Expression of Escherichia coli folylpolyglutamate synthetase in the Chinese hamster ovary cell mitochondrion.
Chinese hamster ovary (CHO) cell transfectants expressing Escherichia coli folylpoly-gamma-glutamate synthetase (FPGS) activity solely in their cytosol lack mitochondrial folylpolyglutamates and are auxotrophic for glycine. Addition of a mammalian mitochondrial leader sequence targeted E. coli FPGS to the mitochondria of these cells. Mitochondrial expression of FPGS restored mitochondrial folylpolyglutamate pools and overcame the glycine requirement. Pteroyltriglutamates functioned as effectively as the longer glutamate chain length folates found in wild type CHO cells in the metabolic cycle of glycine synthesis provided they were located in the mitochondria. Although folypolyglutamates cannot enter the mitochondria, mitochondrial folylpolyglutamates can be released without prior hydrolysis and CHO transfectants expressing E. coli FPGS activity solely in the mitochondria possessed normal cytosolic folylpolyglutamate pools. The proportion of cellular folate in the mitochondrion is governed by competition between mitochondrial and cytosolic FPGS activities.
Lin
B F
BF
Department of Nutritional Sciences, University of California, Berkeley 94720.
Shane
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002455:Cell Division; D006224:Cricetinae; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000096:biosynthesis / Q000378:metabolism; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection / Q000379:methods*
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005492:Folic Acid; D009954:Ornithine Carbamoyltransferase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase