Retrovirally-mediated complementation of the glyB phenotype : cloning of a human gene encoding the carrier for entry of folates into mitochondria †

Transduction of a human placental cDNA retroviral library into glyB cells, a CHO K1 subline deficient in the transport of folates into mitochondria, resulted in complementation of the glycine auxotropy of these cells. A 2.6 kb 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 gly B cells and reinstated folate accumulation in mitochondria of transfected cells. The human origin, chromosomal location, and intron-exon organization of the isolated mitochondrial folate transporter gene were deduced from dbEST and human genome project data. 3 by gest on N ovem er 6, 2017 hp://w w w .jb.org/ D ow nladed from 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), 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 transfer of folates into mitochondria, presumably from the cytosol, is largely unknown as is the release of folates back to the cytosol from the mitochondria. Once folate monoglutamates, the form of folate found in the circulation, enter mammalian cells they are quickly metabolized to polyγ glutamate derivatives by cytosolic folylpolylγ-glutamate synthetase (FPGS), a process needed to promote 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 have demonstrated the penetration into isolated mitochondria by folates in a process that was saturable and temperature dependent (13,14). These studies would support the existence of a transporter responsible for entry of folates into the mitochondria, as does the fact that cells which either lack mitochondrial FPGS (11) or are incapable of accumulation of mitochondrial folates (15) are glycine auxotrophs. Early studies by Puck and his colleagues selected somatic cells auxotrophic for glycine and demonstrated that they fell into four complementation groups, named gly A through D (16,17). Gly A was found to be due to a deficiency in mitochondrial SHMT (17,18). The Gly C and D mutations have not, to our knowledge, been assigned to functions. Gly B cells had normal cytosolic folate metabolism and enzymes and the same mitochondrial SHMT and FPGS as CHO cells, but lacked mitochondrial folates (15) . The most likely candidate for the function missing in glyB cells was a transport protein which faciliated the entry of folates into mitochondria. We have transferred a library of human cDNAs in a retroviral vector into glyB cells and 4 by gest on N ovem er 6, 2017 hp://w w w .jb.org/ D ow nladed from isolated a transduced cell line no longer auxotrophic for glycine. These cells contained a human cDNA which, when rescued by PCR and recloned into a mammalian expression plasmid, complemented the auxotrophy of glyB cells at high frequency and also reinstated folate entry into mitochondria. We conclude that we have isolated the human gene encoding the inner membrane protein responsible for entry of folates into mitochondria. Materials and Methods Cell culture. Cells were grown in 37°C in 5% CO2. The glyB cell line, which was derived from CHO-K1 cells (16,17) , was a generous gift of Dr Lawrence Chasin (Columbia University, NY, NY). HEK293gp packaging cells (a gift from Dr Oliver Bogler of this University) were grown in DMEM, glyB and CHO-K1 in MEM, and CEM in RPMI-1640, all containing 10 % fetal calf serum. For virus production, HEK293gp cells were plated at 5 x10 6 cells on each of twelve 100 mm dishes. Forty eight hours later, 15 μg of plasmid library DNA, 15 μg of pVSVG plasmid (Dr Oliver Bogler), which allows pseudeotyping of the viral envelope proteins (19,20), and 55 μg of Lipofectamine-2000 (Life Technologies) were added to each dish in DMEM. After 20 min at room temperature, the medium was replaced with 25 μM chloroquine in DMEM and 1.5 ml of the lipid-DNA complex was added to each plate. Media was changed to complete DMEM after 5 h at 37 °; the supernatant, containing the collection of viruses from the library, was harvested after 36 and 72 hours and filtered. Transduction efficiency and viral titer were determined by monitoring fluorescent cells on control plates transduced with a virus expressing the enhanced green fluorescent protein (Clontech). GlyB cells (2 x 106) were suspended in 9 ml MEM and 7 ml of viral supernatant and plated on each of ten 150 mm dishes; after 5 hours the medium was replaced with complete MEM. After 48 hours at 37°, selection in glycine-free MEM containing dialyzed serum was applied. After 1014 days, colonies were expanded in glycine-free medium. PCR analysis and rescue of transductants