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 3H-TPP as a substrate and mitochondria isolated from mouse liver and human-derived liver HepG2 cells. Results showed 3H-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 3H-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.

Vitamin B1 in critically ill patients: needs and challenges

“…TMP and free thiamine were the only detectable species in the extracellular fluid. The majority of TPP in the body is found in erythrocytes and accounts for approximately 80% of the total bodies stores [14,[42][43][44]. There is no consensus on reference intervals for thiamine compounds in the human body.…”

The Janus Kinase 2 Inhibitor Fedratinib Inhibits Thiamine Uptake: A Putative Mechanism for the Onset of Wernicke’s Encephalopathy

“…The freshly purified mitochondria were suspended in the storage buffer from the kit to achieve the protein concentration of 10-12 mg/ml. The assay was carried out as reported elsewhere (Hopfer et al, 1973;Subramanian et al, 2013). Briefly, mitochondria (5 ml) were added to the uptake buffer (20 ml) containing 0.1 mM [ 3 H]TPP and then incubated at 37°C.…”

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

“…Previous studies have delineated different physiological and biological aspects of the MTPPT system [29, 41], but little is known about the structure-function relationship of this mitochondrial transporter. Thus, our aim in this study was to address this issue, and for that we employed a comparative protein-docking modeling [36, 37] approach as well as a multiple sequence alignment [39] approach to predict amino acid residues that may be important for function of this transporter.…”

“…All the mutations were introduced in the open reading frame (ORF) of hMTPPT ( SLC25A19 gene; i.e, in a phMTPPT-EGFP) using the Quick change™ II site-directed mutagenesis kit (Santa Clara, CA) and primers (Table-1) as described previously [29]. All mutations were made to alanine (unless otherwise stated) since such a substitution is predicted to have the minimal effect on secondary structure of proteins [30–32].…”

“…The 3 H-TPP uptake on isolated mitochondria was performed as previously described [29]. Briefly, the freshly prepared mitochondrial fraction was suspended in uptake buffer [140mM KCl, 0.3mM EDTA, 5mM MgCl 2 , 10mM MES, 10mM HEPES and 10mM succinate (to maintain the function of mitochondria), pH 7.4] and uptake was performed as described before [29] using rapid filtration method [33]. The mitochondrial suspension (20 μl; containing ~15–20 μg/μl of protein) was then added to 80 μl of uptake buffer containing 0.38 μM of 3 H-TPP and incubated at 37°C for 5 min.…”