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• W2019686818 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. 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. serine hydroxymethyltransferase folylpoly-γ-glutamate synthetase Chinese hamster ovary polymerase chain reaction minimum Eagle's medium human mitochondrial folate transporter gene kilobase kilobase pair transmembrane energy transfer sequence rapid amplification of cDNA ends expressed sequence tag database group of overlapping clones 4-morpholinepropanesulfonic acid In mammalian cells, the processes of folate metabolism are distributed between the cytosolic and mitochondrial compartments (1Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (296) Google Scholar). Mitochondrial folates amount to about 35% of the total cellular pool (2Cook R.J. Blair J.A. Biochem. J. 1979; 178: 651-659Crossref PubMed Scopus (26) Google Scholar, 3Wang F.K. Kock J. Stokstad E.L.R. Biochem. Z. 1967; 346: 458-466PubMed Google Scholar) 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 (4Kamen B.A. Wang M. Streckfuss A.J. Peryea X. Anderson R.G.W. J. Biol. Chem. 1988; 263: 13602-13609Abstract Full Text PDF PubMed Google Scholar, 5Henderson G.B. Strauss B.P. Cancer Res. 1990; 50: 1709-1714PubMed Google Scholar, 6Goldman I.D. Lichtenstein N.S. Oliverio V.T. J. Biol. Chem. 1968; 243: 5007-5017Abstract Full Text PDF PubMed Google Scholar), and two of the transport systems have been cloned (7Dixon K.H. Lanpher B.C. Chiu J. Kelley K. Cowan K.H. J. Biol. Chem. 1994; 269: 17-20Abstract Full Text PDF PubMed Google Scholar, 8Brigle K.E. Spinella M.J. Westin E.H. Goldman I.D. Biochem. Pharmacol. 1994; 47: 337-345Crossref PubMed Scopus (70) Google Scholar). 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-γ-glutamate derivatives by cytosolic folylpoly-γ-glutamate synthetase (FPGS), a process needed to promote the retention of folate cofactors in cells (9Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (289) Google Scholar). FPGS is also present in the mitochondrial compartment of mammalian cells (10Lin B.-F. Huang R.-F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar) translated from transcripts from the FPGS gene, which add a mitochondrial leader sequence to the coding region of the protein found in the cytosol (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar,12Turner F. Andreassi J. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar). Two studies (13Horne D.W. Holloway R.S. Said H.M. J. Nutr. 1992; 122: 2204-2209Crossref PubMed Scopus (44) Google Scholar, 14Cybulski R.L. Fisher R.R. Biochim. Biophys. Acta. 1981; 646: 329-333Crossref PubMed Scopus (28) Google Scholar) 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 (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) or are incapable of accumulation of mitochondrial folates (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar) are glycine auxotrophs.Early studies by Puck and coworkers (16Kao F.T. Puck T.T. Genetics. 1975; 79: 343-352PubMed Google Scholar, 17Kao F.T. Chasin L. Puck T.T. Proc. Natl. Acad. Sci. U. S. A. 1969; 64: 1284-1291Crossref PubMed Scopus (99) Google Scholar) 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 (17Kao F.T. Chasin L. Puck T.T. Proc. Natl. Acad. Sci. U. S. A. 1969; 64: 1284-1291Crossref PubMed Scopus (99) Google Scholar, 18Chasin L.A. Feldman A. Konstam M. Urlaub G. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 718-722Crossref PubMed Scopus (70) Google Scholar). 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 FPGS as CHO cells, but they lacked mitochondrial folates (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar). The most likely candidate for the function missing inglyB cells was a transport protein that facilitated the entry of folates into the mitochondria.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 glyBcells at high frequency and also 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.DISCUSSIONWe hereby report the isolation of a novel gene encoding a protein that 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar). 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 I). This finding 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 (32Scharfe C. Zaccaria P. Hoertnagel K. Jaksch M. Klopstock T. Dembowski M. Lill R. Prokish H. Gerbitz K.D. Neupert W. Mewes H.W. Meitinger T. Nucleic Acids Res. 2000; 28: 155-158Crossref PubMed Scopus (70) Google Scholar).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 (33Saraste M. Walker J.E. FEBS Lett. 1982; 144: 250-254Crossref PubMed Scopus (240) Google Scholar, 34Aquila H. Link T.A. Klingenberg M. EMBO J. 1985; 4: 2369-2376Crossref PubMed Scopus (277) Google Scholar, 35Runswick M.J. Walker J.E. Bisaccia F. Iacobazzi V. Palmieri F. Biochemistry. 