The presence of a 200 base pair fragment of the retroviral ψ packaging signal in genomic DNA was verified by PCR (forward primer (5’A): 5’5 by gest on N ovem er 6, 2017 hp://w w w .jb.org/ D ow nladed from CGCATG GACACCCAGACCAG-3’, reverse primer (3’A): 5’CTAGAGAAGGAGTGAGG-GCTG-3’). The PCR conditions were: 94 °C for 30 sec, 56 °C for 30 sec, 72 °C for 30 sec for 35 rounds. Subsequent PCR amplification of the virally encoded insert in genomic DNA from transductant cell line 10-15 used primers which spanned the multiple cloning site of the retroviral vector (forward primer (5’B): 5’CCGCCCCGTCTCTCCCCCTTG-3’; reverse primer (3’B): 5’CTACAGGTGGGGTCTTTCATTCC-3’). Product was only observed with the following conditions: 100 nM 5’ and 3’ primers, 1x PCR enhancer (Life Technologies), 1.8 μM MgCl 2, 200 nM primers, 100 μM dNTPs, 250 ng genomic DNA, 1x Elongase (Life Technologies) Taq buffer and 2-5 units of Elongase Taq polymerase in a final volume of 50 μl with 35 cyles of PCR at 94 °C for 30 sec, 56 °C for 30 sec, 72 °C for 2 min and 30 sec. A 2.6 kb fragment amplified from the 10-15 cell line was subcloned into the pCR-II Topo vector (Invitrogen) and plasmid from four overnight colonies was sequenced in both directions. The insert sequence was analyzed using the BLAST program (www.ncbi.nlm.nih.gov/BLAST) and was also compared against the human genome project database. GlyB rescue hmft cDNA A PCR derived cDNA devoid of Taq polymerase-generated fidelity errors was subcloned into the NotI site of pcDNA3 (Invitrogen). Purified plasmid was transfected into glyB cells as a calcium phosphate precipitate (11,21). Two days after transfection, 1 mg/ml G418 was added to medium in the presence or absence of glycine. Plates were refed daily for 14 days, then colonies were fixed and stained. Northern Blotting RNA was isolated using the Trizol reagent (Life Technologies). Poly-A(+) RNA was prepared (Qiagen) and 10 μg of poly-A+ mRNA was run in each lane of a MOPSformamide gel (22). A 1.1 kb EcoRI fragment spanning the entire open reading frame of hMFT was 32P labelled by random priming and added to blots in hybridization mix at 2x 10 6 cpm/ml. An overnight 63°C hybridization was followed by two 15 minute room temperature washes in 2xSSPE (22), 0.1% SDS and a 15 minute 0.5x SSPE , 0.1% SDS wash at 63°C. 6 by gest on N ovem er 6, 2017 hp://w w w .jb.org/ D ow nladed from Subcellular distribution of cellular folates. Cell cultures (6 x10 6 per 175 cm2 flask) were grown for 48 hours in MEM supplemented with dialyzed fetal calf serum and 0.3 μCi/ml 3Hfolic acid (Moravek) that had been purified by hplc within one week of use. Cells were washed with 10 ml of PBS, detached with 5 ml of trypsin and 9 ml of PBS containing 5% FCS were added; cells were pelleted by centrifugation at 900 x G for 10 minutes. The pellet was washed twice with 10 ml of PBS containing FCS and resuspended in 2 ml homogenization solution (HMS, 0.25 M sucrose and 1 mM EDTA); a 0.5 ml aliquot of this suspension was counted for total cellular radioactivity. The remaining suspension was homogenized with 25 strokes in a hand-held Dounce homogenizer and centrifuged at 900 xG for 5 minutes. The post-nuclear supernatant (PNS) was removed, and the pellet (containing nuclei and unbroken cells) was resuspended in 2 ml of HMS and again homogenized. The nuclear pellet was collected at 900 xG for 5 minutes, the supernatant was added to the previous PNS, and the pellet was resuspended in 2 ml of HMS and recentrifuged. The supernatant was again added to the PNS fraction and the nuclear pellet was saved on ice. The PNS was centrifuged first at 900 x G for 5 minutes, the pellet was discarded, and the supernatant was centrifuged for 15 minutes at 10,000 x G. The supernatant (the cytosol fraction) and the pellet (the mitochondrial fraction) were saved. The pelleted fractions were dissolved at 37 °C in NaOH, acidified, and added to scintillation vials. Radioactivity in the various fractions was determined by scintillation counting Results Complementation of the gly B mutation. Pioneering studies by Puck and his colleagues established four complementation groups among CHO mutants auxotrophic for glycine (1618). One of these complementation groups, defined by the mutation responsible for the glycine auxotrophy of glyB Chinese hamster cells, had a defect in the ability to accumulate folates in mitochondria (15), a process required for the activity of mammalian mitochondrial SHMT (23). We used the gly B cell line as a recipient for transduction of a human cDNA library const

