Characterization of a gene encoding the human mitochondrial C₁-tetrahydrofolate synthase and submitochondrial localization of the protein
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C1-tetrahydrofolate (THF) synthase is a eukaryotic trifunctional enzyme that possesses the activities 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. These activities catalyze three sequential folate-mediated one-carbon interconversions resulting in the synthesis of formate. In the yeast, Saccharomyces cerevisiae, C1-THF synthase is present in the cytoplasm as well as in the mitochondria. A mammalian cytoplasmic isozyme of C1- THF synthase has been characterized for several years, but the mitochondrial isozyme in mammals had not been previously identified. This dissertation describes the cDNA cloning and characterization of a human mitochondrial C1-THF synthase. Human chromosome 6 carries the gene encoding the mitochondrial isozyme, which spans 236 kilobase pairs. The protein encoded by the full-length cDNA (978 amino acids) is 61% identical to the cytoplasmic isozyme and has an N-terminal mitochondrial targeting sequence. Expression of the full-length cDNA (2934 bp) clone in Chinese Hamster Ovary (CHO) cells showed that the protein is localized in the mitochondria. The gene encoding the mitochondrial C1-THF synthase isozyme is expressed in most of the human tissues examined and is high in placenta, followed by thymus and spleen. The transcript is distinct from that of the cytoplasmic isozyme in size and tissue distribution. The transcriptional start site of the gene was mapped as well. A short transcript representing the 5’-end of the gene was also observed by Northern blot analysis and was shown to arise from alternative splicing. The possibility of the short transcript encoding a functional protein was investigated. A cDNA of the short transcript of C1-THF synthase was cloned into an E. coli expression vector and the recombinant protein (short isoform) was purified. Although catalytically inactive, the purified short isoform was successfully used to make polyclonal antibodies specific for mitochondrial C1-THF synthase. Earlier studies done in our lab had also shown that adult rat liver mitochondria can metabolize carbon 3 of serine into formate by a folate-dependent pathway. To confirm that this one-carbon metabolism is occurring inside the mitochondria, we further explored the sublocalization of human mitochondrial C1-THF synthase. Analysis of mitochondrial subfractions from CHO cells transfected with the fulllength cDNA clone demonstrated that the human mitochondrial C1-THF synthase was associated with the matrix side of the inner mitochondrial membrane. Subfractionation of rat spleen mitochondria showed that mitochondrial C1-THF synthase was localized in the inner membrane in adult mammalian tissues as well. During the course of this work, studies by other members of the lab revealed that the recombinant human mitochondrial C1-THF synthase is monofunctional, possessing only the 10-formyl-THF synthetase activity. Since the human mitochondrial isozyme appears to be an inner membrane protein and also monofunctional, we hypothesized that it might interact with an unidentified enzyme that could provide the missing 5,10- methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities. Initial attempts to identify such an interaction were unsuccessful. In summary, we have identified the gene encoding the human mitochondrial C1-THF synthase, cloned and characterized it as well. The protein encoded by this gene was shown to be localized in the mitochondria, specifically in the inner mitochondrial membrane in a mammalian cell line.