Substrates and functions of the Sideroflexin mitochondrial transporter family

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7. PROJECT SUMMARY/ABSTRACT Metabolic flux generates energy and building blocks to fuel the many processes occurring in cells and organisms. Mitochondria are central organelles in both catabolic and anabolic cellular metabolism, a function for which they require efficient exchange of metabolites with the rest of the cell. Mitochondrial transporters facilitating metabolite exchange across the inner mitochondrial membrane thus play a key role in metabolism and physiology. Alterations in mitochondrial transporters have been linked to diseases including cancer, however, the identities and functions of these transporters are not well understood. One metabolite whose transport into mitochondria is critical for biosynthesis and proliferation is the amino acid serine. I recently identified Sideroflexin1 (SFXN1) as the long-sought transporter responsible for serine uptake into mitochondria in the one-carbon metabolic pathway, which is commonly upregulated in cancer. SFXN1 is part of the conserved carrier family of Sideroflexins consisting of five homologues in mammals. All Sideroflexins localize to mitochondria but vary in their tissue expression patterns and their functions in vivo are unknown. My preliminary data suggest that Sideroflexins, including SFXN1, have other physiologically relevant substrates beside serine, such as alanine and cysteine, and play important roles in metabolism outside of the one-carbon pathway. While SFXN1 and its closest homologue, SFXN3, are functionally redundant, other Sideroflexins can compensate for loss of SFXN1 to varying degrees suggesting a difference in substrate specificities and/or affinities. The most distant homologue, SFXN4, has a function outside of one-carbon metabolism and likely does not transport serine. I hypothesize that since SFXN4 is required for TCA cycle flux and mitochondrial respiration, it is responsible for the uptake of another metabolite with a key role in mitochondrial metabolism, such as glutamine. To broadly identify SFXN1 and SFXN4 candidate substrates, I will use untargeted metabolomics methods and determine whether transport of these substrates occurs in vitro and in cells. I will use Seahorse extracellular flux analysis to determine which respiratory chain complex and thus which electron donor is affected by loss of SFXN4 and perform a negative selection CRISPR screen to probe SFXN4 function in an unbiased way. Finally, I will determine the physiological functions of Sideroflexins using knockout mice. By elucidating the functions of Sideroflexins, an important but largely unstudied family of potentially chemotherapeutically relevant mitochondrial transporters, my research will shed light on the poorly understood role of mitochondrial transport in cellular metabolism and in physiology.