Abstract
The transduction of a human placental cDNA retroviral library into glyB cells, a Chinese hamster ovary K1 subline that is deficient in the transport of folates into mitochondria, resulted in the complementation of glycine auxotrophy of these cells. A 2.6-kilobase pair cDNA insert flanked by retroviral sequences had integrated into genomic DNA in rescued cells. An open reading frame in this cDNA encoded a 35-kDa protein homologous to several inner mitochondrial wall transporters for intermediate metabolites. The subcloned cDNA complemented the glycine auxotrophy of glyB cells and reinstated folate accumulation in the mitochondria of transfected cells. The human origin, chromosomal location, and intron-exon organization of the isolated mitochondrial folate transporter gene were deduced from the expressed sequence tag database and human genome project data.
Highlights
The transduction of a human placental cDNA retroviral library into glyB cells, a Chinese hamster ovary K1 subline that is deficient in the transport of folates into mitochondria, resulted in the complementation of glycine auxotrophy of these cells
The form of folate found in the circulation, enter the mammalian cells they are quickly metabolized to poly-␥-glutamate derivatives by cytosolic folylpoly-␥-glutamate synthetase (FPGS), a process needed to promote the retention of folate cofactors in cells [9]
One of these complementation groups, defined by the mutation responsible for the glycine auxotrophy of glyB Chinese hamster cells, had a defect in its ability to accumulate folates in mitochondria [15], a process required for the activity of mammalian mitochondrial serine hydroxymethyltransferase (SHMT) [23]
Summary
The transduction of a human placental cDNA retroviral library into glyB cells, a Chinese hamster ovary K1 subline that is deficient in the transport of folates into mitochondria, resulted in the complementation of glycine auxotrophy of these cells. Two studies [13, 14] have demonstrated the penetration into isolated mitochondria by folates in a process that was saturable and temperature-dependent These studies would support the existence of a transporter responsible for the entry of folates into the mitochondria as does the fact that cells that either lack mitochondrial FPGS [11] or are incapable of accumulation of mitochondrial folates [15] are glycine auxotrophs. We have transferred a library of human cDNAs in a retroviral vector into glyB cells and isolated a transduced cell line that was no longer auxotrophic for glycine These cells contained a human cDNA that, when rescued by PCR and recloned into a mammalian expression plasmid, complemented the auxotrophy of glyB cells at high frequency and reinstated folate entry into the mitochondria. We conclude that we have isolated the human gene encoding the inner membrane protein that is responsible for the entry of folates into the mitochondria
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