Sideroflexins enable mitochondrial transport of polar neutral amino acids

Abstract

Mitochondria contribute to compartmentalized metabolism in eukaryotic cells, facilitating diverse enzymatic reactions that support cell function. However, this compartmentalization of metabolism necessitates regulated transport of metabolites across the inner mitochondrial membrane. While many proteins enabling mitochondrial membrane transport of metabolites are known, how some metabolites are transported is not known, and several mitochondrial amino acid transporters are largely uncharacterized. The goal of this dissertation is to better understand which proteins in the mitochondrial inner membrane regulate amino acid transport, particularly for substrates that lack known transporters, and how these proteins regulate associated metabolic pathways. Using CRISPR-Cas9-mediated candidate transporter knockouts coupled with assessment of metabolite transport via a mitochondrial swelling assay, we identified SFXN1 as a gene that mediates mitochondrial membrane permeability to polar neutral amino acids, including proline, glycine, taurine, hypotuarine, beta-alanine, and gammaaminobutyric acid (GABA). SFXN2 and SFXN3 partially complemented loss of SFXN1 to enable glycine transport, while SFXN2 and SFXN5 partially complemented loss of SFXN1 to enable GABA transport. Altogether, this work suggests that sideroflexins regulate the delivery of polar neutral amino acids across the inner mitochondrial membrane, many of which lack known carriers, and contributes to a better understanding of how mitochondrial amino acid transport regulates cellular metabolism.