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.

Keywords: Mitochondria; Multiple sequence alignment; Protein-docking modeling; Site-directed mutagenesis; Thiamin pyrophosphate transporter; Uptake.

Figure 1. Amino acid residues in the hMTPPT protein predicted to have potential role in recognition/function

A. Docking model of hMTPPT with TPP created by the docking server and the protein (hMTPPT) was shown as a multi-color cartoon representation and substrate (TPP) as orange colored. The homology model of hMTPPT was generated based on the crystal structure of the mitochondrial ADP/ATP carrier in complex with carboxyatractyloside (PDB ID: 1OKC) [21]. B. Predicted topology model of hMTPPT showing six TMDs with both NH₂ and COOH- terminal oriented towards the cytosol. The location of predicted substrate recognition site and conserved positively-charged residues are shown by yellow and pink colored boxes, respectively. C. MTPPT protein sequence from human (NP_001119594), sumatran orangutan (NP_001127123), chimpanzee (NP_001233547), crab-eating macaque (NP_001306520), bovine (NP_001039352), sheep (NP_001136362), wild boar (NP_001157986), mouse (NP_001239313) and rat (NP_001007675) were aligned by using PRALINE software. Conserved and unconserved residues are indicated by red to blue color, respectively and identities of conserved positively-charged residues (which are located in potentially strategic position based on homology model) in TMD of hMTPPT are shown in boxes.

Figure 1. Amino acid residues in the hMTPPT protein predicted to have potential role in recognition/function

A. Docking model of hMTPPT with TPP created by the docking server and the protein (hMTPPT) was shown as a multi-color cartoon representation and substrate (TPP) as orange colored. The homology model of hMTPPT was generated based on the crystal structure of the mitochondrial ADP/ATP carrier in complex with carboxyatractyloside (PDB ID: 1OKC) [21]. B. Predicted topology model of hMTPPT showing six TMDs with both NH₂ and COOH- terminal oriented towards the cytosol. The location of predicted substrate recognition site and conserved positively-charged residues are shown by yellow and pink colored boxes, respectively. C. MTPPT protein sequence from human (NP_001119594), sumatran orangutan (NP_001127123), chimpanzee (NP_001233547), crab-eating macaque (NP_001306520), bovine (NP_001039352), sheep (NP_001136362), wild boar (NP_001157986), mouse (NP_001239313) and rat (NP_001007675) were aligned by using PRALINE software. Conserved and unconserved residues are indicated by red to blue color, respectively and identities of conserved positively-charged residues (which are located in potentially strategic position based on homology model) in TMD of hMTPPT are shown in boxes.

Figure 2. Effect of mutating residues in the hMTPPT protein predicted to play a role in substrate recognition/interaction on ³H-TPP uptake by mitochondria and on the level of expression of hMTPPT mRNA and protein

A. Uptake of ³H-TPP (0.38 μM; 37 °C) by mitochondria isolated from HepG2 cells stably expressing WT hMTPPT and the mutants Thr²⁹, Arg³⁰, Ile³³, Ser³⁴, Asp³⁷ and Phe²⁹⁸. Uptake was measured after 5 min incubation (buffer, pH 7.4, 37°C) and calculated as described in “Method”. Data are mean ± SE of at least three independent experiments, **P < 0.01. B. The level of mRNA expression of the WT hMTPPT and mutants (Ile³³, Ser³⁴ and Asp³⁷) determined by real-time PCR (RT-PCR) using total RNA isolated from HepG2 cells stably expressing these constructs. Data are presented as mean ± SE of three to four independent experiments after normalization with β-actin. (C) & (D) show Western blot analysis performed using equal amount of whole HepG2 cell homogenate protein and purified isolated mitochondria, respectively (“Methods”). The blots were probed with anti-GFP monoclonal antibody and anti-PDH monoclonal antibody; data was normalized relative to the mitochondrial internal control, PDH. Representative blots are shown in inset. Data are mean ± SE from at least three different samples from three different batches of cells. *P < 0.05 and ** P < 0.01.

Figure 3. Effect of the hMTPPT Ser³⁴Ala, Ser³⁴Thr and Ser³⁴Val mutants on ³H-TPP uptake and on the level of expression of hMTPPT mRNA and protein

A. Uptake of ³H-TPP (0.38 μM) by mitochondria of HepG2 cells stably expressing WT hMTPPT and the Ser³⁴Ala, Ser³⁴Val and Ser³⁴Thr mutants. Uptake was measured after 5 min incubation (buffer, pH 7.4, 37 °C) and calculated as described in “Method”. Data are mean ± SE of at least three independent experiments. **P < 0.01 B. The level of mRNA expression of Ser³⁴Ala, Ser³⁴Thr and Ser³⁴Val mutants compared to WT hMTPPT. The RT-PCR was performed using total RNA isolated from HepG2 cells expressing the WT hMTPPT and mutants. Data are mean ± SE of three to four independent experiments after normalization with β-actin expression. Western blot analyses were performed with an equal amount of whole cell homogenate (C), and purified mitochondria (D) from HepG2 cells expressing WT hMTPPT or mutants (“Methods”). The blot was probed with anti-GFP monoclonal antibody and anti-PDH monoclonal antibody; data was then normalized relative to the mitochondrial internal control, PDH. Representative blots are shown in inset. I and II are indicates the Ser³⁴Ala, Ser³⁴Val, Ser³⁴Thr and His¹³⁷Ala, His¹³⁷Arg, respectively. Data are mean ± SE from at least three different samples from three different batches of cells.

Figure 4. Effect of mutating conserved positively-charged residues in hMTPPT TMDs on ³H-TPP uptake by mitochondria and on the level of mRNA and protein expression of the transporter

A. Uptake of ³H-TPP (0.38μM) by mitochondria of HepG2 cells stably expressing WT hMTPPT and the mutants His⁸², His¹³⁷, Lys²³¹ and Lys ²⁹¹. Freshly isolated mitochondria were incubated in uptake buffer, pH 7.4 at 37°C and ³H-TPP (0.38μM) was added to the incubation buffer to start the uptake assay. Uptake was measured after 5 min incubation and calculated as described in “Method”. Data are mean ± SE of at least three independent experiments. **P < 0.01. B. The level of mRNA expression of the WT hMTPPT and mutants (His¹³⁷ and Lys²⁹¹) determined by real-time PCR (RT-PCR) using total RNA isolated from HepG2 cells stably expressing these constructs. Data are presented as mean ± SE of three to four independent experiments after normalization with β-actin. (C) & (D) show Western blot analysis performed using equal amount of whole HepG2 cell homogenate protein and purified isolated mitochondria, respectively (“Methods”). The blots were probed with anti-GFP monoclonal antibody and anti-PDH monoclonal antibody; data was normalized relative to the mitochondrial internal control, PDH. Representative blots are shown in inset. Data are mean ± SE of at least three different samples from three different batches of cells. ** P < 0.01.