponding to Tyr446 and Trp455 of Gal2 was therefore studied. Phe379 and Trp388 were individually replaced with each of the other 19 amino acids, and the mutant Glut1 transporters were expressed in yeast. The expression level of most mutants was similar to that of the wild-type Glut1, as revealed by immunoblot analysis. Glucose transport activity was assessed by reconstituting a crude membrane fraction of the yeast cells in liposomes. No significant glucose transport activity was observed with any of Trp388 mutants, whereas the Phe379 mutants showed reduced or no activity. These results indicate that the two aromatic amino acids in TM10 of Glut1 are important for glucose transport. However, unlike Gal2, the residue at the cytoplasmic end of TM10 (Trp388, corresponding to Trp455 of Gal2), rather than that in the middle of TM10 (Phe379, corresponding to Tyr446 of Gal2), is essential for transport activity.

A novel, systematic approach was used to identify amino acid residues responsible for substrate recognition in the transmembrane 10 region of the Gal2 galactose transporter of Saccharomyces cerevisiae. A mixture of approximately 25,000 distinct plasmids that encode all the combinations of 12 amino acids in transmembrane 10 that are different in Gal2 and the homologous glucose transporter Hxt2 was synthesized. Selection of galactose transport-positive clones on galactose limited agar plates yielded 19 clones, all of which contained the Tyr446 residue found in Gal2. 14 of the 19 clones contained Trp455 found in Gal2, whereas the other 5 contained Cys455, a residue not found in either Gal2 or Hxt2. When Tyr446 of Gal2 was replaced with any of the other 19 amino acids, no galactose transport activity was observed in the resulting transporters, indicating that Tyr446 plays an essential role in the transport of this sugar. Replacement of 2 amino acids of Hxt2 with the corresponding Tyr446 and Trp455 of Gal2 allowed the modified Hxt2 to transport galactose. The Km of galactose transport for the modified transporter was 8-fold higher than that of Gal2. These results and other evidence unequivocally show that Tyr446 is essential and Trp455 is important for the discrimination of galactose versus glucose.

Rat Glut4 glucose transporter was expressed in the yeast Saccharomyces cerevisiae, but was retained in an intracellular membranous compartment and did not contribute to glucose uptake by intact cells. A crude membrane fraction was prepared and reconstituted in liposome with the use of the freeze-thaw/sonication method. D-glucose-specific, cytochalasin B inhibitable glucose transport activity was observed. Kinetic analysis of D-glucose transport was performed by an integrated rate equation approach. The K(m) under zero-trans influx condition was 12 +/- 1 mM (mean +/- S.E., n = 3) and that under equilibrium exchange condition was 22 +/- 3 mM (n = 4). D-glucose transport was inhibited by 2-deoxy-D-glucose or 3-O-methyl-D-glucose, but not by D-allose, D-fructose or L-glucose. Cytochalasin B, phloretin and phlorizin inhibited D-glucose transport, but neither p-chloromercuribenzoic acid (pCMB) (0-0.1 mM) nor p-chloromercuribenzene sulfonic acid (pCMBS) (0-1.0 mM) inhibited this activity. High concentrations of HgCl2 were required to inhibit D-glucose transport (IC50, 370 microM). Comparing these properties to those of rat Glut1 we found two notable differences; (1) in Glut1, K(m) under zero-trans influx was significantly smaller than that under equilibrium exchange but in Glut4 less than two-fold difference was seen between these two K(m) values; and (2) Glut1 was inhibited with pCMB, pCMBS and low concentrations of HgCl2 (IC50, 3.5 microM), whereas Glut4 was almost insensitive to SH reagents. To examine the role of the exofacial cysteine, we replaced Met-455 of Glut4 (corresponding to Cys-429 of Glut1) with cysteine. The mutated Glut4 was inhibited by pCMB or pCMBS and the IC50 of HgCl2 decreased to 47 microM, whereas K(m), substrate specificity and the sensitivity to cytochalasin B were not significantly changed, indicating that the existence of exofacial cysteine contributed only to increase SH sensitivity in Glut4.

