Symposium: Subcellular Compartmentation of Folate Metabolism

Symposium on the Subcellular Compartmentation of Folate Metabolism1

CONRAD WAGNER Downloaded from https://academic.oup.com/jn/article/126/suppl_4/1228S/4724794 by guest on 29 September 2021 Department of Biochemistry, Vanderbilt University School of Medicine and Department of Veterans Affairs Medical Center, Nashville, TN 37232

This symposium was occasioned by an increased in It was therefore surprising to learn that purification terest in folate metabolism. This is a result of the re of the enzymes involved in one-carbon metabolism and cent discovery of the role of folate nutrition in the development of accurate methods to determine the size incidence of neural tube defects and the realization that of the one-carbon pools showed that the concentration folate deficiency is associated with hyperhomocysti- of the enzymes actually exceeds the concentration of nemia with the attendant increased risk of vascular the one-carbon folate pools. The folate pools consist of disease. When one undertakes to study the role of folate the polyglutamate derivatives of the folate coenzymes and one-carbon units in the cell, it soon becomes clear that have dissociation constants in the 100 nmol/L that metabolism is compartmentalized between the cy- range. These two facts suggest that in the cell most one-carbon derivatives of folate are present bound to tosol and the mitochondria. This is because almost all enzymes or other folate-binding proteins. The concen the folate in the cell is distributed almost equally be tween the two compartments. There are also mito- tration of the free pools of one-carbon folate coenzymes chondrial and cytosolic isoforms of the same enzymes are therefore in the low nmol/L range. This raises the question of whether the rate of one-carbon metabolism and the question arises as to the reasons for this redun is actually controlled by the rate of diffusion of sub dancy. See Figure 1. strates and products from these enzymes or whether The first speaker was Verne Schirch of the Depart there is some other explanation for the transfer of one- ment of Biochemistry of Virginia Commonwealth Uni versity. Schirch discussed the concept that compart- carbon units through a specific metabolic pathway. The concept of organized clusters of enzymes in mentation of folate metabolism may be present even metabolic pathways has been gaining acceptance (Srere within the cytosol itself. The role of the folate coen- 1987). In an enzyme cluster, the products of individual zymes is to carry one-carbon units and within the cyto enzyme catalyzed reactions are transferred directly to sol the folate coenzymes play a role in three major the next enzyme and act as substrates without diffusing biosynthetic pathways (Schirch and Strong 1989). into and out of the solvent. Such transfer of metabolites These are as follows: Î)the pathway of purine biosyn between enzymes is called channeling. Schirch has pu thesis in which folate provides a one-carbon unit at rified and studied the structural and kinetic properties two different steps, 2) the pathway of thymidylate bio of five folate-requiring enzymes that are involved in the synthesis in which a one-carbon unit is transferred to metabolism of 10-formyltetrahydrofoíate. This form of deoxyuridylic acid from 5,10-methylene tetrahydrofo- the coenzyme serves as the donor for carbons 2 and 8 late and simultaneously reduced to form the methyl in purine ring biosynthesis. The first of these enzymes group of thymidylic acid and 3) the formation of methi- is serine hydroxymethyltransferase, which uses the onine from homocysteine and 5-methyltetrahydrofo- third carbon of serine to generate 5,10-methylenetet- late in a vitamin B-12-dependent reaction. It has usu ally been assumed that the concentration of the re duced folates used as substrates for these reactions are 1A summary of manuscripts presented in the Symposium: "Sub- cellular Compartmentation of Folate Metabolism" given at the Ex greater than the concentration of the enzymes (which perimental Biology '95 meeting, Atlanta, GA, on April 10, 1995. This carry out a catalytic function) and that all enzymes symposium was sponsored by the American Institute of Nutrition. that use the same folate one-carbon derivative compete Guest editor for the symposium publication was Conrad Wagner, for a common pool. Vanderbilt University School of Medicine, Nashville, TN.