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), 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)(5)(6), and two of the transport systems have been cloned (7,8).In contrast, the mechanism of transfer of folates into mitochondria, presumably from the cytosol, is largely unknown as is the release of folates back to the cytosol from the mitochondria.Once folate monoglutamates, the form of folate found in the circulation, enter mammalian cells they are quickly metabolized to poly-γ-glutamate derivatives by cytosolic folylpolyl-γ-glutamate synthetase (FPGS), a process needed to promote 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 have demonstrated the penetration into isolated mitochondria by folates in a process that was saturable and temperature dependent (13,14).These studies would support the existence of a transporter responsible for entry of folates into the mitochondria, as does the fact that cells which either lack mitochondrial FPGS (11) or are incapable of accumulation of mitochondrial folates (15) are glycine auxotrophs.
Early studies by Puck and his colleagues selected somatic cells auxotrophic for glycine and demonstrated that they fell into four complementation groups, named gly A through D (16,17).Gly A was found to be due to a deficiency in mitochondrial SHMT (17,18).The Gly C and D mutations have not, to our knowledge, been assigned to functions.Gly B cells had normal cytosolic folate metabolism and enzymes and the same mitochondrial SHMT and FPGS as CHO cells, but lacked mitochondrial folates (15) .The most likely candidate for the function missing in glyB cells was a transport protein which faciliated the entry of folates into mitochondria.
We have transferred a library of human cDNAs in a retroviral vector into glyB cells and isolated a transduced cell line no longer auxotrophic for glycine.These cells contained a human cDNA which, when rescued by PCR and recloned into a mammalian expression plasmid, complemented the auxotrophy of glyB cells at high frequency and also reinstated folate entry into mitochondria.We conclude that we have isolated the human gene encoding the inner membrane protein responsible for entry of folates into mitochondria.

Materials and Methods
Cell culture.Cells were grown in 37°C in 5% CO2.The glyB cell line, which was derived from CHO-K1 cells (16,17) , was a generous gift of Dr Lawrence Chasin (Columbia University, NY, NY).HEK293gp packaging cells (a gift from Dr Oliver Bogler of this University) were grown in DMEM, glyB and CHO-K1 in MEM, and CEM in RPMI-1640, all containing 10 % fetal calf serum.For virus production, HEK293gp cells were plated at 5 x10 6 cells on each of twelve 100 mm dishes.Forty eight hours later, 15 µg of plasmid library DNA, 15 µg of pVSVG plasmid (Dr Oliver Bogler), which allows pseudeotyping of the viral envelope proteins (19,20), and 55 µg of Lipofectamine-2000 (Life Technologies) were added to each dish in DMEM.After 20 min at room temperature, the medium was replaced with 25 µM chloroquine in DMEM and 1.5 ml of the lipid-DNA complex was added to each plate.Media was changed to complete DMEM after 5 h at 37 °; the supernatant, containing the collection of viruses from the library, was harvested after 36 and 72 hours and filtered.Transduction efficiency and viral titer were determined by monitoring fluorescent cells on control plates transduced with a virus expressing the enhanced green fluorescent protein (Clontech).GlyB cells (2 x 10 6 ) were suspended in 9 ml MEM and 7 ml of viral supernatant and plated on each of ten 150 mm dishes; after 5 hours the medium was replaced with complete MEM.After 48 hours at 37°, selection in glycine-free MEM containing dialyzed serum was applied.After 10-14 days, colonies were expanded in glycine-free medium.

