Fig 1.
2-NBDG is incorporated into S. cerevisiae through the hexose transporter Hxt1.
(A) Yeast cells (hxtΔ) expressing either empty vector or Hxt1-HA were first grown in SC-5% glycerol + 2% ethanol medium to mid log phase and shifted to the same medium containing 60 μM of 2-NBDG and incubated for 30 min. Fluorescence microscopy images (left panel) and quantification of relative 2-NBDG fluorescence (right panel, **P < 0.001) were shown. (B) Yeast cells (hxtΔ) expressing Hxt1-HA were grown as described in A. For glucose competition assay, glucose was added to different concentrations (mM) as shown and 2-NBDG uptake was analyzed by fluorescence microscopy. (C) Quantification of relative 2-NBDG uptake in glucose competition assay was shown. FL: Fluorescence, DIC: Differential Interference Contrast.
Fig 2.
The yeast Hxt1 glucose transporter transports glucose in a similar mechanism as the human glucose transporters.
(A) Cartoon for the arrangement of the 12 transmembrane helices and the proposed model of the exofacial glucose-binding sites of Hxt1 protein as viewed from the outside of the cell. For the sake of simplicity, all transmembrane helices were drawn as perfect helices perpendicular to the plane of the membrane. Amino acid residues were grouped according to their chemical properties of side chains (red: neutral polar, purple: acidic or basic polar, green: neutral nonpolar and blue: hydrophobic). Putative glucose-binding residues are shown. (B) Sequence alignment of segments of glucose transporters of yeast (Hxt1-7 and Gal2) and human (Glut1-5) showing proposed amino acids (highlighted in box) predicted to interact with glucose. (C) Western blot analysis of Hxt1-HA levels at the plasma membrane. Yeast cells (WT) expressing indicated Hxt1-HA proteins were grown in SC-2% glucose (Glu) medium to mid log phase and shifted to SC medium containing 2% galactose (Gal) for 6 hr. Membrane fractions were analyzed using anti-HA antibody. Actin was served as loading control. (D) Yeast cells (hxtΔ) expressing either empty vector or indicated Hxt1-HA proteins were spotted on 2% glucose plate supplemented with Antimycin-A (1μg/ml). The first spot of each row represents a count of 5 x 107 cell/ml, which is diluted 1:10 for each spot thereafter. The plate was incubated for 3 days and photographed.
Fig 3.
Glucose transport-defective mutant Hxt1 cannot uptake 2-NBDG.
(A) Yeast cells (hxtΔ) expressing either empty vector or indicated Hxt1-HA proteins were grown as described in Fig. 1A. 2-NBDG uptake by the yeast cells was analyzed by fluorescence microscopy. FL: Fluorescence, DIC: Differential Interference Contrast. (B) Quantification of relative 2-NBDG uptake by the yeast cells was shown (*P < 0.05).
Fig 4.
Mutation at the putative glucose-binding residues of Hxt1 does not affect its localization to the plasma membrane.
(A) Yeast cells (hxtΔ) expressing either empty vector or indicated Hxt1-GFP proteins were spotted on 2% glucose plate supplemented with Antimycin-A (1μg/ml) as described in Fig. 2D. The plate was incubated for 3 days and photographed. (B) Western blot analysis of Hxt1-GFP levels at the plasma membrane. Yeast cells (WT) expressing indicated Hxt1-GFP proteins were grown as described in Fig. 2C. Membrane fractions were analyzed by Western blotting with anti-GFP antibody. Actin was served as loading control. (C) Yeast cells (WT) expressing indicated Hxt1-GFP proteins were grown as described in Fig. 2C and were analyzed by fluorescence microscopy (top). Relative GFP fluorescence in the plasma membrane was quantified (bottom). Relative GFP fluorescence intensities were plotted with the fluorescence of WT cells (2% glucose condition) set to 100%. The data represented were averages of at least 50 cell counts with error bars representing standard deviations (S.D).
Fig 5.
Some of the putative glucose-binding residues of Hxt1 are required for endocytosis.
Yeast cells (WT and end3Δ) expressing indicated Hxt1-GFP proteins were grown as described in Fig. 2D. (A) Western blot analysis of Hxt1-GFP levels at the plasma membrane and (B) fluorescence microscopy of Hxt1-GFP proteins were shown. Actin was served as loading control. (C) Quantification of relative GFP fluorescence in the plasma membrane was performed as described in Fig. 4E. *Results of the Western blot and fluorescence microscopy analysis of wild type Hxt1-GFP protein in yeast cells (WT) depicted in Fig. 4C were shown for comparison.
Table 1.
Plasmids used in this study.