Sgt1 Dimerization Is Required for Yeast Kinetochore Assembly

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1–211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.Spindle microtubules are coupled to the centromeric region of the chromosome by a structural protein complex called the kinetochore (1, 2). The kinetochore is thought to generate a signal that arrests cells during mitosis when it is not properly attached to microtubules, thereby preventing aberrant chromosome transmission to the daughter cells, which can lead to tumorigenesis (3, 4). The kinetochore of the budding yeast Saccharomyces cerevisiae has been characterized thoroughly, genetically and biochemically; thus, its molecular structure is the most well detailed to date. More than 70 different proteins comprise the budding yeast kinetochore, and several of those are conserved in mammals (2).The budding yeast centromere DNA is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEI is bound by Cbf1 (7–9). CDEIII (25 bp) is essential for centromere function (10) and is the site where CBF3 binds to centromeric DNA. CBF3 contains four proteins: Ndc10, Cep3, Ctf13 (11–18), and Skp1 (17, 18), all of which are essential for viability. Mutations in any of the four CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (19, 20). All of the described kinetochore proteins, except the CDEI-binding Cbf1, localize to kinetochores dependent on the CBF3 complex (2). Therefore, the CBF3 complex is the fundamental structure of the kinetochore, and the mechanism of CBF3 assembly is of major interest.We previously isolated SGT1, the skp1-4 kinetochore-defective mutant dosage suppressor (21). Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required for the formation of the Skp1-Ctf13 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction, the tetratricopeptide repeat (TPR)² (21) and the CS (CHORD protein- and Sgt1-specific) motif. We and others (23–26) have found that both domains are important for the interaction with Hsp90. The Sgt1-Hsp90 interaction is required for the assembly of the core kinetochore complex; this interaction is an initial step in kinetochore assembly (24, 26, 27) that is conserved between yeast and humans (28, 29).In this study, we further characterized the molecular mechanism of this assembly process. We found that Sgt1 forms dimers in vivo, and our results strongly suggest that Sgt1 dimerization is required for kinetochore assembly in budding yeast.     

tmRNA Is Essential in Shigella flexneri

Nonstop mRNAs pose a challenge for bacteria, because translation cannot terminate efficiently without a stop codon. The trans-translation pathway resolves nonstop translation complexes by removing the nonstop mRNA, the incomplete protein, and the stalled ribosome. P1 co-transduction experiments demonstrated that tmRNA, a key component of the trans-translation pathway, is essential for viability in Shigella flexneri. tmRNA was previously shown to be dispensable in the closely related species Escherichia coli, because E. coli contains a backup system for trans-translation mediated by the alternative release factor ArfA. Genome sequence analysis showed that S. flexneri does not have a gene encoding ArfA. E. coli ArfA could suppress the requirement for tmRNA in S. flexneri, indicating that tmRNA is essential in S. flexneri because there is no functional backup system. These data suggest that resolution of nonstop translation complexes is required for most bacteria.     

The Lon Protease Is Essential for Full Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 lon mutants are supersusceptible to ciprofloxacin, and exhibit a defect in cell division and in virulence-related properties, such as swarming, twitching and biofilm formation, despite the fact that the Lon protease is not a traditional regulator. Here we set out to investigate the influence of a lon mutation in a series of infection models. It was demonstrated that the lon mutant had a defect in cytotoxicity towards epithelial cells, was less virulent in an amoeba model as well as a mouse acute lung infection model, and impacted on in vivo survival in a rat model of chronic infection. Using qRT-PCR it was demonstrated that the lon mutation led to a down-regulation of Type III secretion genes. The Lon protease also influenced motility and biofilm formation in a mucin-rich environment. Thus alterations in several virulence-related processes in vitro in a lon mutant were reflected by defective virulence in vivo.     

Penicillin-Binding Protein 1 of Staphylococcus aureus Is Essential for Growth

pbpA, a gene encoding penicillin-binding protein (PBP) 1 of Staphylococcus aureus, was cloned in an Escherichia coli MC1061 transformant which grew on a plate containing 512 μg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of pbpA could be disrupted only when RN4220, a methicillin-sensitive S. aureus strain, had additional copies of pbpA in its episome. Furthermore, these episomal copies of pbpA could not be eliminated by an incompatible plasmid when the chromosomal copy of pbpA was disrupted beforehand. Based on these observations, we concluded that pbpA is essential for the growth of methicillin-sensitive S. aureus.     