PCR analysis and rescue of transductants
The presence of a 200 base pair fragment of the retroviral ψ packaging signal in genomic DNA was verified by PCR (forward primer (5'A): 5'- GlyB rescue hmft cDNA A PCR derived cDNA devoid of Taq polymerase-generated fidelity errors was subcloned into the NotI site of pcDNA3 (Invitrogen).Purified plasmid was transfected into glyB cells as a calcium phosphate precipitate (11,21).Two days after transfection, 1 mg/ml G418 was added to medium in the presence or absence of glycine.Plates were refed daily for 14 days, then colonies were fixed and stained.
Northern Blotting RNA was isolated using the Trizol reagent (Life Technologies).Poly-A(+) RNA was prepared (Qiagen) and 10 µg of poly-A+ mRNA was run in each lane of a MOPSformamide gel (22).A 1.1 kb EcoRI fragment spanning the entire open reading frame of hMFT was 32 P labelled by random priming and added to blots in hybridization mix at 2x 10 6 cpm/ml.
Subcellular distribution of cellular folates.Cell cultures (6 x10 6 per 175 cm 2 flask) were grown for 48 hours in MEM supplemented with dialyzed fetal calf serum and 0.3 µCi/ml 3 Hfolic acid (Moravek) that had been purified by hplc within one week of use.Cells were washed with 10 ml of PBS, detached with 5 ml of trypsin and 9 ml of PBS containing 5% FCS were added; cells were pelleted by centrifugation at 900 x G for 10 minutes.The pellet was washed twice with 10 ml of PBS containing FCS and resuspended in 2 ml homogenization solution (HMS, 0.25 M sucrose and 1 mM EDTA); a 0.5 ml aliquot of this suspension was counted for total cellular radioactivity.The remaining suspension was homogenized with 25 strokes in a hand-held Dounce homogenizer and centrifuged at 900 xG for 5 minutes.The post-nuclear supernatant (PNS) was removed, and the pellet (containing nuclei and unbroken cells) was resuspended in 2 ml of HMS and again homogenized.The nuclear pellet was collected at 900 xG for 5 minutes, the supernatant was added to the previous PNS, and the pellet was resuspended in 2 ml of HMS and recentrifuged.The supernatant was again added to the PNS fraction and the nuclear pellet was saved on ice.The PNS was centrifuged first at 900 x G for 5 minutes, the pellet was discarded, and the supernatant was centrifuged for 15 minutes at 10,000 x G.The supernatant (the cytosol fraction) and the pellet (the mitochondrial fraction) were saved.The pelleted fractions were dissolved at 37°C in NaOH, acidified, and added to scintillation vials.Radioactivity in the various fractions was determined by scintillation counting

