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Mitochondrial uptake of thiamin pyrophosphate: physiological and cell biological aspects. PLoS One 2013;8(8):e73503

Date

09/12/2013

Pubmed ID

24023687

Pubmed Central ID

PMC3758298

DOI

10.1371/journal.pone.0073503

Scopus ID

2-s2.0-84883368559   17 Citations

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.

Author List

Subramanian VS, Nabokina SM, Lin-Moshier Y, Marchant JS, Said HM

Author

Jonathan S. Marchant PhD Chair, Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Anion Transport Proteins
Female
Gene Expression Regulation
Green Fluorescent Proteins
Hep G2 Cells
Humans
Kinetics
Membrane Transport Proteins
Mice
Mitochondria, Liver
Mitochondrial Proteins
Mutant Proteins
Mutation
Protein Transport
RNA, Messenger
Recombinant Fusion Proteins
Thiamine Pyrophosphate
Tritium
Xenopus laevis
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a