Human mitochondrial C1-tetrahydrofolate synthase: gene structure, tissue distribution of the mRNA, and immunolocalization in Chinese hamster ovary calls.
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.
Prasannan
Priya
P
Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin TX 78712.
Pike
Schuyler
S
Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin TX 78712.
Peng
Kun
K
Department of Nutritional Sciences, University of California, Berkeley CA 94720.
Shane
Barry
B
Department of Nutritional Sciences, University of California, Berkeley CA 94720.
Appling
Dean R
DR
Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin TX 78712.
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D002896:Chromosomes, Human, Pair 6; D003001:Cloning, Molecular; D018387:Codon, Initiator; D018388:Codon, Terminator; D006224:Cricetinae; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D000939:Epitopes; D005091:Exons; D005574:Formate-Tetrahydrofolate Ligase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007438:Introns; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection
D018387:Codon, Initiator; D018388:Codon, Terminator; D018076:DNA, Complementary; D000939:Epitopes; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D012333:RNA, Messenger; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase