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. 2021 Feb 18:7:598419.
doi: 10.3389/fmolb.2020.598419. eCollection 2020.

Functional Expression of the Human Glucose Transporters GLUT2 and GLUT3 in Yeast Offers Novel Screening Systems for GLUT-Targeting Drugs

Sina Schmidl  1 , Sebastian A Tamayo Rojas  1 , Cristina V Iancu  2 , Jun-Yong Choe  2   3 , Mislav Oreb  1
Affiliations

Functional Expression of the Human Glucose Transporters GLUT2 and GLUT3 in Yeast Offers Novel Screening Systems for GLUT-Targeting Drugs

Sina Schmidl et al. Front Mol Biosci. .

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.

Keywords: GLUT2; GLUT3; Glucose transport inhibitor; drug screening system; hxt0 yeast strain.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Growth of hxt0 cells expressing GLUT3 or GLUT3S66Y on glucose. Cell growth of EBY.S7 (A) or EBY.VW4000 (B) cells in the YEP medium containing 200 µg/ml G418 and 0.2% (w/v) glucose was recorded with the Cell Growth Quantifier (Aquila Biolabs). The cells were transformed with the empty pRS62K vector, pRS62K vector or with plasmids encoding the endogenous Hxt1 the endogenous Hxt1 transporter, GLUT1, GLUT3, or GLUT3S66Y. The average of three biological replicates (continuous lines) and the error (dashed lines) are shown. Apparent maximal growth rates for the growing cells were calculated with the CGQuant software and are listed together with the duration of lag phases in Supplementary Table S5.
FIGURE 2
FIGURE 2
The structural model for GLUT2, GLUT3, and GLUT2 constructs. (A) The structural model was generated in MOE, based on the crystal structure of the outward-facing conformation of human GLUT3 (PDB ID 4ZWC). The color code represents the sequence homology between GLUT2 and GLUT3, ranging from identical residues (dark blue) to very dissimilar substitutions (red). Unique to GLUT2 is the extension in the loop between transmembrane helices H1 and H2, in the extracellular space (shown in red). (B) GLUT2 and GLUT3 sequence alignment in the region flanking the extended extracellular loop of GLUT2 (in red, underlined). The loop deletion constructs of GLUT2 are: GLUT2Δloop, which lacks residues 55-89, GLUT2ΔloopS, which lacks residues 55-88 and has the mutation A89S, and GLUT2K54S_Δloop, which lacks residues 55-89 and has the mutation K54S. For the two latter constructs, the single substitutions to serine are in analogy to S55 of GLUT3 (black bold underlined). The red curved line in the constructs shows the loop excision site. For the GLUT2 constructs, the number for both the original (bold black) and post-deletion (bold red) sequences are shown.
FIGURE 3
FIGURE 3
Growth of hxt0 cells expressing GLUT2 variants on hexoses. Cell growth of EBY.S7 (A,C) or EBY.VW4000 (B) cells containing the empty pRS62K vector, the endogenous Hxt1 transporter (only (A),(B)), GLUT1 (only (A),(B)), GLUT2, GLUT2ΔloopS or GLUT2ΔloopS_Q455R, was recorded with the Cell Growth Quantifier (Aquila Biolabs). The YEP medium was supplemented with 200 μg/ml G418 for plasmid selection and 0.2% (w/v) glucose (A,B) or 2% (w/v) fructose (C). The average of three biological replicates (continuous lines) and the error (dashed lines) are shown. Apparent maximal growth rates for the growing cells were calculated with the CGQuant software and are listed together with the duration of lag phases in Supplementary Table S5.
FIGURE 4
FIGURE 4
Subcellular localization of GLUT2 variants. envyGFP was fused to the C-termini of GLUT2 (wild-type), GLUT2ΔloopS, and GLUT2ΔloopS_Q455R. CEN.PK2-1C cells, expressing the respective envyGFP construct, were grown in low fluorescent SC -URA medium containing 0.2% (w/v) glucose. For immobilization, 0.6% (w/v) low melt agarose was added to the suspension. Localization was analyzed with the Confocal Laser Scanning Microscope (Zeiss LSM 780, Jena, Germany).