Complementation of the gly B mutation. Pioneering studies by Puck and his colleagues
established four complementation groups among CHO mutants auxotrophic for glycine (16)(17)(18).One of these complementation groups, defined by the mutation responsible for the glycine auxotrophy of glyB Chinese hamster cells, had a defect in the ability to accumulate folates in mitochondria (15), a process required for the activity of mammalian mitochondrial SHMT (23).
We used the gly B cell line as a recipient for transduction of a human cDNA library constructed in the pLIB retroviral vector (Clontech).After transduction of 2 x 10 7 glyB cells with this human placental cDNA library, an isolated colony of rapidly and progressively growing glycine prototrophic cells was isolated and cloned.Attempts to isolate revertants from glyB cells had been unsuccessful with similar levels of cells, and control plates transduced with a retrovirus encoding green fluorescent protein, likewise, did not yield colonies in the absence of glycine.
The glyB transductant was expanded and the presence of a DNA sequence of retroviral origin was sought by PCR against genomic DNA.The ψ packaging signal between the viral LTRs flanking the cloning site of this vector was easily detected in DNA from this cell line, designated 10-15, but not in control glyB cells (Fig. 1).Further PCR using primers designed against the upstream flanking ψ sequence and the downstream multiple cloning site cassette indicated the presence of a 2.6 kb DNA sequence (Fig. 1) of human origin (see below). in the open reading frame (Fig. 2B).An ATP binding motif (27) was not found.The sequence immediately surrounding the ATG at nt 118-120 in this sequence formed only a fair match to consensus for a mammalian translational initiation codon (28), about at the level we previously found with a start methionine used by mouse and human cells for translation of mitochondrial folylpolyglutamate synthetase (11,12).It was noted that the sequence upstream of the putative translational start methionine was in frame, leaving open the possibility of additional upstream coding region and another upstream start methionine.To address this possibility, 5'-rapid amplification of cDNA ends (RACE) (29) was applied to cDNA prepared from CEM cell RNA.

Sequence homology with mitochondrial carrier proteins.
As much as an additional 54 codons of upstream sequence was detected in these RACE products, but a translational start codon was not found in any frame.Overall, the primary sequence of the protein encoded by this cDNA, which had a molecular mass of 35,387 daltons, suggested that it was an inner mitochondrial membrane transporter that did not require ATP for function.
A GenBank search revealed similarity of the protein encoded by the open reading frame we now report, which we name hmft, with the sequence of several carriers involved in the transport of metabolic intermediates across the inner mitochondrial membrane.The highest homologies were with the yeast flx 1 mitochondrial carrier protein for flavin nucleotides (Genbank protein accession number NP_012132.1)(Fig.3), carrier proteins involved in ATP/ADP exchange in rodent and human mitochondria (P12236, P51881, Q09073)(Fig.3), the yeast mitochondrial oxaloacetate transport carrier (NP_012802.1), and the mitochondrial uncoupling protein from various mammalian species (e.g., O97649, BAA28832.RNA hybridized well with this probe.Hence, the library transduction experiment appears to have identified a full-length functional mRNA sequence expressed in human cells.

Human genome project entries corresponding to this cDNA.
A search of the human genome project revealed that the gene corresponding to the hmft sequence was found on human by guest on November 6, 2017 http://www.jbc.org/Downloaded from chromosome 8 on contig RP11-1C8 (accession number AC0112213), corresponding to position 8q21.2.The available chromosome 8 sequence allowed an assignment of the organization of this gene into e 8 exons, spred over > 7 kb (Fig. 4C).This agrees with early results from cellcell hybridization experiments which mapped the glyB defect to the long arm of chromosome 8 (30,31).
Functional complementation of the glyB defect by hmft cDNA.The 2.6 kb hmft cDNA was recloned into the mammalian expression vector pcDNA3 and transfected into gly B cells.
Transfectants were screened for neomycin resistance and for glycine prototrophy.About 30 % of the transfectants which expressed G418 resistance demonstrated prototrophy for glycine (Fig

5.
); in contrast, no colonies were found on the minus glycine plates transfected with the pcDNA3 vector alone.The retroviral transductant, cell line 10-15, in which the gly B phenotype was rescued, also had reacquired the ability to translocate folates into mitochondria, as did cell lines cloned from isolated colonies on plates of glyB cells transfected with the hmft cDNA in pcDNA3 (Table 1).Hence, the small protein encoded by the single open reading frame in the hmft cDNA complemented the mitochondrial transport function deficient in gly B cells, and simultaneously relieved the prototrophy of these mutants.

