Porous polyethylene implant fibrovascularization rate is affected by tissue wrapping, agarose coating, and insertion site.

PURPOSE: Often used in facial and ocular reconstruction, biointegratable materials, such as hydroxyapatite and high density porous polyethylene, can be associated with migration, exposure, and infection. Complications are less likely after implants become fibrovascularly integrated. A model was sought to study the influence of multiple factors on the rate of fibrovascular ingrowth into porous implants. METHODS: High density porous polyethylene cubes were implanted into paraspinous skeletal muscles in rabbits. The cubes were explanted at weekly intervals using survival surgery. The number of fibroblasts at the center of each cube was counted, generating a time-dependent standard curve of cell accumulation. Porous polyethylene cubes uncoated, coated with agarose (a plant-derived carbohydrate), or coated with nonperforated sclera (human or rabbit) were implanted into suprascapular adipose and paraspinous skeletal muscle in other rabbits. RESULTS: Fibrovascular ingrowth occurred more rapidly with cube implantation into skeletal muscle versus adipose, with increased surface area contact between implants and muscle, and with removal of muscle capsules. While the rate of fibroblast accumulation decreased in cubes coated with sclera, coating the cubes with agarose increased the fibrous capsule formation without altering the rate of biointegration. CONCLUSIONS: This study provides a novel approach for the study of fibrovascular ingrowth into implants treated under a variety of conditions. Modification of current surgical techniques may increase the rate of porous polyethylene implant biointegration.

Epidermal and fibroblast growth factors enhance fibrovascular integration of porous polyethylene implants.

PURPOSE: Porous implants used in functional and aesthetic reconstruction of the orbit, face, and cranium are less likely to develop complications after they become biointegrated. We investigated whether the administration of exogenous growth factors could increase the rate of implant integration. METHODS: High-density porous polyethylene cubes were placed in dorsal paraspinal muscles of rabbits, and daily transcutaneous injections of saline, epidermal growth factor, or basic fibroblast growth factor were administered directly over the cubes for 10 days. At serial time points up to 10 weeks, cubes were explanted and the fibroblasts present at the center of the cubes were counted. RESULTS: Injections of epidermal growth factor and basic fibroblast growth factor increased the rate at which fibroblasts accumulated in porous polyethylene implants and decreased the time required to achieve a maximal rate of cellular accumulation within the cubes. At 4 weeks, when all cell populations had attained a linear rate of accumulation, cubes previously injected with saline, epidermal growth factor, or basic fibroblast growth factor contained an average of 10, 40, and 80 cells per 0.0156 mm2, at their centers, respectively. CONCLUSIONS: Enhancement of the rate of biointegration of porous polyethylene cubes in rabbits is achievable by repeated, transcutaneous administration of exogenous growth factors.

Growth factors embedded in an agarose matrix enhance the rate of porous polyethylene implant biointegration.

PURPOSE: Repeated injections of epidermal and basic fibroblastic growth factors have been shown to enhance the biointegration rate of implanted porous polyethylene. A study was done to determine whether agarose, introduced at the time of implant placement, might serve as an adequate "single dose" delivery system for endogenous and exogenous growth factors. METHODS: Polyethylene cubes coated with agarose-containing growth factors were implanted into fat and muscle in rabbits. Factors studied included autogenous whole blood, autogenous serum, ascorbic acid, epidermal growth factor, basic fibroblast growth factor, transforming growth factor alpha, and transforming growth factor beta. The rate and character of the fibrovascular ingrowth into implants and surrounding capsule thickness were assessed. RESULTS: Fibroblast infiltration enhanced two- to sixfold with the use of autogenous or allogenic factors introduced in an agarose matrix at the time of cube implantation. CONCLUSIONS: Growth factors studied altered the thickness of the capsule surrounding implants as well as both the vascularity and stromal density within implants.

The X-ray structure of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.

Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD. We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 A resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.

Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ADE16 and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway. Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine. In this work, we show that an ade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy of ade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17 gene products (Ade16p and Ade17p). Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies. Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining. Epitope-tagged constructs were used to study expression of the two isozymes. The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine. Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation.

Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA.

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain.

Isolation, characterization, and functional expression of cDNAs encoding NADH-dependent methylenetetrahydrofolate reductase from higher plants.

