GTP analogs suppress uptake but not transport of d-glucose analogs in Glut1 glucose transporter-expressing Xenopus oocytes

AbstractA Xenopus oocyte expression-co-injection system was used to study the influence of guanine nucleotides on D-glucose uptake. GTP analogs like GTPγS and GppNHp had no effect on 3-O-methylglucose transport determined by zero-trans uptake or equilibrium exchange, but suppressed 2-deoxyglucose uptake into Glutl glucose transporter-expressing oocytes by up to 86%. Both GTP analogs showed concentration dependence of their effectiveness, with GTPγS being more potent than GppNHp. No statistically significant differences were observed between groups of oocytes co-injected with water or GDPβS (250 and 500 μM intracellular concentration). Glut1 transporter expression in plasma membrane was not different between water or GTPγS-co-injected oocytes. Thus, inhibition of hexokinase catalytic activity is the most likely causative factor for down-regulation of 2-deoxyglucose uptake

Ligand-Induced Movements of Inner Transmembrane Helices of Glut1 Revealed by Chemical Cross-Linking of Di-Cysteine Mutants

The relative orientation and proximity of the pseudo-symmetrical inner transmembrane helical pairs 5/8 and 2/11 of Glut1 were analyzed by chemical cross-linking of di-cysteine mutants. Thirteen functional di-cysteine mutants were created from a C-less Glut1 reporter construct containing cysteine substitutions in helices 5 and 8 or helices 2 and 11. The mutants were expressed in Xenopus oocytes and the sensitivity of each mutant to intramolecular cross-linking by two homobifunctional thiol-specific reagents was ascertained by protease cleavage followed by immunoblot analysis. Five of 9 mutants with cysteine residues predicted to lie in close proximity to each other were susceptible to cross-linking by one or both reagents. None of 4 mutants with cysteine substitutions predicted to lie on opposite faces of their respective helices was susceptible to cross-linking. Additionally, the cross-linking of a di-cysteine pair (A70C/M420C, helices 2/11) predicted to lie near the exoplasmic face of the membrane was stimulated by ethylidene glucose, a non-transported glucose analog that preferentially binds to the exofacial substrate-binding site, suggesting that the binding of this ligand stimulates the closure of helices at the exoplasmic face of the membrane. In contrast, the cross-linking of a second di-cysteine pair (T158C/L325, helices 5/8), predicted to lie near the cytoplasmic face of the membrane, was stimulated by cytochalasin B, a glucose transport inhibitor that competitively inhibits substrate efflux, suggesting that this compound recruits the transporter to a conformational state in which closure of inner helices occurs at the cytoplasmic face of the membrane. This observation provides a structural explanation for the competitive inhibition of substrate efflux by cytochalasin B. These data indicate that the binding of competitive inhibitors of glucose efflux or influx induce occluded states in the transporter in which substrate is excluded from the exofacial or endofacial binding site

Side-view of the orientation of helices 2/11 and 5/8 of Glut1 in the endofacial conformation based on homology modeling.

<p>The orientation of the helices is derived from an homology-based model of Glut1 that used the structure of the E. coli Glycerol-3-P Antiporter as the template molecule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#pone.0031412-Huang1" target="_blank">[17]</a>. Side chains of residues that were mutated to cysteines and subjected to chemical cross-linking analysis are identified by their single letter amino acid codes. Residues that exhibited cross-linking are shown in green in ball and stick form and are connected by dotted lines. Distances between cross-linked residues are given in angstroms (Å). Residues that did not exhibit cross-linking are shown in red.</p

Expression of dicysteine mutants in Xenopus oocyte membranes.

<p>Stage 5 <i>Xenopus</i> oocytes were injected with water (Sham) or with 50 ng of mRNA encoding the parental reporter construct (C-Tev) or the indicated dicysteine mutant. Two days post injection total oocyte membranes were prepared and subjected to immunoblot analysis using a rabbit polyclonal ab raised against a peptide corresponding to the C-terminal 15 residues of human Glut1.</p

Chemical Cross-linking of di-C Mutants.

<p>Stage 5 <i>Xenopus</i> oocytes were injected with 50 ng of mRNA encoding the parental reporter construct (C-Tev) or the indicated dicysteine mutants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#pone-0031412-t001" target="_blank">Table 1</a>). After incubation of oocytes for 2 days, cross-linking analysis was conducted on purified oocyte membranes as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#s4" target="_blank">Materials and Methods</a>”. The reactions were quenched by the addition of 2 mM cysteine and oocyte membranes were digested with Tev protease then subjected to SDS-PAGE followed by immunoblotting with rabbit polylclonal ab raised against the C-terminal 15 residues of human Glut1 (red bands) and a mouse monoclonal ab that recognizes an epitope in the N-terminal half of the central cytoplasmic loop (green bands). Note that the full-length ∼54 kD bands were recognized by both antibodies and show up as yellow when the intensity of the detector was increased. “C-TEV” is the control cysteine-less parental construct. “Control” lanes were loaded with membranes that were not subjected to either chemical cross-linking or protease cleavage. “DMSO” lanes were loaded with membranes that were not subjected to chemical cross-linking but were digested with TEV protease. The “o-PDM” and “BMH” lanes were loaded with membranes that were subjected to cross-linking by the respective chemical and then were treated with TEV protease. The ratio of the intensities of the full-length bands in the DMSO lanes to those in the Control lanes thus provide the maximum level of protease cleavage for each mutant. The ratio of the intensities of the full-length bands in the “o-PDM” or ‘BMH” lanes to those in the Control lanes indicate the extent of cross-linking by either reagent. This ratio is termed the cross-linking efficiency or “fraction cross-linked” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031412#pone-0031412-t002" target="_blank">Table 2</a>.</p

Construction of di-cysteine mutants in a C-Less GLUT 1 reporter molecule.

<p>An aglyco Glut1 reporter molecule was created by mutating the consensus site of N-linked glycosylation at N45 to a threonine residue and by inserting a Tev protease cleavage site (ENLYFQG) between residues 247 and 248 in the central cytoplasmic loop of Glut1. This reporter construct was then used to make the above 13 dicysteine mutants in helices 2/11 and helices 5/8.</p