Phase I study of pemetrexed with sorafenib in advanced solid tumors.

PURPOSE: To determine if combination treatment with pemetrexed and sorafenib is safe and tolerable in patients with advanced solid tumors. RESULTS: Thirty-seven patients were enrolled and 36 patients were treated (24 in cohort A; 12 in cohort B). The cohort A dose schedule resulted in problematic cumulative toxicity, while the cohort B dose schedule was found to be more tolerable. The maximum tolerated dose (MTD) was pemetrexed 750 mg/m2 every 14 days with oral sorafenib 400 mg given twice daily on days 1-5. Because dosing delays and modifications were associated with the MTD, the recommended phase II dose was declared to be pemetrexed 500 mg/m2 every 14 days with oral sorafenib 400 mg given twice daily on days 1-5. Thirty-three patients were evaluated for antitumor activity. One complete response and 4 partial responses were observed (15% overall response rate). Stable disease was seen in 15 patients (45%). Four patients had a continued response at 6 months, including 2 of 5 patients with triple-negative breast cancer. EXPERIMENTAL DESIGN: A phase I trial employing a standard 3 + 3 design was conducted in patients with advanced solid tumors. Cohort A involved a novel dose escalation schema exploring doses of pemetrexed every 14 days with continuous sorafenib. Cohort B involved a modified schedule of sorafenib dosing on days 1-5 of each 14-day pemetrexed cycle. Radiographic assessments were conducted every 8 weeks. CONCLUSIONS: Pemetrexed and intermittent sorafenib therapy is a safe and tolerable combination for patients, with promising activity seen in patients with breast cancer.

p53 Deletion or Hotspot Mutations Enhance mTORC1 Activity by Altering Lysosomal Dynamics of TSC2 and Rheb.

UNLABELLED: The activity of mammalian target of rapamycin complex 1 (mTORC1) is frequently enhanced in carcinomas, an effect thought to contribute to the malignant phenotype. Here, it is demonstrated that either deletion or mutation of TP53 in colon or lung carcinoma cells substantially enhances mTORC1 kinase activity by an effect downstream of and independent of AMPK. Mechanistically, it was determined that loss or mutation of p53 decreased expression of TSC2 and Sestrin2 (SESN2). Complementation of p53 null cells with TSC2 or Sestrin2 reduced mTORC1 activity to levels found in p53 wild-type (wt) cells, whereas their genetic depletion enhanced mTORC1 activity in p53 wt cells. However, the primary causal event in enhanced mTORC1 activity upon loss of p53 appeared to be a diminished distribution of TSC2 to lysosomal membranes containing mTOR. Subsequently, there was increased Rheb in the lysosomal compartment, and a higher mTOR association with Raptor. Transfection of TSC2 into p53 null cells replaced TSC2 and diminished Rheb at the lysosome, recapitulating cells with wt p53. In contrast, transfection of Sestrin2 decreased mTOR in lysosomes, but the lower levels of Sestrin2 in p53 null cells did not change lysosomal mTOR. In summary, loss of the transcriptional activity of p53, either by deletion or by key mutations in the DNA-binding domain, diminishes expression of TSC2 and Sestrin2, thus, shifting membrane-bound TSC2 out of lysosomal membranes, increasing lysosomal Rheb and increasing the kinase activity of mTORC1. IMPLICATIONS: This study establishes that loss of p53 function decreases lysosomal TSC2 and increases lysosomal Rheb resulting in hyperactive mTORC1, findings that are consistent with a more malignant phenotype.

AMP-activated Protein Kinase (AMPK) Control of mTORC1 Is p53- and TSC2-independent in Pemetrexed-treated Carcinoma Cells.

The key sensor of energy status in mammalian cells, AMP-activated protein kinase (AMPK), can also be activated by the AMP analog aminoimidazolecarboxamide nucleoside monophosphate (ZMP) generated directly from aminoimidazolecarboxamide ribonucleoside (AICAR) or from inhibition of purine synthesis by the antifolate pemetrexed (PTX), a drug used extensively in the treatment of lung cancers. Despite this common mechanism, signaling downstream of AMPK activated by PTX or AICAR differed. AICAR-activated AMPK inhibited mTORC1 both directly by phosphorylation of the mTORC1 subunit Raptor and indirectly by phosphorylation of the regulator TSC2. In contrast, PTX-activated AMPK inhibited mTORC1 solely through Raptor phosphorylation. This dichotomy was due to p53 function. Transcription of p53 target genes, including TSC2, was activated by AICAR but not by PTX. Although both PTX and AICAR stabilized p53, only AICAR activated Chk2 phosphorylation, stimulating p53-dependent transcription. However, Raptor phosphorylation by AMPK was independent of p53 and was sufficient, after PTX treatment, to inhibit mTORC1. We concluded that PTX effects on mTORC1 were independent of TSC2 and p53 and that the activation of a p53 transcriptional response by AICAR was due to an activation of Chk2 that was not elicited by PTX.