A systematic series of chimeras between Ga12 galactose transporter and Hxt2 glucose transporter in yeast was produced to delineate the essential domain for substrate recognition. A domain of 101 amino acids close to the COOH-terminus that has been previously identified as the critical substrate recognition region was further divided into four subdomains, by introducing five restriction enzyme sites at exactly corresponding locations of both genes without changing coding amino acids. When each of all possible 16 modified genes was expressed, all the galactose transport-active chimeras were found to possess Ga12-derived transmembrane segment (TM) 10. Of the 35 amino acids in the TM1O region, only 12 differ between Ga12 and Hxt2, indicating that these 12 amino acids include the critical residue(s) responsible for the differential recognition of galactose and glucose in these transporters.

We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze-thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4 +/- 0.2 mM (mean +/- S.E.M.) and was inhibited by cytochalasin B (IC50= 0.44 +/- 0.03 microM), HgCl2 (IC50)= 3.5 +/- 0.5 microM), phloretin (IC50= 49 +/- 12 microM) and phloridzin (IC50= 355 +/- 67 microM). To compare these properties with native GLUT1 we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.

The Gal2 galactose transporter takes up galactose in yeast. A homologous glucose transporter from the same organism, Hxt2, was selected, and various chimeras between these two transporters were constructed by making use of homologous recombination in Escherichia coli. Comparison of the galactose transport activities of three series of chimeras enabled us to positively identify a crucial substrate recognition region of 101 amino acids that lies close to the carboxyl terminus of the Gal2 transporter.

GLUT1 is an isoform of facilitated-diffusion glucose transporters and has been shown to be abundant in cells of blood-tissue barriers. Using antibodies against GLUT1, we investigated the immunohistochemical localization of GLUT1 in the rat placenta. Rat placenta is of the hemotrichorial type. Three cell layers (from the maternal blood side inward) cytotrophoblast and syncytiotrophoblasts I and II, lie between the maternal and fetal bloodstreams. GLUT1 was abundant along the invaginating plasma membrane facing the cytotrophoblast and the syncytiotrophoblast I. Also, the infolded basal plasma membrane of the syncytiotrophoblast II was rich in GLUT1. Apposing plasma membranes of syncytiotrophoblasts I and II, however, had only a small amount of GLUT1. Numerous gap junctions were seen between syncytiotrophoblasts I and II. Taking into account the localization of GLUT1 and the gap junctions, we suggest a possible major transport route of glucose across the placental barrier, as follows: glucose in the maternal blood passes freely through pores of the cytotrophoblast. Glucose is then transported into the cytoplasm of the syncytiotrophoblast I via GLUT1. Glucose enters the syncytiotrophoblast II through the gap junctions. Finally glucose leaves the syncytiotrophoblast II via GLUT1 and enters the fetal blood through pores of the endothelial cells.

The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.

The blood-retinal barrier is part of the blood-ocular barrier. Retinal pigment epithelial cells connected by tight junctions serve as an outer blood-retinal barrier, and the nonfenestrated endothelial cells of blood vessels sealed by tight junctions serve as an inner blood-retinal barrier. Using antibodies specific for the erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of facilitated-diffusion glucose transporters, it was found, by ultrastructural cytochemical examination, that GLUT1 in the rat was localized at both the apical and basolateral plasma membranes of retinal pigment epithelial cells. The fenestrated endothelial cells of the underlying choriocapillaries were negative for GLUT1. In the inner retina, GLUT1 was found at both the luminal and contraluminal plasma membranes of endothelial cells. These observations show that GLUT1 is concentrated at the critical plasma membranes of the blood-retinal barrier and may serve as the machinery for glucose transport across the barrier.

