Anne S. Tibbetts

Compartmentalization of mammalian folate-mediated one-carbon metabolism.

The recognition that mitochondria participate in folate-mediated one-carbon metabolism grew out of pioneering work beginning in the 1950s from the laboratories of D.M. Greenberg, C.G. Mackenzie, and G. Kikuchi. These studies revealed mitochondria as the site of oxidation of one-carbon donors such as serine, glycine, sarcosine, and dimethylglycine. Subsequent work from these laboratories and others demonstrated the participation of folate coenzymes and folate-dependent enzymes in these mitochondrial processes. Biochemical and molecular genetic approaches in the 1980s and 1990s identified many of the enzymes involved and revealed an interdependence of cytoplasmic and mitochondrial one-carbon metabolism. These studies led to the development of a model of eukaryotic one-carbon metabolism that comprises parallel cytosolic and mitochondrial pathways, connected by one-carbon donors such as serine, glycine, and formate. Sequencing of the human and other mammalian genomes has facilitated identification of the enzymes that participate in this intercompartmental one-carbon metabolism, and animal models are beginning to clarify the roles of the cytoplasmic and mitochondrial isozymes of these enzymes. Identifying the mitochondrial transporters for the one-carbon donors and elucidating how flux through these pathways is controlled are two areas ripe for exploration.

Mammalian MTHFD2L Encodes a Mitochondrial Methylenetetrahydrofolate Dehydrogenase Isozyme Expressed in Adult Tissues

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH2-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH2-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP+-dependent CH2-THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH2-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH2-THF to formate in adult mammalian mitochondria.

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

The Saccharomyces cerevisiae ADE16and 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 anade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy ofade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17gene 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.

Yeast mitochondrial oxodicarboxylate transporters are important for growth on oleic acid

The yeast genes ODC1 and ODC2 encode members of the Saccharomyces cerevisiae family of mitochondrial transport proteins that transport oxodicarboxylates. In these studies, the ODC1 gene was identified as able, in low-copy, to rescue a yeast strain that is unable to grow on oleic acid but can grow on other nonfermentable carbon sources. ODC2 was shown to be a high-copy suppressor of this mutant. Odc1delta odc2delta double mutants are unable to grow on oleic acid at 36 degrees C. ODC1 mRNA and protein expression is elevated in oleic acid medium as compared to glucose or glycerol. The ODC1 promoter contains sequences required for the oleic acid response. However, regulation of ODC1 does not require the transcription factors Oaf1p and Pip2p, known to mediate oleic acid induction of other genes. These studies provide the first link between these mitochondrial transporters and peroxisomal beta-oxidation.

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNAfMet). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNAfMet, as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNAfMet to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNAfMet. This accessory factor might be unnecessary when formylated Met-tRNAfMet is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Δfmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNAfMet due to loss of the MIS1 gene that encodes the mitochondrial C1-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.