Mammalian mitochondrial and cytosolic folylpolyglutamate synthetase maintain the subcellular compartmentalization of folates.

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.

Phenylalanine-Based Inactivator of AKT Kinase: Design, Synthesis, and Biological Evaluation.

Strategies to inhibit kinases by targeting the substrate binding site offer many advantages, including naturally evolved selectivity filters, but normally suffer from poor potency. In this work we propose a strategy to design and prepare covalent substrate-competitive kinase inhibitors as a method to improve potency. We have chosen AKT as the model kinase for this work. Using the AKT-GSK3ß cocrystal structure and a reactive cysteine near the substrate binding site, we have identified phenylalanine (Phe) as an appropriate scaffold for the covalent inactivator portion of these inhibitors. By synthesizing compounds that incorporate cysteine-reactive electrophiles into phenylalanine and testing these compounds as AKT inhibitors, we have identified Boc-Phe-vinyl ketone as a submicromolar inactivator of AKT. We also show that Boc-Phe-vinyl ketone (1) potently inhibits AKT1 and inhibits cell growth in HCT116 and H460 cells nearly as well as AKT inhibitors GSK690693 and MK-2206, (2) is selective for kinases that possess an activation loop cysteine such as AKT, (3) requires the vinyl ketone for inactivation, (4) has inactivation that is time-dependent, and (5) alkylates Cys310 of AKT as shown by mass spectrometry. Identification of Boc-Phe-vinyl ketone as a covalent inactivator of AKT will allow the development of peptide and small-molecule substrate-competitive covalent kinase inhibitors that incorporate additional substrate binding elements to increase selectivity and potency. This proof-of-principle study also provides a basis to apply this strategy to other kinases of the AGC and CAMK families.

Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure.

The mouse is extensively used to model human folate metabolism and therapeutic outcomes with antifolates. However, the folylpoly-γ-glutamate synthetase (fpgs) gene, whose product determines folate/antifolate intracellular retention and antifolate antitumor activity, displays a pronounced species difference. The human gene uses only a single promoter, whereas the mouse uses two: P2, akin to the human promoter, at low levels in most tissues; and P1, an upstream promoter used extensively in liver and kidney. We deleted the mouse P1 promoter through homologous recombination to study the dual-promoter mouse system and to create a mouse with a humanized fpgs gene structure. Despite the loss of the predominant fpgs mRNA species in liver and kidney (representing 95 and 75% of fpgs transcripts in these tissues, respectively), P1-knockout mice developed and reproduced normally. The survival of these mice was explained by increased P2 transcription due to relief of transcriptional interference, by a 3-fold more efficient translation of P2-derived than P1-derived transcripts, and by 2-fold higher stability of P2-derived FPGS. In combination, all 3 effects reinstated FPGS function, even in liver. By eliminating mouse P1, we created a mouse model that mimicked the human housekeeping pattern of fpgs gene expression.

Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells.

Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to class III RTKs such as PDGFRb and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.

Tetrahydrofolate recognition by the mitochondrial folate transporter.

A mitochondrial carrier family (MCF) of transport proteins facilitates the transfer of charged small molecules across the inner mitochondrial membrane. The human genome has ∼50 genes corresponding to members of this family. All MCF proteins contain three repeats of a characteristic and conserved PX(D/E)XX(K/R) motif thought to be central to the mechanism of these transporters. The mammalian mitochondrial folate transporter (MFT) is one of a few MCF members, known as the P(I/L)W subfamily, that have evolved a tryptophan residue in place of the (D/E) in the second conserved motif; the function of this substitution (Trp-142) is unclear. Molecular dynamics simulations of the MFT in its explicit membrane environment identified this tryptophan, as well as several other residues lining the transport cavity, to be involved in a series of sequential interactions that coordinated the movement of the tetrahydrofolate substrate within the transport cavity. We probed the function of these residues by mutagenesis. The mutation of every residue identified by molecular dynamics to interact with tetrahydrofolate during simulated transit into the aqueous channel severely impaired folate transport. Mutation of the subfamily-defining tryptophan residue in the MFT to match the MCF consensus at this position (W142D) was incompatible with cell survival. These studies indicate that MFT Trp-142, as well as other residues lining the transporter interior, coordinate tetrahydrofolate descent and positioning of the substrate in the transporter basin. Overall, we identified residues in the walls and at the base of the transport cavity that are involved in substrate recognition by the MFT.

Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells.

Pemetrexed (ALIMTA, Lilly) is a folate antimetabolite that has been approved by the U.S. Food and Drug Administration for the treatment of non-small cell lung cancer and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (Nexavar, Bayer), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K, and/or phosphorylated mTOR, in addition to class III receptor tyrosine kinases such as platelet-derived growth factor receptor beta and VEGF receptors, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.

DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria.

Mitochondrial DNA (mtDNA) has been reported to contain 5-methylcytosine (5mC) at CpG dinucleotides, as in the nuclear genome, but neither the mechanism generating mtDNA methylation nor its functional significance is known. We now report the presence of 5-hydroxymethylcytosine (5hmC) as well as 5mC in mammalian mtDNA, suggesting that previous studies underestimated the level of cytosine modification in this genome. DNA methyltransferase 1 (DNMT1) translocates to the mitochondria, driven by a mitochondrial targeting sequence located immediately upstream of the commonly accepted translational start site. This targeting sequence is conserved across mammals, and the encoded peptide directs a heterologous protein to the mitochondria. DNMT1 is the only member of the three known catalytically active DNA methyltransferases targeted to the mitochondrion. Mitochondrial DNMT1 (mtDNMT1) binds to mtDNA, proving the presence of mtDNMT1 in the mitochondrial matrix. mtDNMT1 expression is up-regulated by NRF1 and PGC1α, transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to hypoxia, and by loss of p53, a tumor suppressor known to regulate mitochondrial metabolism. Altered mtDNMT1 expression asymmetrically affects expression of transcripts from the heavy and light strands of mtDNA. Hence, mtDNMT1 appears to be responsible for mtDNA cytosine methylation, from which 5hmC is presumed to be derived, and its expression is controlled by factors that regulate mitochondrial function.

Pemetrexed indirectly activates the metabolic kinase AMPK in human carcinomas.

The chemotherapeutic drug pemetrexed, an inhibitor of thymidylate synthase, has an important secondary target in human leukemic cells, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of purine biosynthesis. The purine intermediate aminoimidazolecarboxamide ribonucleotide (ZMP), which accumulates behind this block, transmits an inhibitory signal to the mTORC1 complex via activation of the cellular energy sensor AMP-activated kinase (AMPK). Given that the PI3K-AKT-mTOR pathway is frequently deregulated during carcinogenesis, we asked whether the indirect activation of AMPK by pemetrexed offers an effective therapeutic strategy for carcinomas with defects in this pathway. Activation of AMPK by ZMP in pemetrexed-treated colon and lung carcinoma cells and the downstream consequences of this activation were strikingly more robust than previously seen in leukemic cells. Genetic experiments demonstrated the intermediacy of AICART inhibition and the centrality of AMPK activation in these effects. Whereas AMPK activation resulted in marked inhibition of mTORC1, other targets of AMPK were phosphorylated that were not mTORC1-dependent. Whereas AMPK activation is thought to require AMPKα T172 phosphorylation, pemetrexed also activated AMPK in carcinoma cells null for LKB1, the predominant AMPKα T172 kinase whose deficiency is common in lung adenocarcinomas. Like rapamycin analogs, pemetrexed relieved feedback suppression of PI3K and AKT, but the prolonged accumulation of unphosphorylated 4E-BP1, a tight-binding inhibitor of cap-dependent translation, was seen following AMPK activation. Our findings indicate that AMPK activation by pemetrexed inhibits mTORC1-dependent and -independent processes that control translation and lipid metabolism, identifying pemetrexed as a targeted therapeutic agent for this pathway that differs significantly from rapamycin analogs.

Therapeutics by cytotoxic metabolite accumulation: pemetrexed causes ZMP accumulation, AMPK activation, and mammalian target of rapamycin inhibition.

