Structure-function characterization of the human mitochondrial thiamin pyrophosphate transporter (hMTPPT; SLC25A19): Important roles for Ile(33), Ser(34), Asp(37), His(137) and Lys(291).

Abstract Thiamin plays a critical role in cellular energy metabolism. Mammalian cells obtain the vitamin from their surroundings, converted it to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria via a carrier-mediated process that involves the MTPPT (product of the SLC25A19 gene). Previous studies have characterized different physiological/biological aspects of the human MTPPT (hMTPPT), but less is known about structural features that are important for its function. Here, we used a protein-docking model (“Phyre2” and “DockingServer”) to predict residues that may be important for function (substrate recognition) of the hMTPPT; we also examined the role of conserved positively-charged residues predicted (“PRALINE”) to be in the trans-membrane domains (TMDs) in uptake of the negatively-charged TPP. Among the six residues predicted by the docking model (i.e., Thr 29 , Arg 30 , Ile 33 , Ser 34 , Asp 37 and Phe 298 ), only Ile 33 , Ser 34 and Asp 37 were found to be critical for function. While no change in translational efficiency/protein stability of the Ser 34 mutant was observed, both the Ile 33 and Asp 37 mutants showed a decrease in this parameter(s); there was also a decrease in the expression of the latter two mutants in mitochondria. A need for a polar residue at position 34 of the hMTPPT was evident. Our findings with the positively-charged residues (i.e., His 82 , His 137 , Lys 231 and Lys 291 ) predicted in the TMD showed that His 137 and Lys 291 are important for function (via a role in proper delivery of the protein to mitochondria). These investigations provide important information about the structure-function relationship of the hMTPPT.