Research Article

SFXN1 is a mitochondrial serine transporter required for one-carbon metabolism

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Science  16 Nov 2018:
Vol. 362, Issue 6416, eaat9528
DOI: 10.1126/science.aat9528

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Mitochondrial serine transporter identified

One-carbon (1C) metabolism is a universal metabolic process that is required for purine synthesis and supports the high levels of proliferation in cancer cells. The transport of serine into mitochondria supplies most of the 1C units needed for biosynthesis. Kory et al. used a genetic screen to identify the long-sought-after mitochondrial serine transporter. Elucidating the key step of serine transport is important for our understanding of metabolism and has potential implications for cancer treatment.

Science, this issue p. eaat9528

Structured Abstract

INTRODUCTION

One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides, amino acids, and lipids. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate.

In dividing mammalian cells, the mitochondrial catabolism of serine supplies most of the one-carbon units needed for biosynthesis. Indeed, many cancers rely on the one-carbon units generated from serine for proliferation, and mitochondrial serine catabolism enzymes are commonly up-regulated in tumors. Given that the entry of serine into mitochondria is a critical step in the generation of one-carbon units, it is surprising that the mitochondrial transporter(s) for serine remains unknown.

RATIONALE

To seek the transporter responsible for serine import into mitochondria, we designed a CRISPR-Cas9–mediated genetic screen in human cells based on the likelihood that loss of mitochondrial serine transport will reduce the proliferation of cells lacking the cytosolic branch of the one-carbon metabolism pathway. Therefore, we aimed to identify genes that are synthetic lethal with serine hydroxymethyl transferase–1 (SHMT1), a key cytosolic enzyme of the pathway. Moreover, we reasoned that even if there are redundant mechanisms for serine transport, we can sensitize cells to its partial inhibition by lowering cytosolic serine concentrations, which is easily achieved by removing exogenous serine. Thus, we sought genes required for the optimal proliferation of cells lacking the cytosolic one-carbon pathway when cultured in serine-free media. We picked the human blood Jurkat and K562 cancer cell lines for our screen because of their high mitochondrial one-carbon metabolism activity and suitability for screening and transduced cells with a lentiviral single guide RNA (sgRNA) library that targets ∼3000 metabolic enzymes, small-molecule transporters, and metabolism-related transcription factors.

RESULTS

This screen yielded genes in the pathways for serine and purine biosynthesis and with known functions in one-carbon metabolism. In both cell lines, only one gene of unknown molecular function scored, sideroflexin 1 (SFXN1), a multipass mitochondrial membrane protein. Sfxn1 was originally identified as the gene mutated in a mouse mutant with anemia and axial skeletal abnormalities, and is part of the sideroflexin family of proteins conserved throughout eukaryotes. In humans, SFXN1 is highly expressed in the blood, liver, and kidney, which are tissues with high one-carbon metabolism activity. STED super-resolution microscopy confirmed that SFXN1 localizes to the inner and not outer mitochondrial membrane, in agreement with a potential role as a metabolite transporter. The proliferation defect of SFXN1-null cells in serine-depleted media was completely reversed by adding formate, the product of the mitochondrial one-carbon pathway, which directly implicates an insufficient supply of one-carbon units in their defective proliferation. Notably, expression of an sgRNA-resistant SFXN1 cDNA restored the proliferation rate of the SFXN1-null cells to that of the wild-type cells.

Serine tracing experiments place SFXN1 in the mitochondrial branch of the one-carbon pathway. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis and have reduced levels of charged folate species. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including mitochondrial dysfunction and being auxotrophic for glycine. Several human SFXN family members can complement SFXN1-3 double loss, as can their yeast and Drosophila orthologs. These results were confirmed when SFXN3 emerged as one of the top synthetic lethal genes with SFXN1 in the absence of exogenous glycine. To test whether SFXN1 can directly transport serine, we purified the FLAG-tagged protein from mammalian cells and reconstituted it into liposomes. Recombinant SFXN1 mediated serine uptake into liposomes. Conversely, serine uptake into mitochondria isolated from SFXN1-null cells was decreased. SFXN1 may have other physiologically relevant transport substrates besides serine, including cysteine and alanine.

CONCLUSION

SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism. As there are multiple sideroflexins and their expression varies across tissues, SFXN1 and its homologs may turn out to be important nodes for regulating the fate of serine in cells. Because SFXN1 is expressed in many cancers and its expression is likely regulated by the Myc transcription factor, it may also have unexplored roles in cancer cell growth.

SFXN1 transports serine into mitochondria and is a component of the one-carbon metabolism pathway.

Serine is converted to formate in mitochondria, which is then exported to the cytosol to generate one-carbon units carried on tetrahydrofolate (THF) for nucleotide synthesis and other processes. In this diagram, the mitochondria are represented by STED super-resolution images of cells stained both for FLAG-SFXN1 and the outer mitochondrial membrane marker Tom20.

Abstract

One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.

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