Folate-dependent enzymes in cultured Chinese hamster ovary cells: Impaired mitochondrial serine hydroxymethyltransferase activity in two additional glycine — auxotroph complementation classes

Rt, Taylor; Ml, Hanna

doi:10.1016/0003-9861(82)90543-4

Cited by 26 publications

(18 citation statements)

References 49 publications

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“…In this study, we show that DHFRL1 complements the previously uncharacterized CHO glycine auxotrophic mutant, glyC. Previous reports have observed that glyC mutants have normal SHMT levels in both the cytosol and mitochondria (15,24). Because the mitochondrial and cytoplasmic folate pools are not in equilibrium, loss of DHFR activity in mitochondria is expected to result in an accumulation of mitochondrial folate as dihydrofolate, thereby depleting THF pools for the SHMT2-catalyzed conversion of serine to glycine.…”

Section: Discussionmentioning

confidence: 51%

Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria

Anderson

Quintero

Stover

2011

Proc. Natl. Acad. Sci. U.S.A.

158

137

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The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity.folate | one-carbon metabolism | thymidine | deoxyribouridine

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Section: Discussionmentioning

confidence: 51%

Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria

Anderson

Quintero

Stover

2011

Proc. Natl. Acad. Sci. U.S.A.

158

137

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“…Two studies (13,14) have demonstrated the penetration into isolated mitochondria by folates in a process that was saturable and temperature-dependent. These studies would support the existence of a transporter responsible for the entry of folates into the mitochondria as does the fact that cells that either lack mitochondrial FPGS (11) or are incapable of accumulation of mitochondrial folates (15) are glycine auxotrophs.…”

mentioning

confidence: 87%

Retrovirally Mediated Complementation of the glyBPhenotype

Titus

Moran

2000

Journal of Biological Chemistry

118

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The transduction of a human placental cDNA retroviral library into glyB cells, a Chinese hamster ovary K1 subline that is deficient in the transport of folates into mitochondria, resulted in the complementation of glycine auxotrophy of these cells. A 2.6-kilobase pair cDNA insert flanked by retroviral sequences had integrated into genomic DNA in rescued cells. An open reading frame in this cDNA encoded a 35-kDa protein homologous to several inner mitochondrial wall transporters for intermediate metabolites. The subcloned cDNA complemented the glycine auxotrophy of glyB cells and reinstated folate accumulation in the mitochondria of transfected cells. The human origin, chromosomal location, and intron-exon organization of the isolated mitochondrial folate transporter gene were deduced from the expressed sequence tag database and human genome project data.In mammalian cells, the processes of folate metabolism are distributed between the cytosolic and mitochondrial compartments (1). Mitochondrial folates amount to about 35% of the total cellular pool (2, 3) and are used as cofactors for a mitochondrial serine hydroxymethyltransferase (SHMT) 1 by the glycine cleavage system and for the synthesis of the formylmethionine initiator of mitochondrial protein synthesis.The transport of folates through the plasma membrane into the cytosol has been extensively studied (4 -6), and two of the transport systems have been cloned (7,8). In contrast, the mechanism of the transfer of folates into mitochondria, presumably from the cytosol, is largely unknown as is the release of folates back into the cytosol from the mitochondria. Once the folate monoglutamates, the form of folate found in the circulation, enter the mammalian cells they are quickly metabolized to poly-␥-glutamate derivatives by cytosolic folylpoly-␥-glutamate synthetase (FPGS), a process needed to promote the retention of folate cofactors in cells (9). FPGS is also present in the mitochondrial compartment of mammalian cells (10) translated from transcripts from the FPGS gene, which add a mitochondrial leader sequence to the coding region of the protein found in the cytosol (11,12). Two studies (13,14) have demonstrated the penetration into isolated mitochondria by folates in a process that was saturable and temperature-dependent. These studies would support the existence of a transporter responsible for the entry of folates into the mitochondria as does the fact that cells that either lack mitochondrial FPGS (11) or are incapable of accumulation of mitochondrial folates (15) are glycine auxotrophs.Early studies by Puck and coworkers (16, 17) selected somatic cells that were auxotrophic for glycine and demonstrated that these cells fell into four complementation groups named glyA, glyB, glyC, and glyD. glyA was found to be attributed to a deficiency in mitochondrial SHMT (17,18). The glyC and glyD mutations have not to our knowledge been assigned to any functions. glyB cells had normal cytosolic folate metabolism and enzymes and had the same mitochondrial SHMT and...

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“…Yet glyB cells, which lack the MFT (15), glyA cells, which have a mutation in the mitochondrial isoform of SHMT (14), or AUXB1 cells transfected with only cytosolic FPGS require only glycine for growth and survival (see Ref. 13 and Fig.…”

Section: Discussionmentioning

confidence: 99%

“…AUXB1 cells stably transfected with cDNA encoding only the cytosolic form of FPGS no longer required purines and thymidine for survival, but still required glycine (13), indicating that purines and thymidine were synthesized using cytosolic folates. In addition, cells with deficiencies in mitochondrial folate metabolism, attributed to either a lack of mitochondrial serine hydroxymethyltransferase (SHMT) activity (glyA cells) (14) or lack of transport of folates through the inner mitochondrial membrane (glyB cells) (15), require glycine (but not thymidine and purine) for survival, demonstrating that glycine metabolism requires a folate-dependent mitochondrial process not complemented by cytosolic metabolism.…”

mentioning

confidence: 99%

Mammalian Mitochondrial and Cytosolic Folylpolyglutamate Synthetase Maintain the Subcellular Compartmentalization of Folates

Lawrence

Titus

Ferguson

et al. 2014

Journal of Biological Chemistry

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Background: Folylpolyglutamates are in the cytosol and mitochondria and the enzyme that makes these compounds, folylpolyglutamate synthetase (FPGS) is in both compartments. Results: Folylpolyglutamates cannot traverse mitochondrial membranes in either direction. Conclusion: Subcellular isoforms of FPGS are required to establish and maintain subcellular folate compartmentalization and function. Significance: Mitochondrial folates are a separate metabolic pool not in equilibrium with cytosol.

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Folate-dependent enzymes in cultured Chinese hamster ovary cells: Impaired mitochondrial serine hydroxymethyltransferase activity in two additional glycine — auxotroph complementation classes

Cited by 26 publications

References 49 publications

Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria

Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria

Retrovirally Mediated Complementation of the glyBPhenotype

Mammalian Mitochondrial and Cytosolic Folylpolyglutamate Synthetase Maintain the Subcellular Compartmentalization of Folates

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