Discussion
We herein report the isolation of a novel gene encoding a protein which facilitates the translocation of folates from the cytosol into the mitochondrial matrix of mammalian cells.The cDNA encoding this protein was isolated from a human library by complementation of a mutant cell line which lacked this function (15).The cDNA complemented both the cellular phenotype, converting transfectants to prototrophy for glycine (Fig. 4), and the biochemical deficiency, allowing folate transport into mitochondria in transfectants (Table 1).This appears to rule out complementation of the auxotrophy by an indirect effect of the expressed gene.
Overall, this manuscript describes the first human protein identified to transport folates or, to our knowledge, any vitamin across the inner mitochondrial membrane (32).
The family of inner mitochondrial membrane transport proteins from yeast and vertebrates consists of several well characterized members which transport cations (glutamine, acylcarnitines/carnitine, spermine, and ornithine), anions (ATP/ADP, oxoglutarate, citrate, pyruvate, dicarboxylates, Pi, aspartate/glutarate, and branched keto acids) and flavins, as well as the H+ ions involved in the uncoupling reaction; each of these proteins is a six TM domain transporter (33)(34)(35)(36)(37).The marked similarities between these proteins have been reviewed (24,25).The active form of the carriers is thought to form homodimers (38) which together create a 12 TM unit similar to plasma membrane transporters such as the reduced folate carrier into the inner membrane (43,44).The hMFT carrier contains three repeat segments each containing a consensus ES motif located precisely after TM domains 1, 3, and 5 (Fig. 2).
Interestingly, the first ES motif , located after TM1, is a perfect match to consensus, while the several proteins homologous to hMFT, the closest related of which were YEL006w and YIL006w (Fig. 3), which were each 27 % identical to hMFT at the amino acid level.However, when we cloned these genes from yeast DNA by PCR and transfected them individually and together into glyB cells in the pcDNA3 vector, they were unable to support the growth of glyB cells under selective conditions (data not shown).A BLAST search performed against the C. elegans and Drosophila genomes using the hMFT protein sequence as probe, identified at least 25 related members in each genome on the basis of homology in the placement of ES motifs in a tripartitite protein and the position of critical amino acids.
It is interesting to note that glyB cells do not accumulate folates in the cytosol to the same extent as do the wild type CHO-K1 cells from which they were derived nor the CHOderived viral transductant or plasmid transfectants with the hmft gene (Table 1).This would appear to support the concept that folate polyglutamates made in the mitochondria can support folate metabolism in the cytosol of mammalian cells, apparently by direct transfer of folate polyglutamates intact from the mitochondria.This transfer had been hypothesized (45) on the basis of the fact that the naturally-occurring transcripts for mitochondrial FPGS (11,12) or, indeed, artificial transcripts constructed by ligation of model mitochondrial leader sequences to human cytosolic FPGS (46) can support both cytosolic and mitochondrial folate metabolism.
At face value, the data of Table 1 suggests that half of the folate pool in the cytosol stems from the activity of mitochondrial FPGS.This raises questions about the ability of folate antimetabolites to use the hMFT and also whether mitochondrial folate polyglutamates use this transporter for efflux to the cytosol.
The fact that mammalian cells which lack folate transport into mitochondria are glycine auxotrophs (15) but can otherwise survive leads to the somewhat surprising conclusion that all mitochondrial folate metabolism can be dispensed with, except for the folate-dependent supply of glycine.The glyB phenotype is not peculiar to these cells: AUXB1 cells, which lack all FPGS activity, can be reconstituted with cDNAs encoding only the cytosolic isoform of FPGS; these transfectants have intact cytosolic folate metabolism but are unable to accumulate folates in the mitochondria and are simple glycine auxotrophs (11,12).Apparently, exogenous glycine can be delivered to the mitochondria, presumably through the cytosol, and can facilely penetrate the outer and inner mitochondrial membranes.Hence, the glycine auxotrophy of glyB cells indicates that cytosolic SHMT cannot supply glycine for mitochondrial metabolism at any appreciable concentration.This is in accord with current thought that cytosolic SHMT is kinetically controlled and funnels serine to glycine for the production of 5,10methylenetetrahydrofolate for cytosolic one carbon metabolism (47); however, other reactions, e.g. de novo purine synthesis and cytosolic protein synthesis, must consume cytosolic glycine as soon as it is produced.It should be noted that the experiments of Taylor and Hanna showed that the glycine auxotrophy of the glyB cells can be reversed by high concentrations of 5formyltetrahydrofolate (15), a fact which has never been explained.Thus, the operation of mitochondrial folate metabolism and the related interconversions of glycine and serine, have yet to be well understood, in spite of decades of experimental work.

