Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Mitochondrial uptake of thiamin pyrophosphate: physiological and cell biological aspects. PLoS One 2013;8(8):e73503 PMID: 24023687 PMCID: PMC3758298

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
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



View this publication's entry at the Pubmed website PMID: 24023687
jenkins-FCD Prod-130 96200611f8481f0aa4f84230b11dd74d063847a3