title: Mitochondrial uptake of thiamin pyrophosphate: physiological and cell biological aspects.
abstract: Mammalian cells obtain vitamin B1 (thiamin) from their surrounding environment and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. Most of TPP is then transported into the mitochondria via a carrier-mediated process that involves the mitochondrial thiamin pyrophosphate transporter (MTPPT). Knowledge about the physiological parameters of the MTPP-mediated uptake process, MTPPT targeting and the impact of clinical mutations in MTPPT in patients with Amish lethal microcephaly and neuropathy and bilateral striatal necrosis are not fully elucidated, and thus, were addressed in this study using custom-made (3)H-TPP as a substrate and mitochondria isolated from mouse liver and human-derived liver HepG2 cells. Results showed (3)H-TPP uptake by mouse liver mitochondria to be pH-independent, saturable (Km = 6.79±0.53 µM), and specific for TPP. MTPPT protein was expressed in mouse liver and HepG2 cells, and confocal images showed a human (h)MTPPT-GFP construct to be targeted to mitochondria of HepG2 cells. A serial truncation analysis revealed that all three modules of hMTPPT protein cooperated (although at different levels of efficiency) in mitochondrial targeting rather than acting autonomously as independent targeting module. Finally, the hMTPPT clinical mutants (G125S and G177A) showed proper mitochondrial targeting but displayed significant inhibition in (3)H-TPP uptake and a decrease in level of expression of the MTPPT protein. These findings advance our knowledge of the physiology and cell biology of the mitochondrial TPP uptake process. The results also show that clinical mutations in the hMTPPT system impair its functionality via affecting its level of expression with no effect on its targeting to mitochondria.
authors.affiliation: Departments of Medicine, Physiology and Biophysics, University of California Irvine, Irvine, California, United States of America ; Department of Veterans Affairs Medical Center, Long Beach, California, United States of America.
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.forename: Svetlana M
authors.initials: SM
authors.lastname: Nabokina
authors.forename: Yaping
authors.initials: Y
authors.lastname: Lin-Moshier
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Marchant
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D000818:Animals; D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D027321:Anion Transport Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D050505:Mutant Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C434255:SLC25A19 protein, human; C515239:Slc25a19 protein, mouse; D014316:Tritium; D049452:Green Fluorescent Proteins; D013835:Thiamine Pyrophosphate
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