Homology modeling and site-directed mutagenesis identify amino acid residues underlying the substrate selection mechanism of human monocarboxylate transporters 1 (hMCT1) and 4 (hMCT4)
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Human monocarboxylate transporters (hMCTs/SLC16As) mediate the transport of monocarboxylic compounds across plasma membranes. Among the hMCTs, hMCT1 and hMCT4 are expressed in various tissues, and transport substrates involved in energy metabolism. Both transporters mediate l-lactate transport, but, although hMCT1 also transports l-5-oxoproline (l-OPro), this compound is minimally transported by hMCT4. Thus, we were interested in the molecular mechanism responsible for the difference in substrate specificity between hMCT1 and hMCT4. Therefore, we generated 3D structure models of hMCT1 and hMCT4 to identify amino acid residues involved in the substrate specificity of these transporters. We found that the substrate specificity of hMCT1 was regulated by residues involved in turnover number (M69) and substrate affinity (F367), and these residues were responsible for recognizing (directly or indirectly) the –NH– moiety of l-OPro. Furthermore, our homology model of hMCT1 predicted that M69 and F367 participate in hydrophobic interactions with another region of hMCT1, emphasizing its potentially important role in the binding and translocation cycle of l-OPro. Mutagenesis experiments supported this model, showing that efficient l-OPro transport required a hydrophobic, long linear structure at position 69 and a hydrophobic, γ-branched structure at position 367. Our work demonstrated that the amino acid residues, M69 and F367, are key molecular elements for the transport of l-OPro by hMCT1. These two residues may be involved in substrate recognition and/or substrate-induced conformational changes.
KeywordsMonocarboxylate transporter hMCT1 hMCT4 l-Lactic acid l-5-Oxoproline Oocyte
Human monocarboxylate transporters
Major facilitator superfamily
Hidden Markov model
This work was supported by the Japan Society for the Promotion of Science (JSPS; KAKENHI Grant Number JP17J00013). We would like to thank Editage (http://www.editage.jp) for English language editing.
YF designed and performed the experiments, analyzed the results, and wrote the first draft of the manuscript. MK, KN, AF, and KI contributed to the writing of the manuscript. All authors reviewed the results and approved the final version of the manuscript.
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Conflict of interest
The authors have no conflicts of interest to declare.
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