Specific SLC25 carriers regulate mitochondrial protein synthesis

A genome-wide knock out screen identified members of the SLC25 family of mitochondrial carrier proteins as important regulators of the rate of de novo mitochondrial protein synthesis. To elucidate this relationship, we generated human cell knockouts for SLC25A25, SLC25A44, SLC25A45, and SLC25A48, which have been shown to exchange ATP and magnesium, branched-chain amino acids, methylated basic amino acids, and choline, respectively. Multi-omic and functional analyses identified that these four carriers are crucial for mitochondrial translation, biogenesis and function of the oxidative phosphorylation system, as well as mitochondrial morphology. Thermostability screens showed that SLC25A48 is specifically stabilised by choline and changes in the mitochondrial metabolome and lipidome indicated defects in choline biosynthetic pathways and remodelling of mitochondrial membranes, both consistent with SLC25A48 being a choline transporter. These results highlight the essential roles of specific SLC25 transporters in maintaining mitochondrial structure and function and show that of mitochondrial translation is sensitive to local concentrations of branched chain amino acids, methylated basic amino acids, ATP, magnesium, and choline. Overall design: Total RNA from CAL51 cells were isolated from three control cells (WT) and three of each knockout (SLC25A44KO, SLC25A45KO, SLC25A48KO) and was sequenced and analysed for differential expression.