Methylenetetrahydrofolate reductase (MTHFR) is the least understood enzyme of folate-mediated one-carbon metabolism in plants. Genomics-based approaches were used to identify one maize and two Arabidopsis cDNAs specifying proteins homologous to MTHFRs from other organisms. These cDNAs encode functional MTHFRs, as evidenced by their ability to complement a yeast met12 met13 mutant, and by the presence of MTHFR activity in extracts of complemented yeast cells. Deduced sequence analysis shows that the plant MTHFR polypeptides are of similar size (66 kDa) and domain structure to other eukaryotic MTHFRs, and lack obvious targeting sequences. Southern analyses and genomic evidence indicate that Arabidopsis has two MTHFR genes and that maize has at least two. A carboxyl-terminal polyhistidine tag was added to one Arabidopsis MTHFR, and used to purify the enzyme 640-fold to apparent homogeneity. Size exclusion chromatography and denaturing gel electrophoresis of the recombinant enzyme indicate that it exists as a dimer of approximately 66-kDa subunits. Unlike mammalian MTHFR, the plant enzymes strongly prefer NADH to NADPH, and are not inhibited by S-adenosylmethionine. An NADH-dependent MTHFR reaction could be reversible in plant cytosol, where the NADH/NAD ratio is 10(-3). Consistent with this, leaf tissues metabolized [methyl-(14)C]methyltetrahydrofolate to serine, sugars, and starch. A reversible MTHFR reaction would obviate the need for inhibition by S-adenosylmethionine to prevent excessive conversion of methylene- to methyltetrahydrofolate.

Saccharomyces cerevisiae expresses two genes encoding isozymes of methylenetetrahydrofolate reductase.

The identification, expression, and assay of two Saccharomyces cerevisiae genes encoding methylenetetrahydrofolate reductases (MTHFR) is described. MTHFR catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine synthesis. The MET12 gene is located on chromosome XVI and encodes a protein of 657 amino acids. The MET13 gene is located on chromosome VII and encodes a protein of 599 amino acids. The deduced amino acid sequences of these two genes are 34% identical to each other and 32-37% identical to the human MTHFR. A phenotype for the single disruption of MET12 was not observed, however, single disruption of MET13 resulted in methionine auxotrophy. Double disruption of both MET12 and MET13 also resulted in methionine auxotrophy. Growth of the methionine auxotrophs was supported by both methionine and S-adenosylmethionine. Transcripts of both MET12 and MET13 were detected in total RNA from wild type cells grown in the presence or absence of methionine. The methionine requirement of the met12 met13 double disruptant was complemented by plasmid-borne MET13, but not MET12 even when a multicopy plasmid was used. Furthermore, overexpression of the human MTHFR in the met12 met13 double disruptant complemented the methionine auxotrophy of this strain. In contrast, overexpression of the Escherichia coli metF gene did not complement the methionine requirement of met12 met13 cells. Assays for MTHFR in crude extracts and expression of the yeast proteins in Escherichia coli verified that both MET12 and MET13 encode functional MTHFR isozymes.

Genetic approaches to the study of protein-protein interactions.

This article describes genetic approaches to the study of heterologous protein-protein interactions, focusing on the yeast Saccharomyces cerevisiae as a useful eukaryotic model system. Several methods are described that can be used to search for new interactions, including extragenic suppression, multicopy suppression, synthetic lethality, and transdominant inhibition. Strategies for screening, genetic characterization, and clone identification are described, along with recent examples from the literature. In addition, genetic methods are discussed that can be used to further characterize a newly discovered protein-protein interaction. These include the creation of mutant libraries of a given protein by chemical mutagenesis or polymerase chain reaction, the production of dominant-negative mutants, and strategies for introducing these mutant alleles back into yeast for analysis. Although these genetic methods are quite powerful, they are often just a starting point for further biochemical or cell biological experiments.

Role of mitochondrial and cytoplasmic serine hydroxymethyltransferase isozymes in de novo purine synthesis in Saccharomyces cerevisiae.

