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PLoS One
2013 Jul 01;87:e67690. doi: 10.1371/journal.pone.0067690.
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Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4).
Sasaki S
,
Kobayashi M
,
Futagi Y
,
Ogura J
,
Yamaguchi H
,
Takahashi N
,
Iseki K
.
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Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4. Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4. In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization. Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.
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Figure 2. Saturation kinetics of hMCT4-mediated L-lactate transport.Uptake of L-lactate was measured with 10 min incubation at 25°C in transport buffer of pH 5.5 or pH 7.5 in the presence of an increasing concentration of L-lactate. hMCT4-specific uptake was calculated by subtracting the uptake in water-injected oocytes from the uptake in hMCT4 cRNA-injected oocytes. Only the hMCT4-specific uptake was used for kinetic analysis. The inset shows an Eadie-Hofstee plot of L-lactate transport activity. Each point represents the mean ± S.E. of three – five experiments.
Figure 3. pH-dependency of hMCT4 activity in the presence of L-lactate.Uptake by oocytes was assayed for 10 min at 25°C in the presence of 0.1 mM L-lactate. Each point represents the mean ± S.E. of three – five experiments. The background uptake values of water-injected oocytes were subtracted.
Figure 4. Effect of DIDS on transport activity via hMCT4.(A) Oocytes were incubated for 10 min at 25°C with transport buffer of pH 5.5 containing 0.1 mM L-lactate in the absence or presence of 0.5 mM DIDS. Data are presented as means ± S.E. of three independent experiments. The background uptake values of water-injected oocytes were subtracted. (B) Oocytes were preincubated at 25°C for 10 min with/without 5 mM PLP (pH 7.5). L-lactate uptake after preincubation was measured. The oocytes were incubated for 10 min at 25°C with transport buffer of pH 7.5 containing 0.1 mM L-lactate in the absence or presence of 0.5 mM DIDS. Data are presented as means ± S.E. of three independent experiments. The background uptake values of water-injected oocytes were subtracted.
Figure 5. Effect of PGO modification on transport activity via hMCT4.Oocytes were preincubated at 25°C for 15 min with 100 mM PGO (pH 8.0) in the absence or presence of 1 M L-lactate. After treatment with PGO, oocytes were rinsed twice with transport buffer (pH 7.4). Oocytes were incubated additionally twice for 5 min with transport buffer. The oocyte were incubated for 10 min at 25°C with transport buffer of pH 5.5 containing 0.1 mM L-lactate. Data are presented as means ± S.E. of three independent experiments. The background uptake values of water-injected oocytes were subtracted.
Figure 6. Sequence alignment and secondary structure of hMCT4.(A) Amino acid sequence alignment of human MCT4 with human MCT1, MCT2 and MCT3, rat MCT4 and mouse MCT4 transporter homologues using ClustalW. Orange bars in hMCT4 above the sequence are the regions predicted to from transmembrane-spanning domains (TMDs) by TMHMM. Polar residues are highlighted in red (acidic residues), blue (basic residues) and green (neutral residues). Identical residues are indicated by asterisks (*) represents under the residue. (B) Putative topology of hMCT4: The conserved basic residues of MCT1-4 are shown in blue.
Figure 7. PGO effect and pH-dependency in hMCT4 mutants.(A) Uptake of 0.1 mM L-lactate was measured before and after treatment with 100 mM PGO in oocytes injected with hMCT4-WT or the indicated mutants. Data are presented as means ± S.E. of three – five experiments. (B) Uptake of L-lactate was measured in hMCT4-WT or -R198Q expressing oocytes with 10 min incubation in transport buffer of pH 5.5 or pH 7.5 in the presence of 0.1 mM L-lactate. Data are presented as means ± S.E. of four – five experiments.
Figure 8. Fluorescence staining of MCT4 in slices of oocytes.Localization of hMCT4 in oocytes injected with hMCT4-WT or the indicated mutants. Oocytes were treated with antibodies against MCT4. Data shown are typical results of three independent experiments.
Figure 1. Accumulation of L-lactate in hMCT4-expressing oocytes.Oocytes were incubated for various periods at 25°C with transport buffer of pH 5.5 or pH 7.5 containing 0.1 mM L-lactate. Control oocytes were injected with the same volume of water instead of hMCT4 cRNA. Each point represents the mean ± S.E. of three – five experiments.
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