Trypanosome Letm1 Protein Is Essential for Mitochondrial Potassium Homeostasis

Letm1 is a conserved protein in eukaryotes bearing energized mitochondria. Hemizygous deletion of its gene has been implicated in symptoms of the human disease Wolf-Hirschhorn syndrome. Studies almost exclusively performed in opisthokonts have attributed several roles to Letm1, including maintaining mitochondrial morphology, mediating either calcium or potassium/proton antiport, and facilitating mitochondrial translation. We address the ancestral function of Letm1 in the highly diverged protist and significant pathogen, Trypanosoma brucei. We demonstrate that Letm1 is involved in maintaining mitochondrial volume via potassium/proton exchange across the inner membrane. This role is essential in the vector-dwelling procyclic and mammal-infecting bloodstream stages as well as in Trypanosoma brucei evansi, a form of the latter stage lacking an organellar genome. In the pathogenic bloodstream stage, the mitochondrion consumes ATP to maintain an energized state, whereas that of T. brucei evansi also lacks a conventional proton-driven membrane potential. Thus, Letm1 performs its function in different physiological states, suggesting that ion homeostasis is among the few characterized essential pathways of the mitochondrion at this T. brucei life stage. Interestingly, Letm1 depletion in the procyclic stage can be complemented by exogenous expression of its human counterpart, highlighting the conservation of protein function between highly divergent species. Furthermore, although mitochondrial translation is affected upon Letm1 ablation, it is an indirect consequence of K⁺ accumulation in the matrix.     

Prdm6 Is Essential for Cardiovascular Development In Vivo

Members of the PRDM protein family have been shown to play important roles during embryonic development. Previous in vitro and in situ analyses indicated a function of Prdm6 in cells of the vascular system. To reveal physiological functions of Prdm6, we generated conditional Prdm6-deficient mice. Complete deletion of Prdm6 results in embryonic lethality due to cardiovascular defects associated with aberrations in vascular patterning. However, smooth muscle cells could be regularly differentiated from Prdm6-deficient embryonic stem cells and vascular smooth muscle cells were present and proliferated normally in Prdm6-deficient embryos. Conditional deletion of Prdm6 in the smooth muscle cell lineage using a SM22-Cre driver line resulted in perinatal lethality due to hemorrhage in the lungs. We thus identified Prdm6 as a factor that is essential for the physiological control of cardiovascular development.     

The Microtubule-Binding Protein Ensconsin Is an Essential Cofactor of Kinesin-1

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Highlights? Ensconsin is required for cargo transport driven by kinesin-1 ? The C terminus of ensconsin activates kinesin-1; its N terminus binds microtubules ? Kinesin-1 mutants without autoinhibitory activity do not require ensconsin     

Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.The excision of intronic sequences from precursor mRNAs is a critical step during eukaryotic gene expression. This reaction is catalyzed by the spliceosome, a macromolecular complex composed of small nuclear ribonucleoproteins (snRNPs) and many additional proteins. Spliceosome assembly and splicing catalysis occur in an ordered multistep process, which includes multiple conformational rearrangements (35). Spliceosomal snRNPs are assembled from snRNAs and protein components, the latter of which fall into two classes: snRNP-specific and common proteins. The common or canonical core proteins are also termed Sm proteins, specifically SmB, SmD1, SmD2, SmD3, SmE, SmF, and SmG (10; reviewed in reference 9), which all share an evolutionarily conserved bipartite sequence motif (Sm1 and Sm2) required for Sm protein interactions and the formation of the heteroheptameric Sm core complex around the Sm sites of the snRNAs (3, 7, 29). Prior to this, the Sm proteins form three heteromeric subcomplexes: SmD3/SmB, SmD1/SmD2, and SmE/SmF/SmG (23; reviewed in reference 34). Individual Sm proteins or Sm subcomplexes cannot stably interact with the snRNA. Instead, a stable subcore forms by an association of the subcomplexes SmD1/SmD2 and SmE/SmF/SmG with the Sm site on the snRNA; the subsequent integration of the SmD3/SmB heterodimer completes Sm core assembly.In addition to the canonical Sm proteins, other proteins carrying the Sm motif have been identified for many eukaryotes. Those proteins, termed LSm (like Sm) proteins, exist in distinct heptameric complexes that differ in function and localization. For example, a complex composed of LSm1 to LSm7 (LSm1-7) accumulates in cytoplasmic foci and participates in mRNA turnover (4, 8, 31). Another complex, LSm2-8, binds to the 3′ oligo(U) tract of the U6 snRNA in the nucleus (1, 15, 24). Finally, in the U7 snRNP, which is involved in histone mRNA 3′-end processing, the Sm proteins SmD1 and SmD2 are replaced by U7-specific LSm10 and LSm11 proteins, respectively (20, 21; reviewed in reference 28).This knowledge is based primarily on the mammalian system, where spliceosomal snRNPs are biochemically well characterized (34). In contrast, for trypanosomes, comparatively little is known about the components of the splicing machinery and their assembly and biogenesis. In trypanosomes, the expression of all protein-encoding genes, which are arranged in long polycistronic units, requires trans splicing. Only a small number of genes are additionally processed by cis splicing (reviewed in reference 11). During trans splicing, a short noncoding miniexon, derived from the spliced leader (SL) RNA, is added to each protein-encoding exon. Regarding the trypanosomal splicing machinery, the U2, U4/U6, and U5 snRNPs are considered to be general splicing factors, whereas the U1 and SL snRNPs represent cis- and trans-splicing-specific components, respectively. In addition to the snRNAs, many protein splicing factors in trypanosomes have been identified based on sequence homology (for example, see references 14 and 19).Recent studies revealed variations in the Sm core compositions of spliceosomal snRNPs from Trypanosoma brucei. Specifically, in the U2 snRNP, two of the canonical Sm proteins, SmD3 and SmB, are replaced by two novel, U2 snRNP-specific proteins, Sm16.5K and Sm15K (33). In this case, an unusual purine nucleotide, interrupting the central uridine stretch of the U2 snRNA Sm site, discriminates between the U2-specific and the canonical Sm cores. A second case of Sm core variation was reported for the U4 snRNP, in which a single protein, SmD3, was suggested to be replaced by the U4-specific LSm protein initially called LSm2, and later called SSm4, based on a U4-specific destabilization after SSm4 knockdown (30). A U4-specific Sm core variation was also previously suggested and discussed by Wang et al. (33), based on the inefficient pulldown of U4 snRNA through tagged SmD3 protein. However, neither of these two studies conclusively demonstrated by biochemical criteria that the specific Sm protein resides in the U4 Sm core; a copurification of other snRNPs could not be unequivocally ruled out.By using a combination of RNase H cleavage, tandem affinity purification, and mass spectrometry, we provide here direct biochemical evidence that in the variant Sm core of the U4 snRNP, only SmD3 is replaced by the U4-specific SSm4. SSm4 is nuclear localized, and the silencing of SSm4 leads to a characteristic phenotype: dramatic growth inhibition, general trans- and cis-splicing defects, a loss of the integrity of the U4 snRNA, as well as a destabilization of the U4/U6 di-snRNP. Furthermore, in vitro reconstitution assays revealed that under stringent conditions, SSm4 is sufficient to specify U4-specific Sm core assembly. In sum, our data establish SSm4 as a specific component of the U4 Sm core and demonstrate its importance in U4/U6 di-snRNP biogenesis, splicing function, and cell viability.     

