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SVCT2 (sodium-vitamin C co-transporter 2) is the major transporter mediating vitamin C uptake in most organs. Its expression is driven by two promoters (CpG-poor exon 1a promoter and CpG-rich exon 1b promoter). In the present study, we mapped discrete elements within the proximal CpG-poor promoter responsible for exon 1a transcription. We identified two E boxes for USF (upstream stimulating factor) binding and one Y box for NF-Y (nuclear factor Y) binding. We show further that NF-Y and USF bind to the exon 1a promoter in a co-operative manner, amplifying the binding of each to the promoter, and is absolutely required for the full activity of the exon 1a promoter. The analysis of the CpG site located at the upstream USF-binding site in the promoter showed a strong correlation between expression and demethylation. It was also shown that exon 1a transcription was induced in cell culture treated with the demethylating agent decitabine. The specific methylation of this CpG site impaired both the binding of USF and the formation of the functional NF-Y-USF complex as well as promoter activity, suggesting its importance for cell-specific transcription. Thus CpG methylation at the upstream USF-binding site functions in establishing and maintaining cell-specific transcription from the CpG-poor SVCT2 exon 1a promoter.

In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24 h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426-1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and D-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15+/-4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.

The transport conformation of the human erythrocyte glucose transporter (GLUT1) modifies rates of proteolytic cleavage of this protein by a variety of enzymes. We investigated the effects of ligand-induced conformational change on the susceptibility to enzymic cleavage of the insulin-sensitive rat adipocyte glucose transporter (GLUT4). A GLUT4-enriched slow sedimenting microsomal fraction was prepared from basal adipocytes and subjected to PAGE and immunoblotting. The GLUT4 protein was detected in these immunoblots with a C-terminal-specific antiserum as an M(r)-46,000-50,000 doublet. GLUT1 protein was not detected by a GLUT1-specific antiserum in these membranes. Tryptic digestion caused loss of the GLUT4 signal in immunoblots in a time- and concentration-dependent fashion. Low-M(r) membrane-bound fragments were not observed in electrophoretic gels, whether detection was attempted by immunoblotting or by counting radioactivity in gel slices following photolabelling with [3H]cytochalasin B. Transport-specific ligands known to induce an outward-facing conformation in the human erythrocyte GLUT1 protein retarded cleavage of the GLUT4 protein by submaximal concentrations of trypsin, whereas ligands known to induce an inward-facing conformation increased the extent of cleavage. The transported substrate D-glucose retarded tryptic cleavage of GLUT4. This result contrasts with the known behaviour of GLUT1, in which D-glucose accelerates cleavage. Cleavage of GLUT4 by thermolysin was also retarded by the outward-binding analogue 4,6-O-ethylidene glucose. These results show that the conformational sensitivity to proteolysis of GLUT4 mirrors that of GLUT1, except that the glucose-loaded GLUT4 has a different steady-state configuration, which may reflect underlying kinetic differences between the two proteins.

Maltose-maleimide was synthesized as a potential affinity label for the facilitative hexose carrier with selectivity for exofacial sulphydryl groups. This reagent, although probably a mixture of isomers, did not significantly penetrate the plasma membrane of human erythrocytes at concentrations below 5 mM at 37 degrees C. When allowed to react to completion, it irreversibly inhibited the uptake of 3-O-methylglucose, with a half-maximal response at about 1.5-2.0 mM-reagent. The rate of transport inactivation was a saturable function of the maltose-maleimide concentration. Studies of reaction kinetics and effects of known transport inhibitors demonstrated that irreversible reaction occurred on the exofacial outward-facing carrier, although not at a site involved in substrate binding. Reaction of intact erythrocytes with [14C]maltose-maleimide resulted in labelling of a broad band 4.5 protein of Mr (average) 45,000-66,000 in electrophoretic gels. This protein was very likely the hexose carrier, since its labelling was inhibited by cytochalasin B. Exofacial band 4.5 labelling was stoichiometric with respect to transport inhibition, yielding an estimated 300,000 carriers/cell. These results suggest that the exofacial sulphydryl which reacts with maltose-maleimide is distinct from the substrate binding site on the hexose carrier, but that it confers substantial labelling selectivity to impermeant maleimides. Additionally, the high efficiency of carrier labelling obtained with maltose-maleimide is useful in quantifying numbers of carriers in whole cells.

S-(Bismaleimidomethyl ether)cysteine (Cys-Mal) was synthesized as a probe for reactive thiol groups on the erythrocyte glucose carrier. Although Cys-Mal entered cells, its reaction with intracellular GSH prevented alkylation of endofacial membrane proteins, limiting its effect to the cell surface at concentrations below 5 mM. Cys-Mal irreversibly inhibited hexose transport half-maximally at 1.5 mM by decreasing the maximal rate of transport, with no effect on the affinity of substrate for the carrier. Reaction occurred with the outward-facing form of the carrier, but did not affect the ability of the carrier to change orientation. In intact cells, several exofacial proteins were labelled by [35S]Cys-Mal, including the band-4.5 glucose carrier, the labelling of which occurred on a single site sensitive to transport inhibitors. The reactive exofacial group was a thiol group, since both transport inhibition and band-4.5 labelling by Cys-Mal were abolished by the thiol-specific and impermeant compound 5,5'-dithiobis(2-nitrobenzoic acid). Selectivity for carrier labelling in cells was increased by a double differential procedure, which in turn allowed localization of the exofacial thiol group to the Mr 18,000-20,000 membrane-bound tryptic carrier fragment. In protein-depleted ghosts the exofacial thiol group was preferentially labelled at low concentrations of [35S]Cys-Mal, whereas with the reagent at 10 mM the Mr 26,000-45,000 tryptic carrier fragment was also labelled. Cys-Mal should be useful in the study of carrier thiol-group location and function.