Glucose is actively absorbed via a Na(+)-dependent active glucose transporter (Na-GT) in the small intestine. We raised a polyclonal antibody against the peptide corresponding to amino acids 564-575 of rabbit intestinal Na-GT, and localized it immunohistochemically in the rat jejunum. By means of immunofluorescence staining, Na-GT was located at the brush border of the absorptive epithelial cells of the intestinal villi. Electron-microscopic examination showed that Na-GT was localized at the plasma membrane of the apical microvilli of these cells. Little Na-GT was found at the basolateral plasma membrane. Along the crypt-villus axis, all of the absorptive epithelial cells in the villus were positive for Na-GT. In addition to the brush border staining, the supranuclear positive staining, which was shown to be the Golgi apparatus by use of electron microscopy, was seen in cells located between the base to the middle of the villus. Cells in crypts exhibited little or no staining for Na-GT. Goblet cells scattered in the intestinal epithelium were negative for Na-GT staining. These observations show that Na-GT is specific to the apical plasma membrane of the absorptive epithelial cells, and that the onset of Na-GT synthesis may occur near the crypt-villus junction.

Aqueous humor, with its unique low concentration of proteins, is produced by the ciliary body and isolated by the blood-aqueous barrier from the body fluid. Glucose in aqueous humor is a major source of nutrients for lens and corneal cells, and is maintained near the plasma level, suggesting a specific glucose transport mechanism in the blood-aqueous barrier. Using antibodies against erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated diffusion glucose transporters, the authors found immunocytochemically that GLUT1 localizes in the epithelial cells of ciliary body and iris. GLUT1 is also found in the endothelial cells of blood vessels in the iris, whereas no labeling is seen in the blood vessels in the ciliary body. In the ciliary body epithelium, the plasma membranes of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells are positive for GLUT1. By the colloidal gold particle counting, the basal infoldings of PE cells show approximately two-fold denser labeling than those of NPE cells. Since PE and NPE cells make up a functional syncytium with numerous gap junctions, the authors suggest that glucose transport in the ciliary body occurs in this manner: glucose diffuses out from blood vessels through the pores of fenestrated endothelial cells, is transported into PE cells by GLUT1 in their plasma membrane, enters NPE cells through gap junctions connecting PE and NPE cells, and is finally transported into the aqueous humor by GLUT1 of NPE cells. The higher density of GLUT1 in PE cells may account for the consumption of glucose by PE and NPE cells in addition to the transepithelial transport.

Glucose is actively taken up from the glomerular filtrate into the tubule cells by the Na(+)-dependent active glucose transporter (GT), and passively crosses the basolateral membrane via facilitated diffusion GT. With the use of antibodies directed against two types of GTs, we show the immunocytochemical localization of the Na(+)-dependent active GT (SGLT1) and the erythrocyte/HepG2-type facilitated diffusion GT (GLUT1). For light microscopic observation, frozen sections were stained by the rhodamine labeling method. Counterstaining with fluorescein-phalloidin and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was employed to facilitate cell type identification. Immunogold staining was carried out on ultra-thin frozen sections for electron microscopy. The antibody to SGLT1 reacted with a 77 KD protein in immunoblotting of a kidney lysate. By immunocytochemistry, SGLT1 was localized in the microvillous plasma membrane in the apical brush borders of the cells of all three proximal tubule segments (S1, S2, and S3). The antibodies to GLUT1, a member of the facilitated diffusion GT family, were raised against human erythrocyte GT or synthetic oligopeptides derived from HepG2 GT, which reacted with a 48 KD protein in immunoblotting of the kidney lysate. GLUT1 was found at the basolateral plasma membranes of S3 proximal tubule cells, cells of the thick limb of Henle's loop, and collecting duct cells. Combined with known physiological data, our findings suggest that SGLT1 in the apical plasma membrane of the proximal tubule cells is responsible for the Na(+)-dependent active reabsorption of glucose from the glomerular filtrate. GLUT1 in the basolateral plasma membrane of S3 cells may transport reabsorbed glucose to the blood vessels. GLUT1 in the basolateral plasma membranes of cells of the thick limb of Henle's loop and of the collecting duct, on the other hand, may nourish these metabolically active cells by facilitating the diffusion of extracellular glucose provided from blood through the basolateral side of the cells.

