Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

• Published in: Biochemical journal Vol. 339; no. 2; pp. 299 - 307
• Main Authors: Kruckeberg, A.L, Ye, L, Berden, J.A, Dam, K. van
• Format: Journal Article
• Language: English
• Published: England 15-04-1999
• Subjects: Base Sequence; Catalysis; Cell Membrane - metabolism; DNA Primers; Endocytosis; glucose; Glucose Transport Proteins, Facilitative; Green Fluorescent Proteins; Luminescent Proteins - genetics; Membrane Proteins - genetics; Membrane Proteins - metabolism; Monosaccharide Transport Proteins - genetics; Monosaccharide Transport Proteins - metabolism; nutrient transport; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; yeasts
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/0264-6021:3390299

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4x10(5) Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 degrees C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.