Human Mitochondrial C1-Tetrahydrofolate Synthase

C1-tetrahydrofolate (THF) synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. The cytoplasmic isozyme of C1-THF synthase is well characterized in a number of mammals, including humans; but a mitochondrial isozyme has been previously identified only in the yeast Saccharomyces. Here, we report the identification and characterization of the human gene encoding a functional mitochondrial C1-THF synthase. The gene spans 236 kilobase pairs on chromosome 6 and consists of 28 exons plus one alternative exon. The gene encodes a protein of 978 amino acids, including an Nterminal mitochondrial targeting sequence. The mitochondrial isozyme is 61% identical to the human cytoplasmic isozyme. Expression of the gene was detected in most human tissues, but transcripts were highest in placenta, thymus, and brain. Two mRNAs were detected, a 3.6-kb transcript and a 1.1-kb transcript, and both transcripts were observed in varying ratios in each tissue. The shorter transcript results from an alternative splicing event, where exon 7 is spliced to exon 8a instead of exon 8. Exon 8a is derived from an exonized Alu sequence, sharing no homology with exon 8 of the long transcript, and encodes just 15 amino acids followed by a stop codon and a polyadenylation signal. This short transcript potentially encodes a bifunctional enzyme lacking 10-formyl-THF synthetase activity. Both transcripts initiate at the same 5 -site, 107 nucleotides upstream of the ATG start codon. The full-length (2934 bp) cDNA fused to a C-terminal V5 epitope tag was expressed in Chinese hamster ovary cells. Immunoblots of subfractionated cells revealed a 107-kDa protein only in the mitochondrial fractions of these cells, confirming the mitochondrial localization of the protein. Yeast cells expressing the full-length human cDNA exhibited elevated 10-formyl-THF synthetase activity, confirming its identification as the human mitochondrial C1-THF synthase. C1-tetrahydrofolate (THF) 1 synthase is a trifunctional enzyme found in eukaryotes that contains the activities 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5) (Fig. 1, reactions 1–3). These activities, along with serine hydroxymethyltransferase (Fig. 1, reaction 4), are central to the interconversion of the one-carbon units carried by the biologically active form of folic acid, THF. The activated one-carbon units are used in a variety of cellular processes, including de novo purine and thymidylate synthesis, serine and glycine interconversion, methionine biosynthesis, and protein synthesis in mitochondria and chloroplasts. In eukaryotic cells, the mitochondrial and cytosolic compartments each contain a parallel set of one-carbon unit-interconverting enzymes (1). For example, in the yeast Saccharomyces cerevisiae, mitochondrial and cytoplasmic isozymes of C1-THF synthase (encoded by the nuclear genes MIS1 and ADE3, respectively) have been purified and characterized (2, 3). Both isozymes exist as homodimers of 100-kDa subunits. Each subunit consists of a C-terminal 10-formyl-THF synthetase domain of 70 kDa and an N-terminal bifunctional dehydrogenase/cyclohydrolase domain of 30 kDa linked via a proteolytically sensitive connector region. This subunit size and domain structure are shared by cytoplasmic isozymes from mammalian and avian sources (4–9). All three activities of C1-THF synthase are found in mammalian mitochondria as well (10, 11). Our studies with intact rat liver mitochondria and mitochondrial extracts demonstrated the ability of these organelles to oxidize carbon 3 of serine to formate by a folate-dependent pathway (Fig. 1, reactions 1–4) (11). However, the existence, structure, and function of the folate-interconverting activities of C1-THF synthase in mammalian mitochondria have been controversial. MacKenzie and co-workers (12, 13) characterized a bifunctional NAD-dependent 5,10-methylene-THF dehydrogenase/5,10-methenylTHF cyclohydrolase, originally isolated from ascites tumor cells. This bifunctional enzyme lacks the large C-terminal domain catalyzing the 10-formyl-THF synthetase activity and thus is unable to produce formate. This enzyme was shown to be a nuclear encoded mitochondrial protein (14, 15), detectable only in transformed mammalian cells and embryonic or nondifferentiated tissues (12). Among adult differentiated tissues, NAD-dependent 5,10-methylene-THF dehydrogenase activity is detectable only in rat adrenal tissue (16), although the mRNA encoding this enzyme is present at low levels in all tissues examined (17). MacKenzie and co-workers (18, 19) have * This work was supported by National Institutes of Health Grants DK61428 (to D. R. A.) and DK42033 (to B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession number(s) AY374130 and AY374131. § Both authors contributed equally to this work. To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Texas, 1 University Station A5300, Austin, TX 78712-0165. Tel.: 512-471-5842; Fax: 512-471-5849; E-mail: [email protected]. 1 The abbreviations used are: THF, tetrahydrofolate; CHO, Chinese hamster ovary; nt, nucleotide(s); SOE, splice overlap extension; HMS, homogenization solution; TBS, Tris-buffered saline; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag; GCS, glycine cleavage system. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 44, Issue of October 31, pp. 43178–43187, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.