FIGURE 5
FIGURE 5
Effect of phloretin and quercetin on GLUT2∆loopS_Q455R and GLUT3S66Y expressed in hxt0 EBY.S7 cells. The relative transport activity (expressed as % of the transport in the absence of the inhibitor) of GLUT2∆loopS_Q455R (A,B) and GLUT3S66Y (D,E) as a function of phloretin (A,D) or quercetin (B,E) concentrations at 10 mM (for GLUT2∆loopS_Q455R, (A,B)) or 1 mM (for GLUT3S66Y, (D,E)) glucose is shown. Error bars represent the standard deviation from three independent measurements. Growth assays with GLUT2∆loopS_Q455R (C) or GLUT3S66Y (F) expressing EBY.S7 cells on YEP medium supplemented with 0.2% (w/v) glucose and G418 (200 μg/ml) in the presence of 50 μM phloretin (light blue) or its absence (dark blue) were recorded with the Cell Growth Quantifier (Aquila Biolabs). The average of two biological replicates (continuous lines) and the error (dashed lines) are shown. Apparent maximal growth rates of the here-depicted experiments, and EBY.S7 cells expressing Hxt1 with and without phloretin as a control, were calculated with the CGQuant software and are listed together with the duration of lag phases in Supplementary Table S6.
FIGURE 6
FIGURE 6
Structural models of GLUT2 and GLUT3 variants with functional transporter expression in hxt0 cells. Inward-facing (A,C,E) and outward-facing (B,D,F) conformations of GLUT2 and GLUT3 based on the crystal structures for the corresponding conformations of GLUT1 (PDB ID 4PYP, inward-facing) or GLUT3 (PDB ID 5C65, outward-facing), showing the locations of the single-site mutations that increased the transport activity of GLUT2loop and GLUT3 expressed in hxt0 yeast cells: S66 in H2 of GLUT3 and Q455 in H11 of GLUT2loop. The transporter N- and C-halves are in magenta and gold, respectively, and the transmembrane helices are labeled as H1-H12. (C,D) Close-up views of (A,B), respectively, focused on Q455R in GLUT2loop, showing the original side-chain (magenta), and the mutant (yellow). (E,F) Close-up views of (A,B), respectively, focused on S66Y in GLUT3. The mutant side chain is shown in yellow. The extracellular loop between helices H1 and H2 (residues 35-54, shown in blue) is close to the mutated side chain Y66 in the outward-facing conformation (F). Compared with the inward-facing conformation, in the outward-facing conformation, the loop spanning residues 35-54 moves at least 1 Å in the direction indicated by the red arrow. Figures were drawn using Open-Source PyMol Version 2.3.0 (The PyMOL Molecular Graphics System, Version 2.3.0 Schrödinger, LLC).
FIGURE 7
FIGURE 7
Mapping the mutations that render different GLUTs active in hxt0 yeast cells. (A,B) The structural model of the outward-facing conformation for GLUT1-3 and GLUT5 showing the single-site mutations that provide or increase transport activity of the respective human GLUT upon expression in hxt0 yeast cells, viewed from the extracellular side. The mutants are generally substitutions to bulkier side chains and fall into two categories. (A) Mutants that disrupt the packing between the N- and C-domains (colored as cyan and wheat, respectively), such as S72I and S76Y of GLUT5, Q455R of GLUT2, and V69M of GLUT1. (B) Mutants that dislocate the loop between TM helices TM1 and TM2 (the part of the loop that needs to move to accommodate the mutations is shown in blue), causing it to move away from the central cavity, such as S66Y of GLUT3 and W65R of GLUT1.
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