Investigating the contribution of substrate collecting antenna to the proton/lactate cotransport (in PfFNT and MCT1)

Some cells rely intensively on the coupled activity of glycolysis and lactate dehydrogenase to constantly generate ATP. This process releases lactate and protons, the accumulation of which would become detrimental to the cell´s survival. Therefore, the removal of these metabolites is critical to maintain the cells energy generation. This is ensured by monocarboxylate transporters (MCT), such as the human MCT and the Plasmodium falciparum formate nitrite transporter (PfFNT).
In the case of human MCT, it has been established that their transport activity was modulated by partner proteins: its chaperone Basigin and carbonic anhydrases enzymes. The surface of such proteins act as proton and substrate collecting antennas, generating microenvironments of greater substrate concentration close to the transport sites. This work set out to investigate how such antennas were involved in the modulation of the monocarboxylate transport functionality of MCT1 and PfFNT.
Initial attempts of expressing fusion constructs of carbonic anhydrase, Basigin chaperone and MCT1 transporter proved unsuccessful. Then, alternative methods of protein production were explored to observe interaction between the transporter and the proton antenna. Moreover, this work identified that C-terminal poly-Histidine tag initially intended for protein purification and identification would affect the transport capacity of such MCT1 transporter.
This work also hypothesized that the PfFNT C-terminal helix, highly conserved among all human-infecting Plasmodia, plays the role of an endogenous proton collecting antenna facilitating the proton/lactate cotransport. Experimental results suggested that this terminus does modulate the substrate transport (radiolabeled lactate influx capacity was increased in acidic extracellular pH upon its deletion), but it remains to be determined whenever this collecting antenna increases the local concentration of protons or lactate.


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