title: Mammalian mitochondrial and cytosolic folylpolyglutamate synthetase maintain the subcellular compartmentalization of folates.
abstract: Folylpoly-γ-glutamate synthetase (FPGS) catalyze the addition of multiple glutamates to tetrahydrofolate derivatives. Two mRNAs for the fpgs gene direct isoforms of FPGS to the cytosol and to mitochondria in mouse and human tissues. We sought to clarify the functions of these two compartmentalized isoforms. Stable cell lines were created that express cDNAs for the mitochondrial and cytosolic isoforms of human FPGS under control of a doxycycline-inducible promoter in the AUXB1 cell line. AUXB1 are devoid of endogenous FPGS activity due to a premature translational stop at codon 432 in the fpgs gene. Loss of folates was not measurable from these doxycycline-induced cells or from parental CHO cells over the course of three CHO cell generations. Likewise, there was no detectable transfer of folate polyglutamates either from the cytosol to mitochondria, or from mitochondria to the cytosol. The cell line expressing cytosolic FPGS required exogenous glycine but not thymidine or purine, whereas cells expressing the mitochondrial isoform required exogenous thymidine and purine but not glycine for optimal growth and survival. We concluded that mitochondrial FPGS is required because folate polyglutamates are not substrates for transport across the mitochondrial membrane in either direction and that polyglutamation not only traps folates in the cytosol, but also in the mitochondrial matrix.
authors.affiliation: From the Departments of Pharmacology and Toxicology and.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lawrence
authors.affiliation: the Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Titus
authors.affiliation: the Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ferguson
authors.affiliation: the Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Heineman
authors.affiliation: the Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298 Microbiology and Immunology, and.
authors.forename: Shirley M
authors.initials: SM
authors.lastname: Taylor
authors.affiliation: From the Departments of Pharmacology and Toxicology and the Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298 rmoran@vcu.edu.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Moran
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000201:enzymology*; D018076:DNA, Complementary / Q000378:metabolism; D005492:Folic Acid / Q000737:chemistry*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D051336:Mitochondrial Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010453:Peptide Synthases / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D020033:Protein Isoforms; D005492:Folic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase
keywords: Cell Compartmentalization; FPGS; Folate; Folylpolyglutamate Synthetase; Intracellular Trafficking; Mitochondrial Transport; Transport