The Synaptonemal Complex Protein ZYP1 Is Required for Imposition of Meiotic Crossovers in Barley

In many cereal crops, meiotic crossovers predominantly occur toward the ends of chromosomes and 30 to 50% of genes rarely recombine. This limits the exploitation of genetic variation by plant breeding. Previous reports demonstrate that chiasma frequency can be manipulated in plants by depletion of the synaptonemal complex protein ZIPPER1 (ZYP1) but conflict as to the direction of change, with fewer chiasmata reported in Arabidopsis thaliana and more crossovers reported for rice (Oryza sativa). Here, we use RNA interference (RNAi) to reduce the amount of ZYP1 in barley (Hordeum vulgare) to only 2 to 17% of normal zygotene levels. In the ZYP1^RNAi lines, fewer than half of the chromosome pairs formed bivalents at metaphase and many univalents were observed, leading to chromosome nondisjunction and semisterility. The number of chiasmata per cell was reduced from 14 in control plants to three to four in the ZYP1-depleted lines, although the localization of residual chiasmata was not affected. DNA double-strand break formation appeared normal, but the recombination pathway was defective at later stages. A meiotic time course revealed a 12-h delay in prophase I progression to the first labeled tetrads. Barley ZYP1 appears to function similarly to ZIP1/ZYP1 in yeast and Arabidopsis, with an opposite effect on crossover number to ZEP1 in rice, another member of the Poaceae.     

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.     

The PS1 Hairpin of Mcm3 Is Essential for Viability and for DNA Unwinding In Vitro

The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3_K499A) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3 _K499A strain. The ATPase activity of Mcm2-7_3K499A was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.     

BUB1 Is Essential for the Viability of Human Cells in which the Spindle Assembly Checkpoint Is Compromised

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Mthfd1 Is an Essential Gene in Mice and Alters Biomarkers of Impaired One-carbon Metabolism

