Abstract
The transport of glucose across the plasma membrane is mediated by members of the glucose transporter family. In this study, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt1) by measuring incorporation of 2-NBDG, a non-metabolizable, fluorescent glucose analog, into the yeast Saccharomyces cerevisiae. We find that 2-NBDG is not incorporated into the hxt null strain lacking all glucose transporter genes and that this defect is rescued by expression of wild type Hxt1, but not of Hxt1 with mutations at the putative glucose-binding residues, inferred from the alignment of yeast and human glucose transporter sequences. Similarly, the growth defect of the hxt null strain on glucose is fully complemented by expression of wild type Hxt1, but not of the mutant Hxt1 proteins. Thus, 2-NBDG, like glucose, is likely to be transported into the yeast cells through the glucose transport system. Hxt1 is internalized and targeted to the vacuole for degradation in response to glucose starvation. Among the mutant Hxt1 proteins, Hxt1N370A and HXT1W473A are resistant to such degradation. Hxt1N370A, in particular, is able to neither uptake 2-NBDG nor restore the growth defect of the hxt null strain on glucose. These results demonstrate 2-NBDG as a fluorescent probe for glucose uptake in the yeast cells and identify N370 as a critical residue for the stability and function of Hxt1.
Highlights
Metastasized tumor cells metabolize large amounts of glucose through glycolysis and produce copious amounts of lactic acid even in the presence of oxygen [1,2]
Previous evidence suggests that Q161, Q282 and W412 of Glut1 may be involved in glucose-binding
Substitution of an asparagine at Q161significantly reduces the affinity for the substrate-binding site of a nontransported glucose analog, suggesting that this residue is critical for transport activity and exofacial ligand binding [44]
Summary
Metastasized tumor cells metabolize large amounts of glucose through glycolysis and produce copious amounts of lactic acid even in the presence of oxygen [1,2]. This phenomenon, termed the Warburg effect, is a hallmark of cancer [3]. In this study, using 2-NBDG, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt). Our results show that Hxt transports 2-NBDG in a mechanism similar to Gluts and, that some of the putative glucose-binding residues of Hxt are involved in endocytosis. Discussed is the possible roles of these residues in the stability and function of Hxt
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