Legends
The integrated cDNA flanked by viral sequences, which was associated with rescue of the gly B phenotype, encoded an open reading frame of 945 base pairs with 1500 bp of apparent 3'-untranslated region and 112 bp upstream of the first methionine codon; the sequence has been submitted to GenBank and can be retrieved under accession number AF283645.A hydropathy plot of the 315 amino acid protein encoded by the single open reading frame in this sequence (Fig. 2B) suggested that the protein consisted of six transmembrane (TM) domains and three interspersed loops facing the mitochondrial matrix.In addition, a motif identified as the energy transfer sequence (ES) commonly found in mitochondrial inner membrane proteins (24-26) was repeated three times

BAA90458. 1 )
(see Discussion).When the sequence of this 2.6 kb cDNA was used to search the expressed sequence tag (dbEST) database, several partial sequences of human origin identical to portions of our clone were identified (Fig 4A); the dbEST sequences stemmed from libraries constructed from several different tissue types (see Legend to Fig.4), indicating a broad pattern of tissue distribution of the corresponding mRNA.When a 1.1 kb EcoR1 fragment of this cDNA was used to probe polyA (+) RNA from CEM human leukemia cells on Northern blots, a 1.5 kb mRNA species was detected, as well as diffuse hybridization between 2.4 and 2.6 kb (Fig.4B).Likewise, the matching dbEST entries initiated either at the 3'-end of the cloned sequence or 1500 bases into the sequence (Fig.4A), corresponding to the pattern seen in the Northern analysis of expressed mRNA transcripts.Neither glyB cell nor CHO cell polyA(+)

( 39 )
and the thiamine carrier(40,41) as well as mitochondrial carriers such as the major intrinsic protein family and the major facilitator superfamily(25).The predicted hMFT protein has several properties which identify it as a new member of the family of inner mitochondrial membrane transport proteins.It has a predicted size within the range for the several previously reported inner mitochondrial membrane transporters (28-34 kDa)(24,25,(33)(34)(35)(36)(37), and has a predicted primary structure suggestive of six TM domains.Inner mitochondrial membrane transporters can be divided into three repeated segments each of about 100 amino acids in length.The repeats consists of 2 TM domains and a mitochondrial ES motif:PX[D/E]X[L/I/V/A/T][R/K]X[L/R/H]-[L/I/V/M/F/Y][Q/G/A/I/V/M](24,26,42) which is found immediately following TM domains 1, 3, and 5.These signals are thought to be required for mitochondrial targeting, translocation across the outer mitochondrial membrane, and insertion second and third motifs do not have a D or E at position 3.The second motif has a W at position 3 and the third has a Q; these same deviations from the overall consensus is seen for the flx-related family members (Fig3).Given that the function of the second and third ES signals is thought to involve binding to inner membrane proteins (TIMs) necessary for tracking to and insertion into the inner mitochondrial membrane(44), this structural difference apparently unique to the flx subfamily is very intriguing.A recent comparison of sequence homologies among 200 recognizable mitochondrial carrier open reading frames (42) identified a small group of proteins which have PIW or PLW in the second energy signature motif; hMFT clearly belongs to this subgroup.Alignment of these repeats in hMFT with other members of the PIW-PLW subgroup of this protein family revealed regions of high homology and several residues that are absolutely conserved among the family members (Fig.3).Thus, in addition to the PX(D/E)XX(R/K) energy signature immediately following TMs 1,3, and 5, there are two other recognizable conserved regions in this limited series: an ES-related weaker consensus of Y(D/E)XX(K/R) is located after TMs 2, 4, and 6, and a (D/E)GX(R/K)G-(L/F)Y(K/R)G motif located immediately before TMs 2, 4, and 6.These homologies help the assignment of TM domains within the sequence and presumably involve interactions essential to transporter membrane positioning and function, such as the charge interactions previously described(42).A closely related sequence can be found in any of several genome databases, and other putative family members were easily identified by computer searches against those genomes sequenced to date.A systematic search of the yeast database (42) identified 35 inner mitochondrial membrane transporters based on homology to known family members of which the function of eight can currently be assigned.BLAST searches of the yeast genome revealed

