Identification of a De Novo Thymidylate Biosynthesis Pathway in Mammalian Mitochondria
Total Page：16
File Type：pdf, Size：1020Kb
Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria Donald D. Andersona,b, Cynthia M. Quinteroa, and Patrick J. Stovera,b,1 aDivision of Nutritional Sciences, Cornell University, 127 Savage Hall, Ithaca, NY 14853; and bGraduate Field of Biochemistry, Molecular and Cellular Biology, Cornell University, Ithaca, NY 14853 Edited by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, and approved July 12, 2011 (received for review March 7, 2011) The de novo and salvage dTTP pathways are essential for maintain- ing 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 pre- sence 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 dihydro- folate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner mem- Fig. 1. De novo and salvage pathway synthesis of dTTP. There are two dis- brane, confirming the presence of this pathway in mitochondria. tinct pathways for dTTP synthesis. In the de novo pathway, SHMT catalyzes Knockdown of DHFRL1 using siRNA eliminated DHFR activity in the conversion of serine and tetrahydrofolate (THF) to methyleneTHF and mitochondria. DHFRL1 expression in CHO glyC, a previously unchar- glycine. TYMS converts methyleneTHF and dUMP to dihydrofolate (DHF) and dTMP. DHF is converted to THF for subsequent rounds of dTMP synthesis acterized mutant glycine auxotrophic cell line, rescued the glycine in an NADPH-dependent reaction catalyzed by DHFR. In the salvage pathway, auxotrophy. De novo thymidylate synthesis activity was dimin- thymidine is phosphorylated by TK to form dTMP. ished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells In this pathway, 5,10-methyleneTHF, a one-carbon donor, is gen- treated with methotrexate, a DHFR inhibitor. De novo thymidylate erated from serine by SHMT and used for the conversion of synthesis in mitochondria prevents uracil accumulation in mito- dUMP to dTMP in a reaction catalyzed by TYMS. The TYMS- chondrial DNA (mtDNA), as uracil levels in mtDNA isolated from catalyzed reaction generates dihydrofolate, which is converted to glyA CHO cells was 40% higher than observed in mtDNA isolated THF in an NADPH-dependent manner by DHFR. The regener- from wild-type CHO cells. These data indicate that unlike other ated THF can then be used for subsequent rounds of thymidylate nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity. biosynthesis. Both de novo and salvage dTMP synthesis have been assumed folate ∣ one-carbon metabolism ∣ thymidine ∣ deoxyribouridine to occur in the cytoplasm to support both nuclear and mtDNA replication, but recent studies have demonstrated nuclear locali- egulation of cellular dTTP pools is essential for faithful repli- zation of TK1 (9) and the de novo dTMP biosynthesis pathway Rcation of nuclear and mitochondrial DNA (mtDNA), with during S-phase and as a result of DNA damage (10, 11). The in- both depletion and expansion of the pool affecting DNA integrity ability of the mitochondrial dTMP salvage pathway to support and human health (1). Depletion of de novo dTMP synthesis mtDNA synthesis, and recent studies indicating that de novo results in deoxyuridine misincorporation into nuclear DNA lead- dTMP synthesis occurs in the nucleus, raised the possibility for a ing to genome instability (2), whereas depletion of mitochondrial requirement for de novo thymidylate biosynthesis in mitochon- dTTP pools, due to mitochondrial deoxyribonucleoside kinases, dria. De novo thymidylate synthesis has been shown to occur deoxyguanosine kinase, or thymidine kinase 2 (TK2) mutations in plant mitochondria through the activities of an SHMT isozyme are associated with mtDNA-depletion syndromes and severe [mitochondrial serine hydroxymethyltransferase (SHMT2)] and a mitochondrial dysfunction in humans (3). Elevations in dTTP bifunctional enzyme containing both TYMS and DHFR (12). pools, as observed in mitochondrial neurogastrointestinal ence- Interestingly, previous studies have localized TYMS to the mito- phalomyopathy (4), an autosomal recessive disease resulting from chondria in mammalian cells (1, 13) and in neurospora crassa decreased levels of cytoplasmic thymidine phosphorylase, results (14). In mammalian cells, mitochondria contain SHMT2, which in mtDNA depletion (5), deletions (6), and site-specific point is required for formate and glycine synthesis (15), but the third mutations (6). enzyme in the pathway, DHFR, does not localize to the mito- There are two distinct pathways for thymidine nucleotide BIOCHEMISTRY chondria (16). synthesis (Fig. 1). The salvage pathway involves the conversion of thymidine to thymidylate, and occurs in the mitochondria and cytoplasm catalyzed by TK2 and thymidine kinase 1 (TK1), re- Author contributions: D.D.A. and P.J.S. designed research; D.D.A. and C.M.Q. performed spectively (7). Salvage pathway synthesis of dTTP is not sufficient research; D.D.A. and P.J.S. analyzed data; and D.D.A. and P.J.S. wrote the paper. to sustain mtDNA replication (3) and therefore requires de novo The authors declare no conflict of interest. dTMP synthesis, which involves the conversion of dUMP to This article is a PNAS Direct Submission. dTMP, catalyzed by the tetrahydrofolate (THF)-dependent en- 1To whom correspondence should be addressed. E-mail: [email protected]. zymes serine hydroxymethyltransferase (SHMT), thymidylate This article contains supporting information online at www.pnas.org/lookup/suppl/ synthase (TYMS), and dihydrofolate reductase (DHFR) (8). doi:10.1073/pnas.1103623108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1103623108 PNAS ∣ September 13, 2011 ∣ vol. 108 ∣ no. 37 ∣ 15163–15168 Downloaded by guest on September 25, 2021 In this study, a de novo dTMP biosynthesis pathway was identified in mammalian mitochondria that includes a unique mitochondrial isozyme of DHFR called DHFRL1, which was previously thought to be a pseudogene (17). De novo dTMP synthesis is shown to be necessary to prevent uracil accumulation in mtDNA. Results Mammalian Mitochondria Contain a De Novo dTMP Synthesis Path- way. Purified intact mitochondria from Chinese hamster ovary (CHO) cells and HepG2 cells (Table 1 and Fig. S1) were capable of catalyzing the formation of 3H-dTMP from unlabeled dUMP, NADPH, and [2,3-3H]-L-serine in vitro. This activity is a unique demonstration of folate-dependent de novo dTMP biosynthesis in mammalian mitochondria. Methotrexate inhibited dTMP production in mitochondria isolated from wild-type CHO cells. Mitochondria isolated from glyA CHO cell mutants, which lack SHMT2, exhibited a 94% reduction in dTMP synthesis capacity, indicating the essentiality of SHMT2 within mitochondria for mitochondrial de novo dTMP synthesis. Disruption of mitochon- dria by sonication-reduced de novo dTMP synthesis activity, in- ′ dicating that compartmentalization within mitochondria, or the Fig. 2. Identification of DHFRL1 and alignment to DHFR. (A)5 RACE was performed to identify DHFR transcripts that encoded a mitochondrial DHFR formation of complexes within mitochondria, may be important isozyme. Using a primer complementary to DHFR sequence, two bands were for de novo dTMP synthesis. Alternatively, the reduced activity obtained following electrophoresis. The bands were isolated, cloned, and may reflect dilution or loss of the endogenous folate cofactor pool sequenced revealing DHFRL1 as an expressed transcript. (B) The sequence following sonication. of DHFR and DHFRL1 were aligned using ClustalW in Lasergene Megalign software. The sequences are 92% identical. Identification of DHFRL1 as an Expressed Gene. To determine if al- ternative human DHFR transcripts are expressed that encode a DHFRL1 was constructed using the crystal structure of human mitochondrial leader sequence, 5′ rapid amplification of cDNA DHFR [Protein Data Bank (PDB) ID code 3GHW] as a tem- ends (RACE) was conducted using total mRNA isolated from plate. The model of DHFRL1 structure and DHFR structure HepG2 cells. Two prominent bands were observed from the 5′ are highly similar with an rms value of 0.0034 (Fig. S2). RACE experiment. Upon sequencing the two bands, both DHFR and DHFRL1 were identified (Fig. 2A). DHFRL1 is annotated as Localization of DHFRL1 and TYMS to Mitochondria. The subcellular a mammalian pseudo gene in Entrez Gene, and expressed se- localization of DHFRL1 and TYMS were determined by confo- quence tags are present (GenBank accession no. DN994912). cal microscopy following the transfection of pCMV6-AC- The DHFR and DHFRL1 primary sequences are 92% identical DHFRL1-GFP and pCMV6-AC-TYMS-GFP constructs into (Fig. 2B). An energy minimized three-dimensional model of HeLa cells. The mitochondrial control vector pTagCFP-mito was also transfected to visualize mitochondria, and DRAQ5 nuclear stain was used to visualize nuclei. Colocalization of the mitochon- Table