1990; 29: 11033-11040Crossref PubMed Scopus (140) Google Scholar, 36Runswick M.J. Powell S.J. Nyren P. Walker J.E. EMBO J. 1987; 6: 1367-1373Crossref PubMed Scopus (181) Google Scholar, 37Aquila H. Misra D. Eulitz M. Klingenberg M. Hoppe-Seyler's Z. Physiol. Chem. 1982; 363: 345-349Crossref PubMed Scopus (184) Google Scholar). The marked similarities among these proteins have been reviewed (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar). The active form of the carriers is thought to form homodimers (38Klingenberg M. Nature. 1981; 290: 449-454Crossref PubMed Scopus (214) Google Scholar), which together create a 12-TM unit similar to plasma membrane transporters, such as the reduced folate carrier (39Dixon K.H. Lanpher B.C. Chiu J. Kelley K. Cowan K. J. Biol. Chem. 1994; 269: 17-20Abstract Full Text PDF PubMed Google Scholar) and the thiamine carrier (40Fleming J.C. Tartaglini E. Steinkamp M.P. Schorderet D.F. Cohen N. Neufeld E.J. Nat. Genet. 1999; 22: 305-308Crossref PubMed Scopus (203) Google Scholar, 41Diaz G.A. Banikazemi M. Oishi K. Desnick R.J. Gelb B.D. Nat. Genet. 1999; 22: 309-312Crossref PubMed Scopus (178) Google Scholar), as well as mitochondrial carriers, such as the major intrinsic protein family and the major facilitator superfamily (25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar). The predicted hMFT protein has several properties, which identify it as a new member of the family of inner mitochondrial membrane transport proteins. The protein has a predicted size within the range for the several previously reported inner mitochondrial membrane transporters (28–34 kDa) (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar, 33Saraste M. Walker J.E. FEBS Lett. 1982; 144: 250-254Crossref PubMed Scopus (240) Google Scholar, 34Aquila H. Link T.A. Klingenberg M. EMBO J. 1985; 4: 2369-2376Crossref PubMed Scopus (277) Google Scholar, 35Runswick M.J. Walker J.E. Bisaccia F. Iacobazzi V. Palmieri F. Biochemistry. 1990; 29: 11033-11040Crossref PubMed Scopus (140) Google Scholar, 36Runswick M.J. Powell S.J. Nyren P. Walker J.E. EMBO J. 1987; 6: 1367-1373Crossref PubMed Scopus (181) Google Scholar, 37Aquila H. Misra D. Eulitz M. Klingenberg M. Hoppe-Seyler's Z. Physiol. Chem. 1982; 363: 345-349Crossref PubMed Scopus (184) Google Scholar) 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 consist of two 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) (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 26Bairoch A. Nucleic Acids Res. 1992; 20: 2013-2018Crossref PubMed Scopus (424) Google Scholar, 42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar), which is found immediately after TM domains 1, 3, and 5. These signals are thought to be required for mitochondrial targeting, translocation across the outer mitochondrial membrane, and insertion into the inner membrane (43Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 44Endres M. Neupert W. Brunner M. EMBO J. 1999; 18: 3214-3221Crossref PubMed Scopus (149) Google Scholar). 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 the consensus, whereas the second and third motifs do not have a Asp or Glu at position 3. The second motif has a Trp at position 3, and the third motif has a Gln at position 3; these same deviations from the overall consensus are seen for the flx-related family members (Fig. 3). Given that the function of the second and third ES signals is thought to involve binding to inner membrane proteins necessary for tracking and insertion into the inner mitochondrial membrane (44Endres M. Neupert W. Brunner M. EMBO J. 1999; 18: 3214-3221Crossref PubMed Scopus (149) Google Scholar), 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 (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar) 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 after 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 is located immediately before TMs 2, 4, and 6. These homologies help the assignment of TM domains within the sequence and presumably involve interactions that are essential to transporter membrane positioning and function, such as the charge interactions previously described (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar).Open reading frames closely related to hMFT were easily identified by computer searches against those genomes sequenced to date. A systematic search of the yeast database (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar) 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 several proteins homologous to hMFT. The most closely related yeast putative transporters 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 intoglyB 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 theCaenorhabditis elegans and Drosophila genomes using the hMFT protein sequence as probe, identified at least 25 related members in each genome based on homology in the placement of ES motifs in a tripartite 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 either the wild type CHO-K1 cells from which they were derived or the CHO-derived viral transductant or plasmid transfectants with the hmft gene (Table I). This observation 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 that are intact from the mitochondria. This transfer had been hypothesized (45Qi H.A.I. Xiao S. Choi Y.J. Tobimatsu T. Shane B. Adv. Enzyme Regul. 