Complementation of a Glucose Transporter Mutant ofSchizosaccharomyces pombe by a Novel Trypanosoma brucei Gene

Abstract

The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host. This is underscored by biochemical switches in its nutritional requirements. In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose. Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis. Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a lambdaEMBL4 genomic library of T. brucei EATRO 164 using a rat liver glucose transporter cDNA clone. Genomic analysis shows that the gene is present as a single copy within the parasite genome. The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily. Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence. It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains. However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants. The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides. The kinetics of glucose uptake reflected those in T. brucei. In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells.