Human mitochondrial C1-tetrahydrofolate synthase: gene structure, tissue distribution of the mRNA, and immunolocalization in Chinese hamster ovary calls

J Biol Chem. 2003 Oct 31;278(44):43178-43187. doi: 10.1074/jbc.M304319200. Epub 2003 Aug 22.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alternative Splicing
  • Amino Acid Sequence
  • Aminohydrolases / biosynthesis*
  • Aminohydrolases / chemistry
  • Aminohydrolases / genetics*
  • Animals
  • Blotting, Northern
  • CHO Cells
  • Chromosomes, Human, Pair 6
  • Cloning, Molecular
  • Codon, Initiator
  • Codon, Terminator
  • Cricetinae
  • Cytoplasm / enzymology
  • Cytoplasm / metabolism
  • DNA, Complementary / metabolism
  • Epitopes
  • Exons
  • Formate-Tetrahydrofolate Ligase / biosynthesis*
  • Formate-Tetrahydrofolate Ligase / chemistry
  • Formate-Tetrahydrofolate Ligase / genetics*
  • Humans
  • Immunoblotting
  • Introns
  • Methylenetetrahydrofolate Dehydrogenase (NADP) / biosynthesis*
  • Methylenetetrahydrofolate Dehydrogenase (NADP) / chemistry
  • Methylenetetrahydrofolate Dehydrogenase (NADP) / genetics*
  • Mitochondria / enzymology*
  • Mitochondria / metabolism
  • Models, Biological
  • Models, Genetic
  • Molecular Sequence Data
  • Multienzyme Complexes / biosynthesis*
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / genetics*
  • Protein Isoforms
  • Protein Structure, Tertiary
  • RNA, Messenger / metabolism
  • Sequence Homology, Amino Acid
  • Subcellular Fractions / metabolism
  • Tissue Distribution
  • Transfection

Substances

  • Codon, Initiator
  • Codon, Terminator
  • DNA, Complementary
  • Epitopes
  • Multienzyme Complexes
  • Protein Isoforms
  • RNA, Messenger
  • formyl-methenyl-methylenetetrahydrofolate synthetase
  • Methylenetetrahydrofolate Dehydrogenase (NADP)
  • Aminohydrolases
  • Formate-Tetrahydrofolate Ligase

Associated data

  • GENBANK/AY374130
  • GENBANK/AY374131