1999; 39: 263-273Crossref PubMed Scopus (15) Google Scholar) based on the fact that the naturally occurring transcripts for mitochondrial FPGS (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Turner F. Andreassi J. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar) or indeed the artificial transcripts constructed by ligation of model mitochondrial leader sequences to human cytosolic FPGS (46Chen L. Qi H. Korenberg J. Garrow T.A. Choi Y. Shane B. J. Biol. Chem. 1996; 271: 13077-13087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) can support both cytosolic and mitochondrial folate metabolism. At face value, the data of Table I suggest that half of the folate pool in the cytosol stems from the activity of mitochondrial FPGS. This fact 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar) 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 glyBphenotype 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 (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Turner F. Andreassi J. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar). Apparently, exogenous glycine can be delivered to the mitochondria, presumably through the cytosol, and can easily penetrate the outer and inner mitochondrial membranes. Hence, the glycine auxotrophy ofglyB cells indicates that cytosolic SHMT cannot supply glycine for mitochondrial metabolism at any appreciable concentration. This finding is in accordance with the current thought that cytosolic SHMT is kinetically controlled and funnels serine to glycine for the production of 5,10-methylenetetrahydrofolate for cytosolic 1-carbon metabolism (47Kastanos E.K. Woldman Y.Y. Appling D.R. Biochemistry. 1997; 36: 14956-14964Crossref PubMed Scopus (84) Google Scholar); 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar) showed that the glycine auxotrophy of the glyB cells can be reversed by high concentrations of 5-formyltetrahydrofolate, a fact that 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 despite decades of experimental work. In mammalian cells, the processes of folate metabolism are distributed between the cytosolic and mitochondrial compartments (1Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (296) Google Scholar). Mitochondrial folates amount to about 35% of the total cellular pool (2Cook R.J. Blair J.A. Biochem. J. 1979; 178: 651-659Crossref PubMed Scopus (26) Google Scholar, 3Wang F.K. Kock J. Stokstad E.L.R. Biochem. Z. 1967; 346: 458-466PubMed Google Scholar) 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 (4Kamen B.A. Wang M. Streckfuss A.J. Peryea X. Anderson R.G.W. J. Biol. Chem. 1988; 263: 13602-13609Abstract Full Text PDF PubMed Google Scholar, 5Henderson G.B. Strauss B.P. Cancer Res. 1990; 50: 1709-1714PubMed Google Scholar, 6Goldman I.D. Lichtenstein N.S. Oliverio V.T. J. Biol. Chem. 1968; 243: 5007-5017Abstract Full Text PDF PubMed Google Scholar), and two of the transport systems have been cloned (7Dixon K.H. Lanpher B.C. Chiu J. Kelley K. Cowan K.H. J. Biol. Chem. 1994; 269: 17-20Abstract Full Text PDF PubMed Google Scholar, 8Brigle K.E. Spinella M.J. Westin E.H. Goldman I.D. Biochem. Pharmacol. 1994; 47: 337-345Crossref PubMed Scopus (70) Google Scholar). 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-γ-glutamate derivatives by cytosolic folylpoly-γ-glutamate synthetase (FPGS), a process needed to promote the retention of folate cofactors in cells (9Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (289) Google Scholar). FPGS is also present in the mitochondrial compartment of mammalian cells (10Lin B.-F. Huang R.-F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar) translated from transcripts from the FPGS gene, which add a mitochondrial leader sequence to the coding region of the protein found in the cytosol (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar,12Turner F. Andreassi J. