The structure-function relationship of functionally distinct but structurally similar hexose transporters from Trypanosoma congolense

We have previously characterized, in Trypanosoma brucei, a multigene family encoding two developmentally regulated glucose transporters that are 80% i dentical at the amino-acid level. We report here the characterization of th e homologous glucose transporters (TcoHT1 and TcoHT2) in Trypanosoma congol ense, an African trypanosome responsible for disease in domestic animals. B oth TcoHT isoforms, which are 92.4% identical, are encoded by a single clus ter of genes containing two copies of TcoHT1 and three copies of TcoHT2 arr anged alternately. Northern blot analysis revealed that TcoHT2 is expressed in all of the adaptive forms, while mRNA encoding TcoHT1 is only present i n the metacyclic and bloodstream forms of T. congolense. When transfected w ith the TcoHT2 gene, Chinese Hamster Ovary cells express a hexose transport er with properties similar to those of the T. congolense procyclic forms (K -m d-glucose = 41 mu m versus 64 mu m). In contrast to TcoHT2, TcoHT1 expre ssed in the Chinese hamster ovary cells appeared to be a relatively low aff inity glucose transporter (K-i d-glucose = 0.8 mm). To determine the region (s) involved in the different apparent affinities for glucose, a chimera an alysis was undertaken on the TcoHT isoforms. This study shows that amino-ac id residues important for d-glucose recognition are located in the central region (between transmembrane domains 3 and 7) and in the C-terminal intrac ellular domain of TcoHT2. Site directed mutagenesis identified Ser193 locat ed within transmembrane helix 4 as a key residue in relaxing the apparent a ffinity of TcoHT1 for glucose.