Identification and functional characterization of the mitochondrial adenine nucleotide carriers of Trypanosoma brucei

The Mitochondrial Carrier Family encloses a group of transmembrane proteins that transport metabolites across the mitochondrial inner membrane. The ADP/ATP carrier is the most widely studied MCF protein. It catalyzes the counter exchange of ADP for ATP in the mitochondrion of all eukaryotes. In the genome of the kinetoplastid parasite Trypanosoma brucei, three putative ADP/ATP carrier sequences (MCP5, MCP15 and MCP16) and one GDP/GTP (MCP13) entries were analyzed by sequence analyses and phylogenetic reconstruction. AACs phylogenetic reconstruction proved a strong association with yeast, funghi and plant clades, whilst separates from those AACs and from metazoans. MCP13 groups with GGCs, seems to be present only on lower eukaryotes and do not seem to present any homologues in metazoans. Gene deletion studies were performed to assess the roles of MCP5, MCP15, MCP16 and 13. A conditional double knockout cell line, with an inducible myc-tagged rescue copy was constructed for MCP5, which proves the essentiality of the protein for the parasite. Growth curves of the mutant cell line proved a growth defect phenotype in various carbon sources conditions. Mitochondrial ATP production assays were performed in the mutant cell line, in presence and absence of the inducible protein, using permeabilized cells with digitonin that confirmed the ADP/ATP transport activity of the carrier. For invitro activity assays, the carriers were cloned and expressed in Escherichia coli and Spodoptera frugiperda, solubilised and reconstituted into liposomes. Unfortunately, the reconstitution was unsuccessful and the conditions and methodologies are discussed