Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria

Abstract

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.

Fig. 1.

De novo and salvage pathway synthesis of dTTP. There are two distinct pathways for dTTP synthesis. In the de novo pathway, SHMT catalyzes the conversion of serine and tetrahydrofolate (THF) to methyleneTHF and glycine. TYMS converts methyleneTHF and dUMP to dihydrofolate (DHF) and dTMP. DHF is converted to THF for subsequent rounds of dTMP synthesis in an NADPH-dependent reaction catalyzed by DHFR. In the salvage pathway, thymidine is phosphorylated by TK to form dTMP.

Fig. 2.

Identification of DHFRL1 and alignment to DHFR. (A) 5′ RACE was performed to identify DHFR transcripts that encoded a mitochondrial DHFR isozyme. Using a primer complementary to DHFR sequence, two bands were obtained following electrophoresis. The bands were isolated, cloned, and sequenced revealing DHFRL1 as an expressed transcript. (B) The sequence of DHFR and DHFRL1 were aligned using ClustalW in Lasergene Megalign software. The sequences are 92% identical.

Fig. 3.

TYMS and DHFRL1 localize to mitochondria. TYMS-GFP fusion proteins (A) and DHFRL1-GFP fusion proteins (B) were expressed in HeLa cells, as well as a mitochondrial marker mito-CFP. (A) TYMS-GFP localizes to the nucleus, cytoplasm, and colocalizes with the mito-CFP mitochondrial marker. (B) DHFRL1-GFP localizes exclusively to mitochondria. (C) TYMS, DHFRL1, and SHMT2 proteins localize to the matrix and inner membrane of fractionated HepG2 cells. Lane 1, mitochondrial extract; lane 2, outer membrane fraction; lane 3, inner membrane space; lane 4, matrix; lane 5, inner membrane. Hsp60 is the inner membrane/matrix marker, Cox I is an inner membrane marker, and Bcl-x_S/L is an outer membrane marker.

Fig. 4.

Knockdown of DHFRL1 inhibits DHFR activity in mitochondrial extracts. Scrambled siRNA and siRNA against DHFRL1 were transfected into HepG2 cells and DHFRL1 protein and activity was measured. (A) Mitochondria from HepG2 cells that were purified for de novo dTMP synthesis assays (Table 1), and DHFRL1 activity experiments exhibited no nuclear or cytosolic contamination using immunoblots against GAPDH and Lamin A. (B) No DHFRL1 was present in mitochondria of HepG2 cells transfected with siRNA against DHFRL1.

Fig. 5.

DHFRL1 cDNA rescues glycine auxotrophy in glyC CHO cells. CHO glyC cells were transfected with cDNAs encoding human DHFRL1-GFP, or a GFP-empty vector control. Stable cells lines were selected for G418 resistance in the presence of 200 μM glycine and 10 mg/L thymidine. For growth assays, cells were cultured with and without glycine and trypan blue exclusion was used to quantify viable cells. Four independent lines were assayed per transfection and experiments were done in triplicate. There was no significant difference in growth among the cells transfected with DHFRL1-GFP with or without glycine. CHO glyC cells transfected with GFP-empty vector and cultured without glycine failed to proliferate over the 72 h time period.