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar). Two studies (13Horne D.W. Holloway R.S. Said H.M. J. Nutr. 1992; 122: 2204-2209Crossref PubMed Scopus (44) Google Scholar, 14Cybulski R.L. Fisher R.R. Biochim. Biophys. Acta. 1981; 646: 329-333Crossref PubMed Scopus (28) Google Scholar) 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 (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) or are incapable of accumulation of mitochondrial folates (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar) are glycine auxotrophs. Early studies by Puck and coworkers (16Kao F.T. Puck T.T. Genetics. 1975; 79: 343-352PubMed Google Scholar, 17Kao F.T. Chasin L. Puck T.T. Proc. Natl. Acad. Sci. U. S. A. 1969; 64: 1284-1291Crossref PubMed Scopus (99) Google Scholar) 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 (17Kao F.T. Chasin L. Puck T.T. Proc. Natl. Acad. Sci. U. S. A. 1969; 64: 1284-1291Crossref PubMed Scopus (99) Google Scholar, 18Chasin L.A. Feldman A. Konstam M. Urlaub G. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 718-722Crossref PubMed Scopus (70) Google Scholar). 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 FPGS as CHO cells, but they lacked mitochondrial folates (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar). The most likely candidate for the function missing inglyB cells was a transport protein that facilitated the entry of folates into the mitochondria. 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 glyBcells at high frequency and also 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. DISCUSSIONWe hereby report the isolation of a novel gene encoding a protein that 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar). 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 I). This finding 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 (32Scharfe C. Zaccaria P. Hoertnagel K. Jaksch M. Klopstock T. Dembowski M. Lill R. Prokish H. Gerbitz K.D. Neupert W. Mewes H.W. Meitinger T. Nucleic Acids Res. 2000; 28: 155-158Crossref PubMed Scopus (70) Google Scholar).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 (33Saraste M. Walker J.E. FEBS Lett. 1982; 144: 250-254Crossref PubMed Scopus (240) Google Scholar, 34Aquila H. Link T.A. Klingenberg M. EMBO J. 1985; 4: 2369-2376Crossref PubMed Scopus (277) Google Scholar, 35Runswick M.J. Walker J.E. Bisaccia F. Iacobazzi V. Palmieri F. Biochemistry. 1990; 29: 11033-11040Crossref PubMed Scopus (140) Google Scholar, 36Runswick M.J. Powell S.J. Nyren P. Walker J.E. EMBO J. 1987; 6: 1367-1373Crossref PubMed Scopus (181) Google Scholar, 37Aquila H. Misra D. Eulitz M. Klingenberg M. Hoppe-Seyler's Z. Physiol. Chem. 1982; 363: 345-349Crossref PubMed Scopus (184) Google Scholar). The marked similarities among these proteins have been reviewed (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar). The active form of the carriers is thought to form homodimers (38Klingenberg M. Nature. 1981; 290: 449-454Crossref PubMed Scopus (214) Google Scholar), which together create a 12-TM unit similar to plasma membrane transporters, such as the reduced folate carrier (39Dixon K.H. Lanpher B.C. Chiu J. Kelley K. Cowan K. J. Biol. Chem. 1994; 269: 17-20Abstract Full Text PDF PubMed Google Scholar) and the thiamine carrier (40Fleming J.C. Tartaglini E. Steinkamp M.P. Schorderet D.F. Cohen N. Neufeld E.J. Nat. Genet. 1999; 22: 305-308Crossref PubMed Scopus (203) Google Scholar, 41Diaz G.A. Banikazemi M. Oishi K. Desnick R.J. Gelb B.D. Nat. Genet. 1999; 22: 309-312Crossref PubMed Scopus (178) Google Scholar), as well as mitochondrial carriers, such as the major intrinsic protein family and the major facilitator superfamily (25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar). The predicted hMFT protein has several properties, which identify it as a new member of the family of inner mitochondrial membrane transport proteins. The protein has a predicted size within the range for the several previously reported inner mitochondrial membrane transporters (28–34 kDa) (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar, 33Saraste M. Walker J.E. FEBS Lett. 1982; 144: 250-254Crossref PubMed Scopus (240) Google Scholar, 34Aquila H. Link T.A. Klingenberg M. EMBO J. 1985; 4: 2369-2376Crossref PubMed Scopus (277) Google Scholar, 35Runswick M.J. Walker J.E. Bisaccia F. Iacobazzi V. Palmieri F. Biochemistry. 1990; 29: 11033-11040Crossref PubMed Scopus (140) Google Scholar, 36Runswick M.J. Powell S.J. Nyren P. Walker J.E. EMBO J. 1987; 6: 1367-1373Crossref PubMed Scopus (181) Google Scholar, 37Aquila H. Misra D. Eulitz M. Klingenberg M. Hoppe-Seyler's Z. Physiol. Chem. 1982; 363: 345-349Crossref PubMed Scopus (184) Google Scholar) 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 consist of two 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) (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 26Bairoch A. Nucleic Acids Res. 1992; 20: 2013-2018Crossref PubMed Scopus (424) Google Scholar, 42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar), which is found immediately after TM domains 1, 3, and 5. These signals are thought to be required for mitochondrial targeting, translocation across the outer mitochondrial membrane, and insertion into the inner membrane (43Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 44Endres M. Neupert W. Brunner M. EMBO J. 1999; 18: 3214-3221Crossref PubMed Scopus (149) Google Scholar). 