0022-3166/96 $3.00 ©1996 American Institute of Nutrition. J. Nutr. 126: 1228S-1234S, 1996. 1228S FOLATE METABOLISM 1229S

ÇytoplMm Mitochondria

l l » Folate -»•DHF-*•THF THF

serine "rco.ine 14 14 12 glycine • glycine

6,10-methylene-THF 5,10-methylene-THF Downloaded from https://academic.oup.com/jn/article/126/suppl_4/1228S/4724794 by guest on 29 September 2021 a^—^- NADP* NADP* C02.NH3 i—^ NADPH NADPH 5,10-methenyl-THF 5,10-me theny 1-THF THF j 4 l' J-J •ormyl-THF-ÕÕNADP* punnes ^~ 10-fonnyl-THF lO-fonnyl ADP + Pi ADP + P 8

formate «*- •*•formate

FIGURE 1 Compartmentation of folate one-carbon metabolism. The numbers refer to enzymatic reactions. They are as follows: 1, dihydrofolate reductase; 2, thymidylate synthase,- 3, 5,10-methylenetetrahydrofolate dehydrogenase,- 4, 5,10-methe- nyltetrahydrofoíate cyclohydrolase; 7,10-formyltetrahydrofoíate dehydrogenase; 8,10-formyltetrahydrofoíate synthase; 9, meth- ionyl-t-RNA formyltransferase; 12, sarcosine dehydrogenase; 13, glycine cleavage enzyme system; 14, serine hydroxymethyltran- sferase; 19, 5,10-methylenetetrahydrofolate reducíase.The activities carried out by reactions 3, 4, 7, 8 and 14 are found in both the cytosoplasm and the mitochondria. rahydrofolate (Schirch 1982). This folate is then con 10-formyltetrahydrof oíatesynthetase (L.Schirch, unpub verted to 10-formyltetrahydrof oíateby the methylene- lished results). In each of these reactions the channeling tetrahydrofolate dehydrogenase and methylenetetrahy- was only efficient for the polyglutamate forms of the drofolate cyclohydrolase activities of the trifunctional coenzyme. Although some evidence for physical associa enzyme Crtetrahydrofolate synthase (Schirch 1978). A tion between serine hydroxymethyltransferase and the third activity of this trifunctional enzyme is 10-formyl 10-formyltetrahydrof oíate dehydrogenase and synthe tetrahydrof oíatesynthetase, which forms 10-formyltet- tase enzymes has been obtained, no clear evidence of rahydrofolate directly from formate in an ATP-depen- how these enzymes might form a cluster has yet been dent reaction. The fifth enzyme purified and studied determined. One significant advantage of channeling of was 10-formyltetrahydrof oíatedehydrogenase (Schirch the reduced folate coenzymes in these reactions is the et al. 1994). This abundant liver enzyme was shown to known increased stability of the enzyme-bound coen bind tetrahydrofolate very tightly by Cook and Wagner zyme. (1982). Together, these five enzymes form two meta The second speaker was Donald W. Home of the bolic cycles that interconvert the third carbon of serine Department of Biochemistry at Vanderbilt University and formate. In rabbit liver the concentration of folate and the Veterans Administration Medical Center in binding sites on these five enzymes is ~30 fimol/L. Nashville. Home addressed the Compartmentation of These enzymes were purified and combined in vitro to folate-linked enzymes and coenzymes and also the is see if they form an enzyme cluster that channels the sue of how folate is transported into the mitochondria. reduced folate coenzymes. It is known that folate-linked enzymes are present Using a variety of kinetic methods evidence was pre in liver mainly in the cytosolic and mitochondrial frac sented that the tetrahydrofolate coenzymes are chan tions (Wagner 1982), and it has been shown that the neled between several of these enzymes. These include folate coenzymes are present also mainly in these two the following: methylenetetrahydrofolate dehydrogenase compartments (Cook and Blair 1979). Furthermore, in and methenyltetrahydrofoíate cyclohydrolase (Schirch vitamin B-12 deficiency induced either by dietary ma 1978); serine hydroxymethyltransferase and 10-formyl- nipulation or by nitrous oxide it is known that folate tetrahydrofolate synthetase (Strong and Schirch 1989); coenzymes accumulate as the 5-methyl derivative at serine hydroxymethyltransferase and 10-formyl tetrahy the expense of other reduced folates. This is called the drofolate dehydrogenase (L. Schirch, unpublished re methyl-trap hypothesis. It occurs because the B-12- sults); and methenyltetrahydrofolate cyclohydrolase and dependent enzyme, methionine synthase (which uses 1230S SUPPLEMENT

5-methyltetrahydrofolate to methylate homocysteine the tetrahydrofolate to generate 10-formyltetrahydrofo- and regenerate methionine and tetrahydrofolate), is in late, which may be oxidized via a mitochondrial ana hibited because of lack of vitamin B-12 or inactivation logue of 10-formyltetrahydrof oíatedehydrogenase. of the enzyme by nitrous oxide and because the en Because Home and his colleagues knew from their zyme, 5,10-methylenetetrahydrofolate reducíase(which previous studies that folylpolyglutamates could not synthesizes 5-methyltetrahydrofolate) is irreversible in readily cross the mitochondrial membrane, they asked vivo (Shane and Stokstad 1985). These facts led Home the question of how folates get into the mitochondria. to wonder whether cytosolic or mitochondrial folates An earlier report stated that only the oxidized folie acid would be affected by nitrous oxide inactivation of me or dihydrofolic acid were taken up into the mitochon thionine synthase. drion (Cybulski and Fisher 1981). However, several as He examined the distribution of folate coenzymes pects of this study (uptake at 0°Cand extramitochon- in liver using HPLC to separate the individual deriva drial concentrations of 50-250 /zmol/L) caused them Downloaded from https://academic.oup.com/jn/article/126/suppl_4/1228S/4724794 by guest on 29 September 2021 tives and the Lactobacillus casei microbiological assay to reinvestigate this problem. Rat liver mitochondria to quantitate the eluted folates. Rats were exposed to were isolated using differential centrifugation. Uptake nitrous oxide and oxygen (8:2) for 18 hs. The livers were of 5-formyl-[3',5',9-3H]tetrahydrofolic acid was deter removed and the cytosolic and particulate fractions mined at 37°Cand at physiological concentrations. (which contain the mitochondria) were obtained by dif Home found that uptake was a saturable process, with ferential centrifugation. They were treated with conju Km = 3.5 /¿mol/L and Vmax = 2.8 pmol/L-mg pro- gase (to hydrolyzed folylpolyglutamates to the folylmo- tein~' •min^1. Uptake was readily inhibited by 5-meth noglutamates) and the individual coenzymes were de yltetrahydrofolate fiso ~4 /zmol/L); however, folie acid termined via HPLC/L. casei assay (Home et al. 1989). and methotrexate were poor inhibitors (I50> 100 ¿tmol/ In controls, liver cytosolic folates were as follows: 5- L). These findings clearly show that under physiologi methyltetrahydrofolate, 45%; 5- and 10-formyltetrahy- cal conditions, mitochondria possess a specific, carrier- drofoíate, 9 and 19%, respectively,- and tetrahydrofo mediated system for the uptake of reduced, folate mo late, 27%. In rats breathing nitrous oxide, cytosolic fo noglutamates (Hörneet al. 1992). lates were as follows: 5-methyltetrahydrofolate, 84%; The third speaker was Robert J. Cook of the Bio 5- and 10-formyltetrahydrofoíate, 2.1 and 9.1%, respec chemistry Department of Vanderbilt University. He fo tively; and tetrahydrofolate, 4.7%. Mitochondrial fo cused his talk on the aspects of folate metabolism that lates were as follows: 5-methyltetrahydrofolate, 7.3%; take place within liver mitochondria. 5- and 10-formyltetrahydrof oíate,11.5 and 33.1%, re Four folate-dependent enzymes have been purified spectively,- and tetrahydrofolate, 48.1%. The mitochon from mitochondria. Dimethylglycine dehydrogenase drial folates did not change in animals exposed to ni (DMGDH; EC 1.5.99.2) and sarcosine dehydrogenase trous oxide. Thus, inactivation of methionine synthase (SARDH; EC 1.5.99.1) are covalent flavoenzymes that by nitrous oxide resulted in accumulation of 5-methyl catalyze successive oxidative demethylations in cho- tetrahydrofolate so that it was almost the only folate line degradation, which is exclusive to liver mitochon coenzyme present in liver cytosol. Also, there was lit dria (MacKenzie 1955). The N-methyl groups from di- tle, if any, transport of the endogenous folylpolygluta methylglycine and sarcosine are oxidized to formalde mates between the cytosolic and mitochondrial com hyde that interacts with tetrahydrofolate to give 5,10- partments. methylenetetrahydrofolate (Witwer and Wagner 1981). These findings may help explain the results of Eells Glycine cleavage enzyme (GCE; EC 2.1.2.10) is a multi- et al. (1985) and others who showed that the ring-2- subunit enzyme complex that uses tetrahydrofolate to carbon of histidine cannot be oxidized to CO2 in rats oxidize glycine to CO2/ NH3 and 5,10-methylenetet exposed to nitrous oxide, whereas formate can be oxi rahydrofolate (Yoshida and Kikuchi 1970). These three dized, albeit at ~50% of control rates. Oxidation of enzymes catalyze irreversible reactions in vivo. The ring-2-carbon of histidine requires cytosolic tetrahy fourth enzyme is mitochondrial serine hydroxymethyl- drofolate to accept a formimino group from formimi- transferase (m-SHMT; EC 2.1.2.1) that interconverts noglutamate in the catabolic pathway for histidine. serine and glycine and 5,10-methylenetetrahydrof oíate. After enzymatic conversion of 5-formiminotetrahydro- The function of m-SHMT is to supply glycine and one- fola te to 10-formyltetrahydrof oíatethe latter is oxi carbon units, in the form of 5,10-methylenetetrahy dized by the cytosolic enzyme 10-formyltetrahydrofo- drofolate, from serine and tetrahydrofolate (Schirch late dehydrogenase to CO2 and tetrahydrofolate. Be 1982). All four enzymes require tetrahydrofolate and cause almost all the cytosolic folate is in the 5-methyl generate 5,10-methylenetetrahydrofolate as products of form in nitrous oxide exposed rats no tetrahydrofolate their reactions. How tetrahydrofolate is regenerated is available for histidine oxidation. In the case of for has been an open question. mate, the cytosolic contribution to its oxidation is also Yoshida and Kikuchi (1970) and Lewis et al. (1978) absent in nitrous oxide-exposed animals. The mito showed that serine, sarcosine and glycine could all be chondrial tetrahydrofolate is unaffected by nitrous ox oxidized to CO2 or converted to formate by mitochondria. ide, however, and formate reacts enzymatically with They speculated that mitochondrial forms of Ci-tetrahy- FOLATE METABOLISM 1231S drofolate synthase (C,-SYNTH: trifunctional: 5,10-meth- sequence, nor does the N-terminal sequence of the pro ylenetetrahydrofolate dehydrogenase (EC 1.5.1.5); 5,10- tein. Moreover, NEUT2 mice have no liver cytosolic methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9); 10FTHFDH because of a 30-kb deletion in the gene for 10-formyltetrahydrofoíate synthetase (EC 6.3.4.3) and 10- cytosolic 10FTHFDH (Champion et al. 1994) but Cook, formyltetrahydrofolate dehydrogenase (10FTHFDH; EC in unpublished studies, showed that mitochondria 1.5.1.6) were responsible for these reactions but no at from NEUT2 mice do have the activity to oxidize 10- tempt was made to isolate or purify these enzymes from formyltetrahydrofolate to tetrahydrofolate, NADPH+ mitochondria (Lewis et al. 1978, Yoshida and Kikuchi and CO2. Fractionation of a high speed supernatant 1970). d-SYNTH has two domains: the dehydrogenase (100,000 Xg) of sonicated, purified rat liver mitochon and cyclohydrolase domain, which converts 5,10-methyl- dria on DEAE-cellulose, revealed a single peak of activ enetetrahydrofoíate to 10-formyltetrahydrof oíate,and the ity that, in the presence of NADP, was able to oxidize 10-formyltetrahydrof oíate synthetase domain, which 10-formyltetrahydrof oíateto tetrahydrofolate with the Downloaded from https://academic.oup.com/jn/article/126/suppl_4/1228S/4724794 by guest on 29 September 2021 synthesizes 10-formyltetrahydrof oíatefrom tetrahydrofo concomitant production of NADPH+. This enzyme late. formate and ATP (Appling 1991).The reactions cata also catalyzed the NADPH+-dependent oxidation of lyzed by C i-SYNTH are all reversible under suitable met the stable analogue of 10-formyltetrahydrof oíate, 10- abolic conditions (Appling 1991). 