One-carbon units are essential to a variety of anabolic processes which yield necessary cellular components including purines, pyrimidines, amino acids, and lipids. Serine hydroxymethyltransferase (SHMT) is the major provider of one-carbon units in the cell. The other product of this reaction is glycine. Both of these metabolites are required in de novo purine biosynthesis. In Saccharomyces cerevisiae, mitochondrial and cytoplasmic SHMT isozymes are encoded by distinct nuclear genes (SHM1 and SHM2). Molecular genetic analyses have begun to define the roles of these two isozymes in folate-mediated one-carbon metabolism [McNeil, J. B., et al. (1996) Genetics 142, 371-381]. In our study, the SHM1 and SHM2 genes were disrupted singly and in combination to investigate the contributions of the two SHMT isozymes to the production of glycine and one-carbon units required in purine biosynthesis. Cell subfractionation experiments indicated that while only 5% of total activity was localized in the mitochondria, the specific activity in that compartment was much higher than in the cytoplasm. Growth and 13C NMR experiments indicate that the two isozymes function in different directions, depending on the nutritional conditions of the cell. When yeast was grown on serine as the primary one-carbon source, the cytoplasmic isozyme was the main provider of glycine and one-carbon groups for purine synthesis. When grown on glycine, the mitochondrial SHMT was the predominant isozyme catalyzing the synthesis of serine from glycine and one-carbon units. However, when both serine and glycine were present, the mitochondrial SHMT made a significant contribution of one-carbon units, but not glycine, for purine synthesis. Finally, NMR data are presented that suggest the existence of at least two sites of de novo purine biosynthesis in growing yeast cells, each being fed by distinct pools of precursors.

Saccharomyces cerevisiae expresses two genes encoding isozymes of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase.

We have isolated and cloned two Saccharomyces cerevisiae genes which encode isozymes of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, the ninth step of the de novo purine biosynthesis pathway. This reaction involves the formylation of AICAR using 10-formyltetrahydrofolate as the formyl donor. ADE16 is located on chromosome XII and encodes an open reading frame of 591 amino acids. ADE17 is located on chromosome XIII and encodes an open reading frame of 592 amino acids. The deduced amino acid sequences of the two genes are 84% identical to each other and are 60-63% identical to the chicken and human bifunctional AICAR transformylase/IMP cyclohydrolase amino acid sequences. Disruption of the two chromosomal yeast genes resulted in adenine auxotrophy, while the expression of either gene alone was sufficient to support growth without adenine. In vitro assays of AICAR transformylase activity demonstrated the lack of IMP production in the double disruptant strain. S. cerevisiae is the only organism known thus far to possess isozymes of this protein. Because it is likely that the proteins encoded by ADE16 and ADE17 also contain IMP cyclohydrolase activity, these two genes complete the set of clones and mutants for the entire de novo purine biosynthesis pathway in yeast.

Crystallization of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses three isozymes of 5,10-methylenetetrahydrofolate dehydrogenase (MTD). The NAD-dependent enzyme is the first monofunctional form found in eukaryotes. Here we report its crystallization in a form suitable for high-resolution structure. The space group is P4(2)2(1)2 with cell constants a = b = 75.9, c = 160.0 A, and there is one 36 kDa molecule in the asymmetric unit. Crystals diffract to 2.9 A resolution.

Site-directed mutagenesis of a highly conserved aspartate in the putative 10-formyl-tetrahydrofolate binding site of yeast C1-tetrahydrofolate synthase.

C1-tetrahydrofolate (THF) synthase is a eukaryotic trifunctional protein possessing the activities 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase. Although the 10-formyl-THF synthetase reaction (a reversible ATP-dependent formylation of THF) has been studied extensively, little is known about specific residues involved in the catalytic mechanism. In this study, we have examined the role of a highly conserved aspartate residue, Asp449 of yeast cytoplasmic C1-THF synthase. Asp449 is part of a putative folate binding site found in many proteins that bind 10-formyl-THF. The corresponding aspartate has been identified as a critical catalytic residue in Escherichia coli and human GAR transformylase, which catalyzes a 10-formyl-THF-dependent formyl transfer. In order to determine if Asp449 has a similar catalytic role in the 10-formyl-THF synthetase reaction, three mutant proteins were produced by site-directed mutagenesis in which Asp449 of yeast cytoplasmic C1-THF synthase was changed to Asn, Glu, or Ala. The mutant proteins were expressed in yeast, purified, and characterized with respect to kinetic properties and enzyme stability. All three of the mutant enzymes retained substantial 10-formyl-THF synthetase activity, indicating that Asp449 is not a critical catalytic residue. However, our data suggest that it does play a role in folate binding, probably by contributing to the proper conformation of the active site. Thus, these results suggest that the 10-formyl-THF binding site differs significantly between the GAR transformylase and 10-formyl-THF synthetase families, and that the conserved aspartate plays different roles in the two enzymes.

