Trypanosomes are purine auxotrophs, salvaging these molecules from their hosts who synthesise them. The first step in purine salvage is transport of the purine nucleoside or nucleobase across the plasma membrane. One of the high affinity adenosine transporters, P2, is also implicated in drug uptake, loss of the P2 transporter leads to resistance to certain trypanocidal drugs, e.g. melarsoprol. Trypanasoma equiperdum is closely related to T. brucei. It is confined to horses where it is transmitted by venereal contact due to its inability to differentiate
into the procyclic form. Cloning of the P2 gene from T. equiperdum was carried out using PCR, based on the T. brucei sequence. Sequencing of a number of clones suggested two alleles were present with two changes at the amino acid level and three silent mutations.
Expression of these genes has been carried out in S. cerevisiae, which has no endogenous adenosine transport. In addition we have exploited a haploid yeast strain that lacks the FCY2 gene. In yeast FCY2 is responsible for purine nucleobase transport including adenine. Using
this yeast strain we have been able to show directly that adenine is a substrate of the P2 transporter confirming earlier predictions from inhibitor studies carried out in trypanosomes. Work is now continuing on the characterisation of these clones in the yeast expression system with respect to trypanocidal drugs thought to be internalised via this route. A PCR mutant isolated during cloning, with two point mutations, was found to be non-functional. One of these V12D, was within the first predicted transmembrane region. Site-directed mutagenesis
of this residue was found to restore function. Supported by the BBSRC