In search of possible diverse roles of glucose transporters (GT's), we examined whether any GT's are present in blood-tissue barriers where selective flow of glucose from blood to tissue cells is critically important. We found in rat that the erythrocyte/HepG2-type GT is localized in all the limiting plasma membranes known to serve as blood-tissue barriers, whether the barriers are endothelial type (brain, iris, inner retina, peripheral nerve) or epithelial type (choroid plexus, ciliary body, outer retina, peripheral nerve, placenta), except for plasma membranes in testis and thymus where no appreciable amount of the GT was found. The erythrocyte/HepG2-type GT may play a vital role for the entry of glucose into these firmly guarded tissues.

The asparagine-linked sugar chain of glucose transporter from human erythrocytes was quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted to radioactive oligosaccharides by NaB3H4 reduction after N-acetylation and fractionated by anion-exchange column chromatography and Bio-Gel P-4 column chromatography after sialidase treatment. Structural study of each oligosaccharide by exo- and endoglycosidase digestion and methylation analysis indicated that the glycoprotein contains a high-mannose-type oligosaccharide, Man9.GlcNAc.GlcNAc, and biantennary complex-type oligosaccharides with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3) Man beta beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores and the poly-N-acetyllactosamine composed of about 16 N-acetyllactosaminyl units as their outer chains. These structural features of the sugar moiety of glucose transporter are quite different from those of two major intrinsic glycoproteins of human erythrocytes, glycophorin A and band 3.

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.

Microsomal vesicles prepared from rat adipocytes were immuno-adsorbed to formaldehyde-fixed Staphylococcus aureus cells (Pansorbin) coated with anti-human-erythrocyte-glucose-transporter IgG. More than 75% of the glucose transporter detected was precipitated. The glucose transporter was about 10-fold enriched by the adsorption procedure. On insulin treatment, the insulin receptor in plasma membranes was internalized and the receptor in the microsome fraction increased 5-fold. Thirty-five % of the insulin receptor in the microsome fraction was recovered with the glucose-transporter-containing vesicles. These observations indicate that on insulin treatment a considerable portion of the microsome vesicles containing the insulin receptor fuses or becomes tightly associated with ones containing the glucose transporter.

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44-55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.

The notion of an insulin-dependent translocation of the glucose transporter in rat adipocytes was confirmed by immunoblotting and reconstitution of glucose transport activity of subcellular fractions. Quantitatively, however, significantly different results were obtained with these two techniques; when compared with reconstitution, immunoblotting detected translocation of a larger amount of the transporter from a low density microsome fraction to a plasma membrane fraction. The acidtropic agents chloroquine and dibucaine, which have been reported to inhibit the recycling of various receptors, were utilized to study the detailed translocation mechanism of the glucose transporter and the insulin receptor. These acidtropic agents caused accumulation of 125I-insulin in a subcellular fraction probably corresponding to lysosomes. They did not, however, significantly affect either the insulin-induced activation of glucose transport or the recycling of the transporter and the insulin receptor as detected by immunoblotting. About 50% of radioactivity released from adipocytes which were allowed to internalize insulin was due to intact insulin, and chloroquine did not change the release rate of intact insulin, raising the possibility of receptor-mediated exocytosis of insulin. The release of degraded insulin decreased with chloroquine treatment. The results are consistent with the idea that these acidtropic agents mainly act to inhibit degradation of insulin in lysosomes, and their effect on the recycling of the glucose transporter and the insulin receptor is minimal, indicating that the recycling of these membrane proteins proceeds irrespective of organelle acidification. Electron micrographs showed vesicles underneath the plasma membranes, with sizes similar to those of the low density microsome fraction where the internalized glucose transporter and the insulin receptor were located.