title: 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.
authors.affiliation: Department of Medical Research, VA Medical Center, Long Beach, CA 90822, United States; Departments of Medicine and Physiology/Biophysics, University of California, Irvine, CA 92697, United States.
authors.forename: Subrata
authors.initials: S
authors.lastname: Sabui
authors.affiliation: Department of Medical Research, VA Medical Center, Long Beach, CA 90822, United States; Departments of Medicine and Physiology/Biophysics, University of California, Irvine, CA 92697, United States.
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.affiliation: Department of Medical Research, VA Medical Center, Long Beach, CA 90822, United States; Departments of Medicine and Physiology/Biophysics, University of California, Irvine, CA 92697, United States.
authors.forename: Rubina
authors.initials: R
authors.lastname: Kapadia
authors.affiliation: Department of Medical Research, VA Medical Center, Long Beach, CA 90822, United States; Departments of Medicine and Physiology/Biophysics, University of California, Irvine, CA 92697, United States. Electronic address: hmsaid@uci.edu.
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D001692:Biological Transport; D017124:Conserved Sequence; D056945:Hep G2 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055550:Protein Stability; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; C434255:SLC25A19 protein, human; D013835:Thiamine Pyrophosphate
keywords: Mitochondria; Multiple sequence alignment; Protein-docking modeling; Site-directed mutagenesis; Thiamin pyrophosphate transporter; Uptake