10FTHFDH catalyzes formyl-5,8-dideazafolate as described for cytosolic the NADP+-dependent oxidation of 10-formyltetrahy- 10FTHFDH (Krupenko et al. 1995). The partially pure drofolate to tetrahydrofolate, CO2, and NADPH+. Cr mitochondrial enzyme did not oxidize propanol nor did SYNTH and 10FTHFDH would provide a route for recy it cross-react with anti-lOFTHFDH serum in dot blots, cling 5,10-methylenetetrahydrofolate back to tetrahy indicating that it is a different protein. drofolate and would explain the presence of the large 10- The identification of mitochondrial enzyme activi formyltetrahydrofolate pool found in mitochondria. ties able to convert 5,10-methylenetetrahydrofolate to Recently, Barlowe and Appling (1988) showed the 10-formyltetrahydrof oíate and tetrahydrofolate illus presence of CrSYNTH and 10FTHFDH activities in trates how folates are recycled and provides an explana mitochondria. It was also shown that the third carbon tion for the composition of the mitochondrial folate of serine and the N-methyl group of sarcosine could pool (Home et al. 1989). These activities are also able be converted to formate that could be utilized in the to incorporate formate into 10-formyltetrahydrof oíate cytosolic biosynthesis of purines (Barlowe and Appling and oxidize it to CO2, which explains how rats were 1988). All three d-SYNTH activities were demon able to oxidize formate when their cytosolic folates strated, but the nature of the enzyme) s) is yet to be were trapped as 5-methyltetrahydrofolate (Eells et al. determined. Analogies have been drawn to the yeast 1982) as detailed by Home. system that has both a cytosolic and a mitochondrial The fourth speaker was Barry Shane of the Depart Ci-SYNTH, encoded by separate nuclear genes (Ap ment of Nutritional Science of the University of Cali pling 1991). fornia at Berkeley. Shane addressed the question of how Subsequent studies with isolated mitochondria have the mitochondrial and cytosolic folate pools are regu shown that the third carbon of serine could be either lated. The first section of his presentation dealt with converted to formate or oxidized to CO2 depending on the enzyme folylpoly-y-glutamate synthetase. the metabolic state of the mitochondria (Garcia-Marti- Folylpolyglutamate synthetase (FPGS) catalyzes the nez and Appling 1993). It was further shown that mito stepwise addition of glutamate residues to cellular fo chondrial extracts plus NADP+ were able to oxidize lates and antifolates to form the physiological active 10-formyltetrahydrof oíateto tetrahydrofolate, NADPH+ coenzymatic forms of the vitamin and more potent and CO2. The nature of the enzyme that catalyzes this anti-folate agents. Metabolism of folate to polygluta- reaction is presently being studied by Cook. The hy mate forms is also required for tissue retention of fo pothesis that this enzyme is a mitochondrial form of late. Chinese hamster ovary (CHO) cell mutants that cytosolic 10FTHFDH (Barlowe and Appling 1988, lack FPGS activity (AUXB1 )have greatly reduced folate Lewis et al. 1978, Yoshida and Kikuchi 1970) has merit. pools, because of an inability to retain folates and are The observations presented by Cook indicate that it is auxotrophic for methionine, glycine, purines and thy- a different enzyme, however. Rat liver cytosolic midine. Wild-type CHO cells normally contain hexa- 10FTHFDH has two functional domains (Cook et al. and heptaglutamates, and mammalian tissues contain 1991). The N-terminal domain shows identity to glyci- a range of polyglutamates varying in glutamate chain namide ribonucleotide transformylase (GAR-TF: EC length from the pentaglutamate to the decaglutamate. 2.1.2.2) and L-methionyl-tRNA formyltransferase (Met- To understand why mammalian tissues synthesize tRNA-FT: EC 2.1.2.9), whereas the C-terminal domain these long-chain polyglutamate species and to define is 48% identical to a series of NAD+-dependent alde the role of FPGS in cellular and subcellular folate accu hyde dehydrogenases (ALDH: EC 1.2.1.3) and is able to mulation, Shane developed a number of mammalian oxidize propanal in the presence of NADP (Cook et al. cell models transfected with various folylpolygluta- 1991). The 5'-untranslated cDNA for rat liver cytosolic mate synthetase genes and containing altered folate 10FTHFDH shows no apparent mitochondrial leader coenzyme distributions. These cells have also been 1232S SUPPLEMENT