Pemetrexed represents the first antifolate cancer drug to be approved by the Food and Drug Administration in 20 years; it is currently in widespread use for first line therapy of mesothelioma and non-small cell lung cancer. Pemetrexed has more than one site of action; the primary site is thymidylate synthase. We now report that the secondary target is the downstream folate-dependent enzyme in de novo purine synthesis, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The substrate of the AICART reaction, ZMP, accumulated in intact pemetrexed-inhibited tumor cells, identifying AICART as the step in purine synthesis that becomes rate-limiting after drug treatment. The accumulating ZMP causes an activation of AMP-activated protein kinase with subsequent inhibition of the mammalian target of rapamycin (mTOR) and hypophosphorylation of the downstream targets of mTOR that control initiation of protein synthesis and cell growth. We suggest that the activity of pemetrexed against human cancers is a reflection of its direct inhibition of folate-dependent target proteins combined with prolonged inhibition of the mTOR pathway secondary to accumulation of ZMP.

Concentration-dependent processivity of multiple glutamate ligations catalyzed by folylpoly-gamma-glutamate synthetase.

Folylpoly-gamma-glutamate synthetase (FPGS, EC 6.3.2.17) is an ATP-dependent ligase that catalyzes formation of poly-gamma-glutamate derivatives of reduced folates and antifolates such as methotrexate and 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDAH 4PteGlu 1). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether single or multiple glutamate residues are added following each folate binding event. A very sensitive high-performance liquid chromatography method has been used to analyze the multiple ligation reactions onto radiolabeled DDAH 4PteGlu 1 catalyzed by FPGS to distinguish between distributive or processive mechanisms of catalysis. Reaction time courses, substrate trapping, and pulse-chase experiments were used to assess folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS can catalyze the processive addition of approximately four glutamate residues to DDAH 4PteGlu 1. The degree of processivity was determined to be dependent on the concentration of the folate substrate, thus suggesting a mechanism for the regulation of folate polyglutamate synthesis in cells.

A mouse gene that coordinates epigenetic controls and transcriptional interference to achieve tissue-specific expression.

The mouse fpgs gene uses two distantly placed promoters to produce functionally distinct isozymes in a tissue-specific pattern. We queried how the P1 and P2 promoters were differentially controlled. DNA methylation of the CpG-sparse P1 promoter occurred only in tissues not initiating transcription at this site. The P2 promoter, which was embedded in a CpG island, appeared open to transcription in all tissues by several criteria, including lack of DNA methylation, yet was used only in dividing tissues. The patterns of histone modifications over the two promoters were very different: over P1, histone activation marks (acetylated histones H3 and H4 and H3 trimethylated at K4) reflected transcriptional activity and apparently reinforced the effects of hypomethylated CpGs; over P2, these marks were present in tissues whether P2 was active, inactive, or engaged in assembly of futile initiation complexes. Since P1 transcriptional activity coexisted with silencing of P2, we sought the mechanism of this transcriptional interference. We found RNA polymerase II, phosphorylated in a pattern consistent with transcriptional elongation, and only minimal levels of initiation factors over P2 in liver. We concluded that mouse fpgs uses DNA methylation to control tissue-specific expression from a CpG-sparse promoter, which is dominant over a downstream promoter masked by promoter occlusion.

Transducing particles of Staphylococcus aureus pathogenicity island SaPI1 are comprised of helper phage-encoded proteins.

The relationship between the composition of SaPI1 transducing particles and those of helper phage 80alpha was investigated by direct comparison of virion proteins. Twelve virion proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry; all were present in both 80alpha and SaPI1 virions, and all were encoded by 80alpha. No SaPI1-encoded proteins were detected. This confirms the prediction that SaPI1 is encapsidated in a virion assembled from helper phage-encoded proteins.

Probing the mechanism of the hamster mitochondrial folate transporter by mutagenesis and homology modeling.