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 the consensus, whereas the second and third motifs do not have a Asp or Glu at position 3. The second motif has a Trp at position 3, and the third motif has a Gln at position 3; these same deviations from the overall consensus are seen for the flx-related family members (Fig. 3). Given that the function of the second and third ES signals is thought to involve binding to inner membrane proteins necessary for tracking and insertion into the inner mitochondrial membrane (44Endres M. Neupert W. Brunner M. EMBO J. 1999; 18: 3214-3221Crossref PubMed Scopus (149) Google Scholar), 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 (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar) 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 after 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 is located immediately before TMs 2, 4, and 6. These homologies help the assignment of TM domains within the sequence and presumably involve interactions that are essential to transporter membrane positioning and function, such as the charge interactions previously described (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar).Open reading frames closely related to hMFT were easily identified by computer searches against those genomes sequenced to date. A systematic search of the yeast database (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar) 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 several proteins homologous to hMFT. The most closely related yeast putative transporters 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 intoglyB 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 theCaenorhabditis elegans and Drosophila genomes using the hMFT protein sequence as probe, identified at least 25 related members in each genome based on homology in the placement of ES motifs in a tripartite 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 either the wild type CHO-K1 cells from which they were derived or the CHO-derived viral transductant or plasmid transfectants with the hmft gene (Table I). This observation 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 that are intact from the mitochondria. This transfer had been hypothesized (45Qi H.A.I. Xiao S. Choi Y.J. Tobimatsu T. Shane B. Adv. Enzyme Regul. 1999; 39: 263-273Crossref PubMed Scopus (15) Google Scholar) based on the fact that the naturally occurring transcripts for mitochondrial FPGS (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Turner F. Andreassi J. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar) or indeed the artificial transcripts constructed by ligation of model mitochondrial leader sequences to human cytosolic FPGS (46Chen L. Qi H. Korenberg J. Garrow T.A. Choi Y. Shane B. J. Biol. Chem. 1996; 271: 13077-13087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) can support both cytosolic and mitochondrial folate metabolism. At face value, the data of Table I suggest that half of the folate pool in the cytosol stems from the activity of mitochondrial FPGS. This fact 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar) 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 glyBphenotype 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 (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Turner F. Andreassi J. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar). Apparently, exogenous glycine can be delivered to the mitochondria, presumably through the cytosol, and can easily penetrate the outer and inner mitochondrial membranes. Hence, the glycine auxotrophy ofglyB cells indicates that cytosolic SHMT cannot supply glycine for mitochondrial metabolism at any appreciable concentration. This finding is in accordance with the current thought that cytosolic SHMT is kinetically controlled and funnels serine to glycine for the production of 5,10-methylenetetrahydrofolate for cytosolic 1-carbon metabolism (47Kastanos E.K. Woldman Y.Y. Appling D.R. Biochemistry. 