used to study the role of different folate derivatives in experiments indicated that mitochondrial folylpolyglu- the various metabolic cycles of one-carbon metabolism tamates can be released without prior hydrolysis and (Lin and Shane 1994, Lowe et al. 1993). CHO transfectants expressing E. coli FPGS activity CHO AUXB1 mutants transfected with the Esche- solely in the mitochondria possessed normal cytosolic richia coli FPGS gene (AUX-coli] express the E. coli folylpolyglutamate pools. In AUX-coli-mcoli cells mi protein in the cytosol and metabolize folates primarily tochondrial folate accumulation did not appear to be to triglutamates rather than the hexa- and heptagluta- limited by mitochondrial folate transport but was gov mates normally found in wild-type CHO cells (CHO- erned by competition between mitochondrial and cyto WT). The triglutamates that accumulate in AUK-coli solic FPGS activity. Elevated expression of cytosolic are retained approximately as effectively as the longer FPGS activity led to a mitochondrial folate deficiency polyglutamate derivatives in CHO-WT cells. Metabo that could only be overcome by expression of very high lism of folate to the triglutamate appears to be suffi levels of mitochondrial FPGS. Downloaded from https://academic.oup.com/jn/article/126/suppl_4/1228S/4724794 by guest on 29 September 2021 cient for effective intracellular retention and accumu Similar results were observed when AUXB1 cells lation of the vitamin. were transfected with a human FPGS cDNA (Garrow To evaluate the metabolic effectiveness of different et al. 1992). Expression of the human cDNA encoding polyglutamate chain length folates in the various meta a mature FPGS protein restored cytosolic FPGS activity bolic cycles of one-carbon metabolism, CHO cells were in these cells and overcame the cell's requirement for cultured in medium lacking purines, thymidine or gly thymidine and purines, but the cells remained auxotro- cine, and the levels of intracellular folate that supported phic for glycine, reflecting the absence of a folate pool half maximal growth rates were assessed. In AUX-coL' in the mitochondria. Expression of a human cDNA en cells, which contain triglutamates, similar intracellular coding a FPGS with a mitochondrial leader sequence folate concentrations to CHO-WT supported growth in restored FPGS activity in the mitochondria, and the thymidine and purine-free medium, but the folate re cells contained normal mitochondrial folate pools and quirement for growth in medium lacking glycine was were prototrophic for glycine. The human FPGS gene increased ~ 100-fold. Glycine is synthesized from serine has Spl binding sites in its promoter region. Different via the serine hydroxymethyltransferase (SHMT) reac forms of the protein are generated by alternate tran tion. Mammalian cells contain two isozymes of this scription start sites and differential splicing resulting enzyme, one cytosolic and one mitochondria!. Cells de in protein with and without mitochondrial leader se fective in the mitochondrial isozyme require glycine quences. for growth. AUX-coL', which expresses E. coli FPGS in Mammalian tissues contain mitochondrial and cyto the cytosol, lacked mitochondrial folates despite pos solic isozymes of SHMT, encoded by separate genes sessing normal cytosolic folate pools. The inability of (Garrow et al. 1993). GlyA cells lack mitochondrial these transfectants to grow in the absence of glycine SHMT but possess normal cytosolic SHMT activity could have been due to lack of mitochondrial folate and require glycine for growth. Overexpression of hu rather than a metabolic ineffectiveness of pteroyltriglu- man SHMT in the cytosol of these cells does not over tamates in the cytosol. When E. coli FPGS was targeted come the glycine requirement, demonstrating that the to the mitochondria of AUXB1 and AUX-coli cells using inability to synthesize glycine in the cytosol was not a modified E. coli FPGS gene construct preceded by a due to insufficient cytosolic SHMT. GlyA cells are de mammalian mitochondrial leader sequence, the trans ficient in mitochondrial one-carbon forms of folate and fectants expressed the E. coli enzyme in their mito this one-carbon deficit is also seen in the cytosol, indi chondria (AUX-coli-mcoli and AUX-mcoi) and accu cating that mitochondrial metabolism supplies one- mulated mitochondrial folate. In these cells, pteroyl- carbon moieties for cytosolic pathways. Formate has triglutamates functioned as effectively as the longer been proposed as a shuttle of one-carbon units from glutamate chain length folates found in wild-type CHO the mitochondria and addition of formate to GlyA cells cells in the metabolic cycle of glycine synthesis pro normalizes cytosolic folate distributions. GlyA cells vided they were located in the mitochondria (Lin and require increased folate for purine synthesis in the cy Shane 1994, Lowe et al. 1993). tosol. Addition of formate to the medium of GlyA or AUX-coJi cells lacked mitochondrial folate despite CHO-WT cells greatly decreases the folate requirement possessing high levels of cytosolic folate, and folylpo- for purine synthesis. lyglutamates cannot enter the mitochondria of mam In summary, model CHO cells obtained by transfect- malian cells. As indicated above, targeting of the E. coli ing CHO mutants with the E. coli and human FPGS FPGS to the mitochondria of these cells (AUX-colz- genes have proven useful for assessing the role of folyl- mcoli] restored mitochondrial folate pools, indicating polyglutamates in one-carbon metabolism and for de that mitochondrial folate accumulation is dependent lineating how folate intracellular stores are regulated. on mitochondrial FPGS activity. However, AUX-mcoL Cells expressing enzymes in specific subcellular com cells, which express E. coli FPGS activity in their mito partments, expressing enzymes with different sub chondria but not in their cytosol, also possessed normal strate specificities, and expressing enzyme activity at cytosolic and mitochondrial folate pools. Pulse chase different levels, all in a common background, in this FOLATE METABOLISM 1233S