Metabolic role of cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses two cytosolic 5,10-methylenetetrahydrofolate (CH2-THF) dehydrogenases that differ in their redox cofactor specificity: an NAD-dependent dehydrogenase encoded by the MTD1 gene and an NADP-dependent activity as part of the trifunctional C1-THF synthase encoded by the ADE3 gene. The experiments described here were designed to define the metabolic roles of the NAD- and NADP-dependent CH2-THF dehydrogenases in one-carbon interconversions and de novo purine biosynthesis. Growth studies showed that the NAD-dependent CH2-THF dehydrogenase is interchangeable with the NADP-dependent CH2-THF dehydrogenase when flow of one-carbon units is in the oxidative direction but that it does not participate significantly when flux is in the reductive direction. 13C NMR experiments with [2-13C]glycine and unlabeled formate confirmed the latter conclusion. Direct measurements of cellular folate coenzyme levels revealed substantial levels of 10-formyl-THF (CHO-THF), the one-carbon donor used in purine synthesis, in the purine-requiring ade3 deletion strain. Thus, CHO-THF is necessary but not sufficient for de novo purine synthesis in yeast. Disruption of the MTD1 gene in this strain resulted in undetectable CHO-THF, indicating that the NAD-dependent CH2-THF dehydrogenase was responsible for CHO-THF production in the ade3 deletion strain. Finally, we examined the ability of wild-type and catalytically-inactive domains of the cytoplasmic C1-THF synthase to complement the adenine auxotrophy of the ade3 deletion strain. Both the dehydrogenase/cyclohydrolase (D/C) domain and the synthetase domain could functionally replace the full-length protein, but, at least for the D/C domain, complementation was not dependent on catalytic activity. These results reveal a catalytic role for the NAD-dependent CH2-THF dehydrogenase in the oxidation of cytoplasmic one-carbon units and indicate that the cytoplasmic C1-THF synthase plays both catalytic and noncatalytic roles in de novo purine biosynthesis in yeast.

13C NMR analysis of the use of alternative donors to the tetrahydrofolate-dependent one-carbon pools in Saccharomyces cerevisiae.

Serine is generally accepted as the major one-carbon donor in folate-mediated one-carbon metabolism in most cells. Previous work from our laboratory with the yeast Saccharomyces cerevisiae has demonstrated that glycine and formate can also provide one-carbon units. Under normal growth conditions, it is likely that cells utilize serine, glycine, formate, and perhaps other one-carbon donors simultaneously, but to differing degrees. In the present work, we have used 13C NMR to monitor how yeast cells distribute alternative, competing one-carbon sources into various pools. Cells were grown with [2-13C]glycine and unlabeled formate or folinic acid (leucovorin, 5-formyl-tetrahydrofolate) as competing one-carbon sources. The relative contribution of each one-carbon donor to the three oxidation states of the tetrahydrofolate-bound one-carbon pool [5-methyl-tetrahydrofolate (CH3-THF), 5,10-methylene-THF (CH2-THF), and 10-formyl-THF (10-CHO-THF)] was determined by analysis of two metabolic end products of one-carbon metabolism, choline and adenine. Glycine-derived 13C-labeled one-carbon units are incorporated into these two metabolites; dilution of the 13C indicates competition by the unlabeled one-carbon source. The results reveal that the contribution from formate, folinic acid, and glycine is different for each of the one-carbon pools. Formate competed most dramatically at the 10-CHO-THF pool, with decreasing competition into the CH2-THF and CH3-THF pools. In a mutant strain lacking cytosolic CH2-THF dehydrogenase activity, a distinct shift toward the use of glycine instead of formate as the source of one-carbon units for the more reduced pools (CH2-THF and CH3-THF) was observed, while 10-CHO-THF pools were not affected. In contrast, the formyl group of folinic acid competed almost exclusively at the 10-CHO-THF level, with barely detectable dilution of the CH2-THF and CH3-THF pools in wild-type cells. The mutant strain exhibited essentially identical results, confirming that 5-formyl-THF enters the active one-carbon pool at the level of 10-CHO-THF, presumably via 5,10-methenyl-THF. Furthermore, donation of one-carbon units by folinic acid was observed only when cells were depleted of THF by treatment with the dihydrofolate reductase inhibitor methotrexate. These results reveal that the state of equilibrium between one-carbon pools in a growing cell depends on the source of the one-carbon units. This work illustrates the power of 13C NMR for examining the in vivo utilization of alternative one-carbon donors under a variety of conditions.

