ABSTRACT
AIMS: Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons. METHODS: The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (Gm) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using 14C-malate. RESULTS: OGC increases proton Gm only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism. CONCLUSION: OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.
Subject(s)
2,4-Dinitrophenol , Fatty Acids , Animals , 2,4-Dinitrophenol/pharmacology , Mice , Fatty Acids/metabolism , Humans , Malates/metabolism , Mitochondria/metabolism , Ion Transport/drug effects , Membrane Potential, Mitochondrial/drug effects , Protons , Ketoglutaric Acids/metabolism , Organic Anion Transporters/metabolism , Organic Anion Transporters/genetics , Membrane Transport ProteinsABSTRACT
Uncoupling protein 3 (UCP3) belongs to the mitochondrial carrier protein superfamily SLC25 and is abundant in brown adipose tissue (BAT), the heart, and muscles. The expression of UCP3 in tissues mainly dependent on fatty acid oxidation suggests its involvement in cellular metabolism and has drawn attention to its possible transport function beyond the transport of protons in the presence of fatty acids. Based on the high homology between UCP2 and UCP3, we hypothesized that UCP3 transports C4 metabolites similar to UCP2. To test this, we measured the transport of substrates against phosphate (32Pi) in proteoliposomes reconstituted with recombinant murine UCP3 (mUCP3). We found that mUCP3 mainly transports aspartate and sulfate but also malate, malonate, oxaloacetate, and succinate. The transport rates calculated from the exchange of 32Pi against extraliposomal aspartate and sulfate were 23.9 ± 5.8 and 17.5 ± 5.1 µmol/min/mg, respectively. Using site-directed mutagenesis, we revealed that mutation of R84 resulted in impaired aspartate/phosphate exchange, demonstrating its critical role in substrate transport. The difference in substrate preference between mUCP2 and mUCP3 may be explained by their different tissue expression patterns and biological functions in these tissues.