case the CHO cell, has allowed the development of pathway can supply 100% of the required one-carbon kinetic models for assessing the role of FPGS in folate units for de novo purine biosynthesis, but blockage of retention and in the cytotoxicity of antifolates. Mito- the cytoplasmic pathway at the dehydrogenase step chondrial FPGS activity is required for mitochondrial slows the growth of the cells. accumulation of folate. Metabolism of folate to the trig- Appling has also used 13CNMR to directly visualize lutamate is sufficient to allow normal cellular folate the flow of one-carbon units from donors into products retention and synthesis of glycine, purines and thymi- such as purines and choline (Pasternack et al. 1994). In dylate. Mitochondrial folate metabolism provides one- these experiments, the 2-carbon of glycine, labeled with carbon units for cytosolic metabolism. 13C,is used as one-carbon donor. Glycine is catabolized The fifth and final speaker of the symposium was exclusively in mitochondria via the glycine cleavage system in which the 2-carbon is donated to THF to

Dean R. Appling of the Department of Chemistry and Downloaded from https://academic.oup.com/jn/article/126/suppl_4/1228S/4724794 by guest on 29 September 2021 Biochemistry of the University of Texas at Austin. Ap yield 5,10-methylene-THF. Cells are grown for several pling addressed the use of molecular genetic and NMR generations in [2-13C]glycine and then acid extracts are approaches to the study of folate compartmentation. prepared and analyzed by 13C NMR spectroscopy. In In eukaryotes, the mitochondrial and cytosol com wild-type cells, three resonances are seen in the purines partments each contain a parallel set of one-carbon- adenine and guanine. The C5 position is labeled due to unit interconverting enzymes (Appling 1991) (see Fig. the direct, folate-independent, incorporation of an in 1). The metabolic role(s) of the mitochondrial enzymes tact glycine molecule in the second step of purine syn and the degree of intercompartmental one-carbon flow thesis. The C8 and C2 positions acquire a 13C from are not well understood. Appling has begun to explore 10-formyl-THF at the GAR and AICAR transformylase the yeast Saccharomyces cerevisiae as a model system reactions (steps 3 and 9, respectively). As in the previous for understanding the subcellular compartmentation of example, the 10-formyl-THF used for purine synthesis folate-mediated one-carbon metabolism in eukaryotes. can come either from the oxidation of cytoplasmically Although the metabolic pathways are essentially iden generated 5,10-methylene-THF or from formate pro tical in yeast and mammals, the molecular genetics duced in the mitochondria. Blockage of the cytoplasmic available in the yeast system offers several powerful 5,10-methylene-THF dehydrogenase reactions, as in advantages. strain MWY4.4, allows determination of which route Appling's group has obtained clones and mutants is being used. 13Cspectra of extracts of MWY4.4 grown in virtually all of the tetrahydrofolate-interconverting in [2-13C]glycine are