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J. Biol. Chem., Vol. 278, Issue 44, 43178-43187, October 31, 2003

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Human Mitochondrial C₁-Tetrahydrofolate Synthase

GENE STRUCTURE, TISSUE DISTRIBUTION OF THE MRNA, AND IMMUNOLOCALIZATION IN CHINESE HAMSTER OVARY CELLS^*

Priya Prasannan, Schuyler Pike, Kun Peng¶, Barry Shane¶, and Dean R. Appling||

From the Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712-0165 and the ^¶Department of Nutritional Sciences, University of California, Berkeley, California 94720

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 N-terminal 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 up-stream 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.

Received for publication, April 24, 2003 , and in revised form, August 20, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY374130 and AY374131.

^* 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.

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: dappling{at}mail.utexas.edu.

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