Abstract
Human GLUT2 and GLUT3, members of the GLUT/SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.
Highlights
Transport of hexoses across plasma membranes marks the first and rate-limiting step of energy metabolism in cells of all domains of life
Amplification of the open reading frame (ORF) with oligonucleotides having 30-40 base pair overhangs to the applied promoter (HXT7-1–329) or terminator (CYC1) region, respectively, and co-transformation with the linearized host plasmid (p426H7) in EBY.VW4000, EBY.S7, and SDY.022 cells allowed for the plasmid assembly via homologous recombination (Oldenburg et al, 1997; Boles and Oreb, 2018)
Re-transformation of the native GLUT3 and the modified GLUT3S66Y construct in EBY.S7 cells resulted in growth on glucose medium, proving that no further mutations, except for the fgy1 mutation, in the yeast strain were essential for this phenotype
Summary
Transport of hexoses across plasma membranes marks the first and rate-limiting step of energy metabolism in cells of all domains of life. GLUT2 is the primary GLUT isoform found in the liver It mediates glucose transport in the kidney, intestine, pancreatic β-cells and the central nervous system (Fukumoto et al, 1988; Thorens, 2015). It exhibits a low affinity for glucose (KM ∼17 mM (Uldry et al, 2002)) and even lower for fructose, galactose, and mannose (KM ∼76, ∼92, and ∼125 mM, respectively (Mueckler and Thorens, 2013)). In murine pancreatic β-cells, GLUT2 mediates glucose stimulated insulin secretion, thereby regulating blood glucose levels. The absence of this function impairs glucose homeostasis leading to diabetes (Ohtsubo et al, 2005). Loss of GLUT2 function causes the Fanconi Bickel Syndrome (Santer et al, 1997), a rare autosomal disease with various symptoms like hepatomegaly, tubular nephropathy, glucose and galactose intolerance, fasting hypoglycemia, rickets, and retarded growth (Santer et al, 2002)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
