Structure - function characterization of the human mitochondrial thiamin pyrophosphate transporter (hMTPPT; SLC25A19): important roles for Ile33 Ser34 Asp37 His137 and Lys291

摘要

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., Thr²⁹, Arg³⁰, Ile³³, Ser³⁴, Asp³⁷ and Phe²⁹⁸), only Ile³³, Ser³⁴ and Asp³⁷ were found to be critical for function. While no change in translational efficiency/protein stability of the Ser³⁴ mutant was observed, both the Ile³³ and Asp³⁷ 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., His⁸², His¹³⁷, Lys²³¹ and Lys²⁹¹) predicted in the TMD showed that His¹³⁷ and Lys²⁹¹ 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