Many essential proteins require pyridoxal 5'-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5'-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5'-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5'-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5'-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5'-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5'-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia.

Center for Cancer Research Massachusetts General Hospital Boston MA USA

Children's Hospital of Eastern Ontario Research Institute Ottawa ON Canada

Dana Farber Cancer Institute Boston MA USA

David H Koch Institute for Integrative Cancer Research MIT Cambridge MA USA

Department of Biological Engineering MIT Cambridge MA USA

Department of Biology MIT Cambridge MA USA

Department of Brain and Cognitive Sciences MIT Cambridge MA USA

Department of Dermatology Cutaneous Biology Research Center Massachusetts General Hospital Harvard Medical School Boston MA USA

Department of Psychiatry Yale School of Medicine New Haven CT USA

Harvard MIT MD PhD Program Boston MA USA

Harvard T H Chan School of Public Health Boston MA USA

Institute of Organic Chemistry and Biochemistry IOCB Prague Czechia

The Picower Institute for Learning and Memory MIT Cambridge MA USA

UMass Chan Medical School Program in Molecular Medicine Worcester MA USA

Whitehead Institute for Biomedical Research Cambridge MA USA

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