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1^gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1^gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.Folate-mediated one-carbon (1C)³ metabolism is compartmentalized in the cytoplasm, mitochondria, and nucleus of mammalian cells (1). In the cytoplasm, 1C metabolism functions to carry and chemically activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine (2) (see Fig. 1). Methionine can be adenosylated to form S-adenosylmethionine (AdoMet), the major cellular methyl group donor required for the methylation of DNA, RNA, histones, small molecules, and lipids. Nuclear 1C metabolism functions to synthesize thymidylate from dUMP and serine during S phase through the small ubiquitin-like modifier-dependent translocation of cytoplasmic serine hydroxymethyltransferase (cSHMT), dihydrofolate reductase, and thymidylate synthase into the nucleus (3).Open in a separate windowFIGURE 1.Folate-mediated one-carbon metabolism occurs in the mitochondria, nucleus, and cytoplasm. Mitochondrial-derived formate traverses to the cytoplasm where it is incorporated into the folate-activated one-carbon pool through the activity of FTHFS and utilized in the synthesis of purines, thymidylate, and the methylation of homocysteine to methionine. Methionine can be converted to a methyl donor through its adenosylation to AdoMet. Thymidylate biosynthesis occurs in the cytoplasm and nucleus. The one-carbon unit is labeled in bold. GCS, glycine cleavage system; mSHMT, mitochondrial serine hydroxymethyltransferase; mMTHFD, mitochondrial methylenetetrahydrofolate dehydrogenase; mMTHFC, mitochondrial methenyltetrahydrofolate cyclohydrolase; mFTHFS, mitochondrial formyltetrahydrofolate synthetase; MTHFD, methylenetetrahydrofolate dehydrogenase; MTHFC, methenyltetrahydrofolate cyclohydrolase; FTHFS, formyltetrahydrofolate synthetase; MTHFR, methylenetetrahydrofolate reductase; TS, thymidylate synthase; DHFR, dihydrofolate reductase; and cSHMT, cytoplasmic serine hydroxymethyltransferase.Serine, through its conversion to glycine by SHMT, is a primary source of 1Cs for nucleotide and methionine synthesis (4). SHMT generates 1Cs in the cytoplasm, mitochondria, and nucleus, although the generation of 1Cs through SHMT activity in the cytoplasm is not essential in mice, indicating the essentiality of mitochondria-derived 1Cs for cytoplasmic 1C metabolism (5). In mitochondria, the hydroxymethyl group of serine and the C2 carbon of glycine are transferred to tetrahydrofolate (THF) to generate 5,10-methylene-THF by the mitochondrial isozyme of SHMT and the glycine cleavage system, respectively (6). The 1C carried by methylene-THF is oxidized and hydrolyzed to generate formate by the NAD-dependent methylene-THF dehydrogenase (MTHFD) and methenyl-THF cyclohydrolase (MTHFC) activities encoded by a single gene, Mthfd2 (7), and 10-formyl-THF synthetase (FTHFS) activity, encoded by Mthfd1L (8) (see Fig. 1).In the cytoplasm, the product of the Mthfd1 gene, C1THF synthase, is a trifunctional enzyme that contains NADP-dependent MTHFD and MTHFC activities on the N-terminal domain of the protein, and FTHFS activity on the C-terminal domain (9). These three activities collectively catalyze the interconversion of THF, 10-formyl-THF, 5,10-methenyl-THF, and 5,10-methylene-THF (10) (Fig. 1). The ATP-dependent FTHFS activity of C1THF synthase condenses mitochondria-derived formate with THF to form 10-formyl-THF, which is required for the de novo synthesis of purines (9). The MTHFC and MTHFD activities convert 10-formyl-THF to methylene-THF (11). Methylene-THF is utilized in the de novo synthesis of thymidylate or, alternatively, can be irreversibly reduced by methylene-THF reductase to 5-methyl-THF, which is used in the remethylation of homocysteine to methionine (12).Impairments in 1C metabolism, due to insufficient folate cofactors and/or single nucleotide polymorphisms in genes that encode folate-dependent enzymes, are associated with numerous pathologies and developmental anomalies, including cancers, cardiovascular disease, and neural tube defects. The causal mechanisms underlying the folate-pathology relationship(s) remains to be established. However, a number of hypotheses have been proposed related to the role of 1C metabolism in genome stability and gene expression. Decreased thymidylate synthesis results in increased uracil misincorporation into DNA and decreased rates of cell division, causing double strand breaks in DNA and genomic instability (13). Decreased AdoMet synthesis alters methylation patterns in CpG islands in DNA and can result in histone hypomethylation, which can alter gene expression (2). Proliferating cells also require the de novo synthesis of purines to maintain rates of DNA synthesis (14).It has been shown that the gene product of Mthfd2, mitochondrial MTHFC/MTHFD is essential in mice, and Mthfd2 deficiency results in embryonic lethality (15). This protein is required for the generation of formate from serine in the mitochondria of embryonic cells. Here, we have investigated the essentiality of the Mthfd1 gene in mice and the effect of altered Mthfd1 gene expression on biomarkers of cytoplasmic 1C metabolism. Our data demonstrate that Mthfd1 is an essential gene in mice and that Mthfd1-deficient mice are a model for the study of folate-associated pathologies.