Figure 1 .
Figure 1.Detection of viral insertion in 10-15 cells.Cell line 10-15, a clone that progressively grew on plates of glyB cells transduced with viruses produced from a human placental library was isolated and mass-cultured as described in the text.A. PCR amplification of a 200 bp fragment of the viral psi (ψ) packaging signal, using primers placed at positions 5'A and 3'A, detected integrated virus in 10-15 cells (Lane 1) but not glyB cells (Lane 2).B. Detection of a 2.6 Kb cDNA insert in integrated viral sequences by PCR using primers located at positions 5'B and 3'B in 10-15 cells (Lane 4) but not glyB cells (Lane 5).Lane 3 demonstrates the detection of 2 fmol of a control virus with a 2.2 kb insert added to glyB genomic DNA.The band shown in lane 4 was cloned and sequenced.The molecular mass standards (STD lane) were a mixture of λ HinDIII and ΦX174/HaeIII fragments.

Figure 2 .
Figure 2. Sequence and structure of the hMFT protein.(A) Predicted amino acid sequence of the cDNA rescued by PCR from 10-15 cells.The predicted transmembrane domains (TM) are overlined and the energy signals are boxed.(B) Hydropathy plot of the hMFT protein.Predicted TM domains are indicated in roman numerals.In this figure, codon number (abscissa) is plotted against the hydrophobicity (positive values) or hydrophilicity (negative values of a sliding window of residues, using the TopPred 2 algorithm (48).

Figure 3 .
Figure 3. Sequence alignment of hMFT with the human mitochondrial ATP/ADP exchanger (hAAC), the uncharacterized yeast mitochondrial protein yil006w, and the yeast inner mitochondrial membrane flavin transporter (flx1).The boxed peptides represent the energy signal motifs.Sequences were aligned using the Clustal V alignment routine (49) of the DNA* program.The initial 28-60 codons, which showed low homology are not shown.Dashes indicate gaps in the alignment, and residues identical to consensus are shaded in black.

Figure 4 .
Figure 4. Transcripts from and intron-exon structure of the hmft gene.A. The positions of dbEST entries identical to the hmft cDNA are mapped to the length of the cDNA insert in virus

Figure 5 .
Figure 5. Complementation of the glycine auxotrophy of glyB cells by transfection of the hmft cDNA.After transfection with either pcDNA3 or the hmrt cDNA cloned into the pcDNA3 vector, plates were cultured either in medium containing G418 (-glycine) or glycine plus G418, and colonies were fixed and stained after 14 days.The plates shown are representative of triplicate dishes from each of two experiments.

Table 1
Distribution of3H-folates in mitochondrial and cytosolic fractions of CHO derivatives.The indicated cell lines were grown for 48 hours in MEM medium containing 0.3 µCi/ml of purified 3 H-folic acid.A fraction of the cells were harvested and washed, and total radioactive folates determined by liquid scintillation counting (Total).The remaining cells were homogenized, a Nuclear and Unbroken Cells fraction was separated and the supernatant fraction was further fractionated into Mitochondria and Cytosol components as described in Materials and Methods.Three cultures were used for each cell line in each of two experiments; 15-30 million cells were in the cultures harvested for analysis.