essentially identical to spectra from enzymes in Saccharomyces. Analysis of the nutritional the wild-type cells. Thus, in the mutant cells, glycine requirements and metabolic defects in this set of mu is being cleaved in mitochondria to produce 5,10-meth tants allows one to define the route by which one- ylene-THF, which is then converted to formate for carbon units move between cytoplasm and mitochon transport into the cytoplasm and subsequent activation dria. The donation of serine-derived one-carbon units to 10-formyl-THF for purine synthesis. Additional 13C for de novo purine biosynthesis illustrates the strategy. NMR experiments with mutant strains lacking either The third carbon of serine can enter the active one- the cytoplasmic or mitochondrial SHMT isozymes have carbon pool as 5,10-methylenetetrahydrofolate (THF) revealed that serine donates most of its one-carbon either in the cytoplasm or mitochondria via the serine units in the mitochondria. hydroxymethyltransferase (SHMT) isozymes present in Closer inspection of the spectra from the acid ex each compartment. In the cytoplasmic pathway, the tracts of [2-13C]glycine-grown cells revealed additional one-carbon unit is oxidized to 10-formyl-THF via an 13Cresonances that were identified as carbons 1, 2 and NAD- or NADP-dependent 5,10-methylene-THF dehy- 4(methyl) of choline (Pasternack et al. 1994). Competi drogenase. The 10-formyl-THF thus formed can then tion experiments with unlabeled serine and methio- be utilized in two reactions of the cytoplasmic de novo nine demonstrated that the labeling occurred during purine synthesis pathway. In the mitochondrial path the biosynthesis of phosphatidylcholine. Choline Cl way, the serine-derived one-carbon unit is oxidized to derives from 5,10-methylene-THF, choline C2 from di 10-formyl-THF as before but must then be converted rect incorporation of glycine and choline C4 (N-meth- to formate for exit out of the mitochondria. This for yls) from 5-methyl-THF via S-adenosylmethionine mate can then be activated to 10-formyl-THF for purine (Carman and Henry 1989). This pathway involves en synthesis by the cytoplasmic 10-formyl-THF synthe- zymes localized to the endoplasmic reticulum as well tase (Barlowe and Appling 1988). Yeast mutants lacking as mitochondria. the two cytoplasmic 5,10-methylene-THF dehydroge- These approaches have revealed a process in which nases were constructed to determine whether the mito one-carbon units generated in mitochondria from ser chondrial pathway, by itself, could support the flow of ine or glycine are utilized in nonmitochondrial path one-carbon units from serine to purines (Pasternack et ways such as purine and phosphatidylcholine biosyn al. 1994). This strain, MWY4.4, grows with a 6-h dou thesis. These two pathways allow one to follow the bling time in media lacking purines, compared with 2 distribution of one-carbon units into three different ox h for its wild-type parent. Clearly, the mitochondrial idation states (formyl-, méthylène-and methyl-THF). 1234S SUPPLEMENT

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