Whole-cell detection by 13C NMR of metabolic flux through the C1-tetrahydrofolate synthase/serine hydroxymethyltransferase enzyme system and effect of antifolate exposure in Saccharomyces cerevisiae.

Folate-mediated one-carbon metabolism is critical for the synthesis of numerous cellular constituents required for cell growth. A potential source of one-carbon units is formate. This one-carbon unit is activated to 10-formyltetrahydrofolate via the synthetase activity of the trifunctional enzyme C1-tetrahydrofolate (THF) synthase for use in purine synthesis or can be further reduced to 5,10-methylene-THF by the dehydrogenase activity of the same enzyme. 5,10-Methylene-THF is used by serine hydroxymethyltransferase (SHMT) in the synthesis of serine. Recently, 13C NMR has been used to establish that the C1-THF synthase/SHMT enzyme system is the only route from formate to serine in vivo in the yeast Saccharomyces cerevisiae [Pasternack et al. (1992) Biochemistry 31, 8713-8719]. In vitro studies have considered the kinetics of the C1-THF synthase/SHMT enzyme system in the catalytic conversion of formate to serine [Strong et al. (1987) J. Biol. Chem. 262, 12519-12525]. In the present work, we begin to study the kinetics of this two-enzyme system in its natural environment. Provision of [13C]formate and direct detection of an intracellular accumulating pool of [3-13C]serine by 13C NMR of whole cells allow us to monitor the rate of flux through this enzyme system in vivo. The rate of accumulation of soluble [3-13C]serine under [13C]formate-saturating conditions is 13.0 +/- 1.2 microM/min relative to an external standard of serine in D2O. The extracellular formate concentration at half-maximal flux was determined to be 900 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

13C NMR analysis of intercompartmental flow of one-carbon units into choline and purines in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the three-carbon of serine is normally the major one-carbon donor, although glycine and formate can substitute for serine. The second carbon of glycine enters via the glycine cleavage system in the mitochondria and can satisfy all cellular one-carbon requirements. It remains unresolved, however, as to the route by which these mitochondrial one-carbon units supply cytosolic anabolic processes. In the present work, we have used yeast mutants blocked at selected sites and 13C NMR to trace the incorporation of glycine-derived mitochondrial 5,10-methylenetetrahydrofolate into nonmitochondrial synthesis of choline and purines. Label incorporation into choline traces the methylation pathway of choline synthesis from production of serine to methylation of phosphatidylethanolamine. The active one-carbon unit of S-adenosylmethionine involved in methylation reactions originates almost solely from C3 of serine. On the other hand, flow of mitochondrial one-carbon units to 10-formyltetrahydrofolate for purine synthesis is shown to occur via both serine and formate. Formate transport accounts for at least 25% of the total, even during growth with sufficient serine to provide for the one-carbon requirements of the cell. This work shows that the synthetase function of the cytosolic C1-tetrahydrofolate synthase plays a critical role in the processing of mitochondrial one-carbon units to 10-formyltetrahydrofolate pools. In addition, this study provides evidence of two pools of glycine within the mitochondria and establishes a system of analyzing flux into the different folate derivatives.

Characterization of the folate-dependent mitochondrial oxidation of carbon 3 of serine.

The folate-dependent catabolism of serine was studied in intact rat liver mitochondria and soluble extracts from sonicated mitochondria. Formate and CO2 are both known to be products of the mitochondrial oxidation of carbon 3 of serine. The present work tests the proposal [Barlowe, C. K., & Appling, D. R. (1988) Biofactors 1, 171-176] that carbon 3 of serine is first oxidized to 10-formyltetrahydrofolate, which can be either oxidized to CO2 or converted to formate. Oxidation of carbon 3 of serine to formate and CO2 was shown to be dependent on the respiratory state of the mitochondria. Formate production was greatest in state-3 (actively respiring) mitochondria and lowest in uncoupled mitochondria. In contrast, CO2 production was greatest in uncoupled mitochondria and lowest in respiratory-inhibited mitochondria. Formate production appeared to be favored when high concentrations of NADP+ and ADP were present, but there was no clear correlation between the NADP+:NADPH redox state and CO2 production. In soluble mitochondrial extracts, CO2 production depended on NADP+ and tetrahydrofolate, whereas formate production required ADP in addition to NADP+ and the reduced folate cofactor. Unlike CO2 production, however, formate production showed a complete dependence on a polyglutamylated form of the folate cofactor. These experiments support the proposed folate-mediated serine oxidation as a major pathway for the flux of one-carbon units through mitochondria.