The mitochondrial folate transporter (MFT) was previously identified in human and hamster cells. Sequence homology of this protein with the inner mitochondrial membrane transporters suggested a domain structure in which the N- and C-termini of the protein are located on the mitochondrial intermembrane-facing surface, with six membrane-spanning regions interspersed by two intermembrane loops and three matrix-facing loops. We now report the functional significance of insertion of the c-myc epitope into the intermembrane loops and of a series of site-directed mutations at hamster MFT residues highly conserved in orthologues. Insertional mutagenesis in the first predicted intermembrane loop eliminated MFT function, but the introduction of a c-myc peptide into the second loop was without effect. Most of the hamster MFT residues studied by site-directed mutagenesis were remarkably resilient to these mutations, except for R249A and G192E, both of which eliminated folate transport activity. Homology modeling, using the crystal structure of the bovine ADP/ATP carrier (AAC) as a scaffold, suggested a similar three-dimensional structure for the MFT and the AAC. An ion-pair interaction in the AAC thought to be central to the mechanism of membrane penetration by ADP is predicted by this homology model to be replaced by a pi-cation interaction in MFT orthologues and probably also in other members of the family bearing the P(I/L)W motif. This model suggests that the MFT R249A and G192E mutations both modify the base of a basket-shaped structure that appears to constitute a trap door for the flux of folates into the mitochondrial matrix.

Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications.

Pemetrexed is a new-generation antifolate, approved for the treatment of mesothelioma and non-small cell lung cancer, currently being evaluated for the treatment of a variety of other solid tumors. This review traces the history of antifolates that led to the development of pemetrexed and describes the unique properties of this agent that distinguish it from other antifolates. These include (a) its very rapid conversion to active polyglutamate derivatives in cells that build to high levels and are retained for long intervals to achieve prolonged and potent inhibition of its major target enzyme thymidylate synthase, (b) its high affinity for three folate transporters, and (c) its marked sensitivity to the level of physiologic folates in cells. The latter results in the unique and paradoxical finding that when transport mediated by the major folate transporter (the reduced folate carrier) is impaired, pemetrexed activity is preserved. This is due to concurrent contraction of competing cellular physiologic folates and utilization of a novel second transport carrier for which pemetrexed has high affinity, recently identified as the proton-coupled folate transporter (PCFT). Laboratory studies are reviewed that raise the possibility of new approaches to the use of folic acid supplementation in clinical regimens with pemetrexed.

Modifications of human histone H3 variants during mitosis.

Phosphorylation of histone H3 is a hallmark event in mitosis and is associated with chromosome condensation. Here, we use a combination of immobilized metal affinity chromatography and tandem mass spectrometry to characterize post-translational modifications associated with phosphorylation on the N-terminal tails of histone H3 variants purified from mitotically arrested HeLa cells. Modifications observed in vivo on lysine residues adjacent to phosphorylated Ser and Thr provide support for the existence of the "methyl/phos", binary-switch hypothesis [Fischle, W., Wang, Y., and Allis, C. D. (2003) Nature 425, 475-479]. ELISA with antibodies selective for H3 at Ser10, Ser28, and Thr3 show reduced activity when adjacent Lys residues are modified. When used together, mass spectrometry and immunoassay methods provide a powerful approach for elucidation of the histone code and identification of histone post-translational modifications that occur during mitosis and other specific cellular events.

Folate metabolism in plants: an Arabidopsis homolog of the mammalian mitochondrial folate transporter mediates folate import into chloroplasts.

The distribution of folates in plant cells suggests a complex traffic of the vitamin between the organelles and the cytosol. The Arabidopsis thaliana protein AtFOLT1 encoded by the At5g66380 gene is the closest homolog of the mitochondrial folate transporters (MFTs) characterized in mammalian cells. AtFOLT1 belongs to the mitochondrial carrier family, but GFP-tagging experiments and Western blot analyses indicated that it is targeted to the envelope of chloroplasts. By using the glycine auxotroph Chinese hamster ovary glyB cell line, which lacks a functional MFT and is deficient in folates transport into mitochondria, we showed by complementation that AtFOLT1 functions as a folate transporter in a hamster background. Indeed, stable transfectants bearing the AtFOLT1 cDNA have enhanced levels of folates in mitochondria and can support growth in glycine-free medium. Also, the expression of AtFOLT1 in Escherichia coli allows bacterial cells to uptake exogenous folate. Disruption of the AtFOLT1 gene in Arabidopsis does not lead to phenotypic alterations in folate-sufficient or folate-deficient plants. Also, the atfolt1 null mutant contains wild-type levels of folates in chloroplasts and preserves the enzymatic capacity to catalyze folate-dependent reactions in this subcellular compartment. These findings suggest strongly that, despite many common features shared by chloroplasts and mitochondria from mammals regarding folate metabolism, the folate import mechanisms in these organelles are not equivalent: folate uptake by mammalian mitochondria is mediated by a unique transporter, whereas there are alternative routes for folate import into chloroplasts.

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.