Mechanism, proton co-transport, and directionality of the formate-nitrite transporter family (FNT)

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The main focus of the first project phase was on the cell-free production and characterization of difficult membrane proteins. Our work on the putative formate-nitrite-transporter (PfFNT) from malaria parasites led to an unexpected breakthrough in the malaria research field. PfFNT has turned into the currently most important candidate as the long-sought lactate-transporter of the parasites und represents a highly promising novel drug target. The work programme of this submission has been adapted according to the recommendations of the reviewers, i.e. removal of the uncertain protein crystallization and shifting of the focus on the study of the basic, largely unknown mechanisms of the formate-nitrite-transporter family with respect to anion transport, proton co-transport, and directionality. We will clone two new FNT proteins from Bacillus thuringiensis (BtFNT) and Entamoeba histolytica (EhFNT), biochemically characterize and compare their properties to established bacterial and plasmodial FNTs. This selection is due to two reasons: 1. B. thuringiensis is closely related to extremely pathogenic Bacillus species, such as the etiologic agent of anthrax, and E. histolytica causes amoebiasis and produces acetate as a metabolic end product, which may be expelled by EhFNT. Both FNTs, thus, are of therapeutic interest. 2. BtFNT and EhFNT exhibit an exchange to glutamine or asparagine, respectively, of a highly conserved histidine within the transport channel. The histidine is central in the current models on the FNT transport mechanism, which postulate protonation of a passing anion by a proton relay comprising the mentioned histidine plus an equally conserved threonine and a fixed water molecule. The amides glutamine and asparagine can neither act as proton acceptors or donors. Apparently, nature has evolved variations, which we want to exploit to gain detailed insight into the protonation mechanism of the FNTs, into proton co-transport, and directionality. We will generate mutants of the critical histidine and threonine residues, use and establish assay systems based on yeast and proteoliposomes, visualize proton co-transport by fluorescent dyes, and set up membrane systems in which the orientation of the FNT proteins is uniform or in which we will selectively inhibit transport in one direction in order to analyze directionality.