1997; 36: 14956-14964Crossref PubMed Scopus (84) Google Scholar); 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar) showed that the glycine auxotrophy of the glyB cells can be reversed by high concentrations of 5-formyltetrahydrofolate, a fact that 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 despite decades of experimental work. We hereby report the isolation of a novel gene encoding a protein that 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar). 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 I). This finding 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 (32Scharfe C. Zaccaria P. Hoertnagel K. Jaksch M. Klopstock T. Dembowski M. Lill R. Prokish H. Gerbitz K.D. Neupert W. Mewes H.W. Meitinger T. Nucleic Acids Res. 2000; 28: 155-158Crossref PubMed Scopus (70) Google Scholar). 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 (33Saraste M. Walker J.E. FEBS Lett. 1982; 144: 250-254Crossref PubMed Scopus (240) Google Scholar, 34Aquila H. Link T.A. Klingenberg M. EMBO J. 1985; 4: 2369-2376Crossref PubMed Scopus (277) Google Scholar, 35Runswick M.J. Walker J.E. Bisaccia F. Iacobazzi V. Palmieri F. Biochemistry. 1990; 29: 11033-11040Crossref PubMed Scopus (140) Google Scholar, 36Runswick M.J. Powell S.J. Nyren P. Walker J.E. EMBO J. 1987; 6: 1367-1373Crossref PubMed Scopus (181) Google Scholar, 37Aquila H. Misra D. Eulitz M. Klingenberg M. Hoppe-Seyler's Z. Physiol. Chem. 1982; 363: 345-349Crossref PubMed Scopus (184) Google Scholar). The marked similarities among these proteins have been reviewed (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar). The active form of the carriers is thought to form homodimers (38Klingenberg M. Nature. 1981; 290: 449-454Crossref PubMed Scopus (214) Google Scholar), which together create a 12-TM unit similar to plasma membrane transporters, such as the reduced folate carrier (39Dixon K.H. Lanpher B.C. Chiu J. Kelley K. Cowan K. J. Biol. Chem. 1994; 269: 17-20Abstract Full Text PDF PubMed Google Scholar) and the thiamine carrier (40Fleming J.C. Tartaglini E. Steinkamp M.P. Schorderet D.F. Cohen N. Neufeld E.J. Nat. Genet. 1999; 22: 305-308Crossref PubMed Scopus (203) Google Scholar, 41Diaz G.A. Banikazemi M. Oishi K. Desnick R.J. Gelb B.D. Nat. Genet. 1999; 22: 309-312Crossref PubMed Scopus (178) Google Scholar), as well as mitochondrial carriers, such as the major intrinsic protein family and the major facilitator superfamily (25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar). The predicted hMFT protein has several properties, which identify it as a new member of the family of inner mitochondrial membrane transport proteins. The protein has a predicted size within the range for the several previously reported inner mitochondrial membrane transporters (28–34 kDa) (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 25Kuan J. Saier M.H. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 209-233Crossref PubMed Scopus (153) Google Scholar, 33Saraste M. Walker J.E. FEBS Lett. 1982; 144: 250-254Crossref PubMed Scopus (240) Google Scholar, 34Aquila H. Link T.A. Klingenberg M. EMBO J. 1985; 4: 2369-2376Crossref PubMed Scopus (277) Google Scholar, 35Runswick M.J. Walker J.E. Bisaccia F. Iacobazzi V. Palmieri F. Biochemistry. 1990; 29: 11033-11040Crossref PubMed Scopus (140) Google Scholar, 36Runswick M.J. Powell S.J. Nyren P. Walker J.E. EMBO J. 1987; 6: 1367-1373Crossref PubMed Scopus (181) Google Scholar, 37Aquila H. Misra D. Eulitz M. Klingenberg M. Hoppe-Seyler's Z. Physiol. Chem. 1982; 363: 345-349Crossref PubMed Scopus (184) Google Scholar) 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 consist of two 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) (24Palmieri F. FEBS Lett. 1994; 346: 48-54Crossref PubMed Scopus (307) Google Scholar, 26Bairoch A. Nucleic Acids Res. 1992; 20: 2013-2018Crossref PubMed Scopus (424) Google Scholar, 42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar), which is found immediately after TM domains 1, 3, and 5. These signals are thought to be required for mitochondrial targeting, translocation across the outer mitochondrial membrane, and insertion into the inner membrane (43Sirrenberg C. Endres M. Folsch H. Stuart R.A. Neupert W. Brunner M. Nature. 1998; 391: 912-915Crossref PubMed Scopus (242) Google Scholar, 44Endres M. Neupert W. Brunner M. EMBO J. 1999; 18: 3214-3221Crossref PubMed Scopus (149) Google Scholar). 