Retrovirally Mediated Complementation of the glyBPhenotype

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.In mammalian cells, the processes of folate metabolism are distributed between the cytosolic and mitochondrial compartments (1). Mitochondrial folates amount to about 35% of the total cellular pool (2, 3) and are used as cofactors for a mitochondrial serine hydroxymethyltransferase (SHMT) 1 by the glycine cleavage system and for the synthesis of the formylmethionine initiator of mitochondrial protein synthesis.The transport of folates through the plasma membrane into the cytosol has been extensively studied (4 -6), and two of the transport systems have been cloned (7,8). In contrast, the mechanism of the transfer of folates into mitochondria, presumably from the cytosol, is largely unknown as is the release of folates back into the cytosol from the mitochondria. Once the folate monoglutamates, 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). FPGS is also present in the mitochondrial compartment of mammalian cells (10) translated from transcripts from the FPGS gene, which add a mitochondrial leader sequence to the coding region of the protein found in the cytosol (11,12). 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.Early studies by Puck and coworkers (16, 17) selected somatic cells that were auxotrophic for glycine and demonstrated that these cells fell into four complementation groups named glyA, glyB, glyC, and glyD. glyA was found to be attributed to a deficiency in mitochondrial SHMT (17,18). The glyC and glyD mutations have not to our knowledge been assigned to any functions. glyB cells had normal cytosolic folate metabolism and enzymes and had the same mitochondrial SHMT and...

Methenyltetrahydrofolate Synthetase Regulates Folate Turnover and Accumulation

“…Consistent with this notion, less folate entered into this stable pool when cells were labeled with [ 3 H]folic acid (Fig. 3A), a folate derivative that cannot traverse the mitochondrial membrane until it is reduced to tetrahydrofolate in the cytoplasm (4,20,43). Alternatively, the stable pool may represent folate molecules that have been processed to their polyglutamate form by the enzyme folylpolyglutamate synthetase (44).…”

Identification of the Mitochondrial NAD+ Transporter in Saccharomyces cerevisiae

“…To our knowledge, none of these proteins has been characterized biochemically. However, NP_110407.2 (named hMFT), the closest human relative of Ndt1p and Ndt2p (31 and 30% identical amino acids, respectively), has been reported to be the mitochondrial carrier for folate (49). This conclusion was based on the finding that hMFT re-instates folate in the mitochondria of transfected glyB cells (49 the exchange is much higher than that of the uniport.…”

“…However, NP_110407.2 (named hMFT), the closest human relative of Ndt1p and Ndt2p (31 and 30% identical amino acids, respectively), has been reported to be the mitochondrial carrier for folate (49). This conclusion was based on the finding that hMFT re-instates folate in the mitochondria of transfected glyB cells (49 the exchange is much higher than that of the uniport. However, the uniport reaction can be essential in special conditions, for example when cells divide or must meet higher energetic demands associated with specific metabolic states and growth conditions.…”

“…The findings of the present report add two new members, Ndt1p and Ndt2p, to the group of already identified mitochondrial transporters (Flx1p (34), Tpc1p (51), and human MFT (49)) that import essential coenzymes (FAD, thiamine pyrophosphate, and folate, respectively) from the cytosol to the mitochondrial matrix.…”

γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

“…MRPs contribute to drug resistance or increase drug efficacy depending on polyglutamylation of antifolates and intracellular folate concentrations (Assaraf 2006). In addition, the SLC25A32 gene encodes a folate transporter that shuttles folate from the cytoplasm into the mitochondria (Titus and Moran 2000). A reduced, monoglutamylated form of cytoplasmic folate (probably tetrahydrofolate or 5-formyltetrahydrofolate) is transported to the mitochondria by the mitochondrial folate transporter, followed by the mitochondrial FPGS-induced polyglutamylation resulting in mitochondrial folate accumulation (Chen et al 1996).…”