SLC25A45 mediates mitochondrial import of methylated mitochondrial amino acids and carnitine biosynthesis

Solute carriers (SLCs) regulate cellular and organismal metabolism by transporting small molecules and ions across membranes, yet the physiological substrates of ~20% remain elusive. To address this, we developed a supervised machine learning platform to predict and prioritize gene-metabolite associations. This approach identifies UNC93A and SLC45A4 as candidate plasma membrane transporters for acetylglucosamine and polyamines, respectively. Additionally, we uncover SLC25A45 as a mitochondrial transporter linked to serum levels of methylated basic amino acids, products of protein catabolism. Mechanistically, SLC25A45 is necessary for the mitochondrial import of methylated basic amino acids, including ADMA and TML, the latter serving as a precursor for carnitine synthesis. In line with this observation, SLC25A45 loss impairs hepatic carnitine synthesis and upregulation of carnitine-containing metabolites under fasted conditions. By facilitating mitochondrial TML import, SLC25A45 connects protein catabolism to carnitine production, sustaining ß-oxidation during fasting. Altogether, our study identifies putative substrates for three SLCs and provides a resource for transporter deorphanization. Overall design: 7-week-old male Slc25a45-/- vs. Slc25a45+/-, Slc25a45+/+ mice were transferred into a clean cage with or without the food access for 24 hours. At the beginning of experiment as well as 24 hours after, the blood was collected for serum analysis. Subsequently, the liver tissues were harvested, immediately flash frozen and stored at -80°C for subsequent metabolomic profiling by liquid chromatography-mass spectrometry (LC-MS). Total RNA was extracted from the livers harvested as described above from Slc25a45-/- vs. Slc25a45+/-, Slc25a45+/+ mice using the RNeasy Mini Kit (Qiagen #74104) in accordance with the manufacturer's protocol. Library preparation and RNA-sequencing was performed by Genewiz.