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 the consensus, whereas the second and third motifs do not have a Asp or Glu at position 3. The second motif has a Trp at position 3, and the third motif has a Gln at position 3; these same deviations from the overall consensus are seen for the flx-related family members (Fig. 3). Given that the function of the second and third ES signals is thought to involve binding to inner membrane proteins necessary for tracking and insertion into the inner mitochondrial membrane (44Endres M. Neupert W. Brunner M. EMBO J. 1999; 18: 3214-3221Crossref PubMed Scopus (149) Google Scholar), 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 (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar) 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 after 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 is located immediately before TMs 2, 4, and 6. These homologies help the assignment of TM domains within the sequence and presumably involve interactions that are essential to transporter membrane positioning and function, such as the charge interactions previously described (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar). Open reading frames closely related to hMFT were easily identified by computer searches against those genomes sequenced to date. A systematic search of the yeast database (42Nelson D.R. Felix C.M. Swanson J.M. J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (168) Google Scholar) 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 several proteins homologous to hMFT. The most closely related yeast putative transporters 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 intoglyB 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 theCaenorhabditis elegans and Drosophila genomes using the hMFT protein sequence as probe, identified at least 25 related members in each genome based on homology in the placement of ES motifs in a tripartite 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 either the wild type CHO-K1 cells from which they were derived or the CHO-derived viral transductant or plasmid transfectants with the hmft gene (Table I). This observation 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 that are intact from the mitochondria. This transfer had been hypothesized (45Qi H.A.I. Xiao S. Choi Y.J. Tobimatsu T. Shane B. Adv. Enzyme Regul. 1999; 39: 263-273Crossref PubMed Scopus (15) Google Scholar) based on the fact that the naturally occurring transcripts for mitochondrial FPGS (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Turner F. Andreassi J. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar) or indeed the artificial transcripts constructed by ligation of model mitochondrial leader sequences to human cytosolic FPGS (46Chen L. Qi H. Korenberg J. Garrow T.A. Choi Y. Shane B. J. Biol. Chem. 1996; 271: 13077-13087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) can support both cytosolic and mitochondrial folate metabolism. At face value, the data of Table I suggest that half of the folate pool in the cytosol stems from the activity of mitochondrial FPGS. This fact 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar) 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 glyBphenotype 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 (11Freemantle S.J. Taylor S.M. Krystal G. Moran R.G. J. Biol. Chem. 1995; 270: 9579-9584Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Turner F. Andreassi J. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar). Apparently, exogenous glycine can be delivered to the mitochondria, presumably through the cytosol, and can easily penetrate the outer and inner mitochondrial membranes. Hence, the glycine auxotrophy ofglyB cells indicates that cytosolic SHMT cannot supply glycine for mitochondrial metabolism at any appreciable concentration. This finding is in accordance with the current thought that cytosolic SHMT is kinetically controlled and funnels serine to glycine for the production of 5,10-methylenetetrahydrofolate for cytosolic 1-carbon metabolism (47Kastanos E.K. Woldman Y.Y. Appling D.R. Biochemistry. 1997; 36: 14956-14964Crossref PubMed Scopus (84) Google Scholar); 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 (15Taylor R.T. Hanna M.L. Arch. Biochem. Biophys. 1982; 217: 609-623Crossref PubMed Scopus (20) Google Scholar) showed that the glycine auxotrophy of the glyB cells can be reversed by high concentrations of 5-formyltetrahydrofolate, a fact that 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 despite decades of experimental work. We thank Professors Larry Chasin for the original gift of the glyB cells, Oliver Bogler for suggestions and advice on retroviral transfer technologies and packaging cells, Glen VanTuyle for advice on mitochondrial isolations and purifications, Robert Tombes for help with fluorescence microscopy, Verne Schirch for insight into mitochondrial folate metabolism, and Shirley Taylor for continued advice on the project and for her critique of the manuscript." @default.
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• W2019686818 title "Retrovirally Mediated Complementation of the glyBPhenotype" @default.
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