title: Assessing glucose uptake through the yeast hexose transporter 1 (Hxt1).
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.
authors: Department of Biochemistry and Molecular Medicine, The George Washington University School of Medicine and Health Science, 2300 Eye Street, NW, Washington, D. C., 20037, United States of America.
authors: Adhiraj
authors: A
authors: Roy
authors: Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, 1015 Life Science Circle, Blacksburg, Virginia 24061, United States of America.
authors: Angela D
authors: AD
authors: Dement
authors: Department of Biology, Indiana State University, 200N 7th St, Terre Haute, Indiana 47809, United States of America.
authors: Kyu Hong
authors: KH
authors: Cho
authors: Department of Biochemistry and Molecular Medicine, The George Washington University School of Medicine and Health Science, 2300 Eye Street, NW, Washington, D. C., 20037, United States of America.
authors: Jeong-Ho
authors: JH
authors: Kim
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D003847:Deoxyglucose / Q000031:analogs & derivatives / Q000378:metabolism; D004795:Enzyme Stability; D005947:Glucose / Q000378:metabolism*; D051246:Glucose Transport Proteins, Facilitative / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051272:Glucose Transporter Type 1 / Q000378:metabolism; D006801:Humans; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D051246:Glucose Transport Proteins, Facilitative; D051272:Glucose Transporter Type 1; C090594:HXT1 protein, S cerevisiae; C495677:SLC2A1 protein, human; D029701:Saccharomyces cerevisiae Proteins; D003847:Deoxyglucose; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose