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Updated: May 8, 2026
Site-Specific Lysine Lactylation via Genetic Code Expansion in E. coli and Mammalian Cells
Published on: February 24, 2026
Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4)
Shotaro Sasaki1, Masaki Kobayashi, Yuya Futagi
1Laboratory of Clinical Pharmaceutics and Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Background:
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.
Experimental Approach:
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.
Results:
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.
Conclusions:
Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.
Insights
Monocarboxylate transporter 4 (MCT4) transports lactate. Arginine-278 is crucial for MCT4
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Monocarboxylate transporters (MCTs) are vital for cellular metabolism and are attractive cancer targets.
- Limited understanding of human monocarboxylate transporter 4 (hMCT4) function and structure compared to MCT1.
- Identifying key amino acids in hMCT4 is essential for developing targeted cancer therapies.
Purpose of the Study:
- To elucidate the molecular mechanism of L-lactate transport by hMCT4.
- To identify specific amino acid residues critical for hMCT4 function.
Main Methods:
- L-lactate transport assays using Xenopus laevis oocytes expressing hMCT4.
- Chemical modification of hMCT4 with arginine-specific agents (phenylglyoxal).
- Site-directed mutagenesis of conserved arginine residues in hMCT4 and assessment of L-lactate uptake.
Main Results:
- Phenylglyoxal treatment abolished hMCT4 transport activity, which was rescued by L-lactate.
- L-lactate uptake was abolished in hMCT4 mutants at Arg-278 (R278) without affecting transporter localization.
- Specific arginine residues, particularly Arg-278 in transmembrane domain 8, are critical for hMCT4 function.
Conclusions:
- Arg-278 is a key residue for L-lactate recognition and transport by hMCT4.
- This finding provides insights into the molecular mechanism of hMCT4 and potential therapeutic strategies.
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