A mutation inactivating the mitochondrial inner membrane folate transporter creates a glycine requirement for survival of chinese hamster cells.
A mutant Chinese hamster ovary cell line, glyB, that required exogenous glycine for survival and growth was reported previously (Kao, F., Chasin, L., and Puck, T. T. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 1284-1291). We now report that the defect in glyB cells causative of this phenotype is a point mutation in an inner mitochondrial membrane protein required for transport of folates into mitochondria. The CHO mitochondrial folate transporter (mft) was sequenced and compared with that from glyB cells. The hamster sequence was nearly identical to that of the recently reported human mitochondrial folate transporter. The corresponding cDNA from glyB cells contained a single nucleotide change that introduced a glutamate in place of the glycine in wild-type hamster MFT at codon 192 in a predicted transmembrane domain. Transfection of the wild-type hamster cDNA into glyB cells allowed cell survival in the absence of glycine and the accumulation of folates in mitochondria, whereas transfection of the Glu-192 cDNA did not. Genomic sequence analysis and fluorescence in situ hybridization demonstrated a single mutated allele of the mft gene in glyB cells, whereas there were two alleles in CHO cells. We conclude that we have defined the cause of the glyB auxotrophy and that the glyB mft mutation identified a region of this mitochondrial folate carrier vital to its transport function.
McCarthy
Erin A
EA
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Titus
Steven A
SA
Taylor
Shirley M
SM
Jackson-Cook
Colleen
C
Moran
Richard G
RG
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002470:Cell Survival / Q000502:physiology*; D003062:Codon; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005492:Folic Acid / Q000378:metabolism*; D015870:Gene Expression; D018698:Glutamic Acid; D005998:Glycine / Q000502:physiology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D026901:Membrane Transport Proteins / Q000737:chemistry; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017354:Point Mutation; D017385:Sequence Homology; D014162:Transfection
D003062:Codon; D018076:DNA, Complementary; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; C417506:SLC25A32 protein, human; D018698:Glutamic Acid; D005492:Folic Acid; D005998:Glycine