Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters

Abstract

Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.