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Volume 271, Number 22, Issue of May 31, 1996 pp. 13077-13087
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Purification and Properties of Human Cytosolic Folylpoly--glutamate Synthetase and Organization, Localization, and Differential Splicing of Its Gene

(Received for publication, February 28, 1996)

Linda Chen , Hong Qi , Julie Korenberg ^§ , Timothy A. Garrow , Yun-Jung Choi and Barry Shane

From the  Department of Nutritional Sciences, University of California, Berkeley, California 94720 and the ^§ Ahmanson Department of Pediatrics and Medical Birth Defects Center, Cedars-Sinai Medical Center, UCLA, Los Angeles, California 90048

Human cytosolic folylpolyglutamate synthetase (FPGS) was expressed in Escherichia coli and purified to homogeneity. Tetrahydrofolate and dihydrofolate were the most effective substrates, while 5-substituted folates were poor substrates. Most pteroyldiglutamates were better substrates than monoglutamates.

The human FPGS gene spans 12 kilobases and contains 15 exons and 14 introns. A single FPGS gene was located to chromosome region 9q34.1. Four exon 1 variants were identified, each of which was spliced to exon 2. The exon 1 variant corresponding to the isolated cDNA contains two ATG codons and multiple transcription start sites in this region generates mitochondrial and cytosolic FPGS (Freemantle, S. J., Taylor, S. M., Krystal, G., and Moran, R. G. (1995) J. Biol. Chem. 270, 9579-9584). Exons 1B and 1C, generated by alternate splicing in intron 1, and exon 1A, which is 5 to exon 1 and may encode an additional mitochondrial isoform, are preceded by a number of potential promoter sites.

Chinese hamster ovary cell transfectants expressing FPGS activity in the mitochondria contained normal mitochondrial and low cytosolic folylpolyglutamate pools. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, while cytosolic FPGS activity is needed for establishment of normal cytosolic folate pools. The reconstructed FPGS gene restored normal cytosolic and mitochondrial folate metabolism in hamster cells.

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