Tetrahydrofolate Recognition by the Mitochondrial Folate Transporter

Abstract

A mitochondrial carrier family (MCF) of transport proteins facilitates the transfer of charged small molecules across the inner mitochondrial membrane. The human genome has ϳ50 genes corresponding to members of this family. All MCF proteins contain three repeats of a characteristic and conserved PX(D/E)XX(K/R) motif thought to be central to the mechanism of these transporters. The mammalian mitochondrial folate transporter (MFT) is one of a few MCF members, known as the P(I/L)W subfamily, that have evolved a tryptophan residue in place of the (D/E) in the second conserved motif; the function of this substitution (Trp-142) is unclear. Molecular dynamics simulations of the MFT in its explicit membrane environment identified this tryptophan, as well as several other residues lining the transport cavity, to be involved in a series of sequential interactions that coordinated the movement of the tetrahydrofolate substrate within the transport cavity. We probed the function of these residues by mutagenesis. The mutation of every residue identified by molecular dynamics to interact with tetrahydrofolate during simulated transit into the aqueous channel severely impaired folate transport. Mutation of the subfamily-defining tryptophan residue in the MFT to match the MCF consensus at this position (W142D) was incompatible with cell survival. These studies indicate that MFT Trp-142, as well as other residues lining the transporter interior, coordinate tetrahydrofolate descent and positioning of the substrate in the transporter basin. Overall, we identified residues in the walls and at the base of the transport cavity that are involved in substrate recognition by the MFT.Folate metabolism is essential for mammalian cell survival and consists of reaction sequences that occur in the mitochondrial and cytosolic compartments. In dividing cells, cytosolic folate metabolism uses one-carbon units for purine nucleotide and thymidylate synthesis, whereas mitochondrial folate metabolism primarily drives the supply of formate to the cytoplasm for these processes. Mitochondrial folates are also used as cofactors for mitochondrial glycine synthesis, and cells deficient in mitochondrial folate metabolism are auxotrophic for glycine despite the fact that enzymes capable of glycine synthesis are present in both compartments (1-5). An example of such glycine auxotrophy is the Chinese hamster ovary (CHO)-derived glyB cell, which retains wild-type activity of all the enzymes involved in mitochondrial folate metabolism but is unable to accumulate mitochondrial folates (1-3). Previously, we used complementation of glycine auxotrophy with a retroviral cDNA library to isolate the gene defective in glyB cells; it encoded a protein that transported folates into mitochondria, the mitochondrial folate transporter (MFT) 2 (3). In glyB cells, the MFT open reading frame has a G192E missense mutation that diminishes mitochondrial folate levels by ϳ99% and effectively eliminates the transport of mitochondrial folates (2, 3). Transfection of cDNAs encoding ...

SLC25 Family Member Genetic Interactions Identify a Role for HEM25 in Yeast Electron Transport Chain Stability

“…Another SLC25 family member, SLC25A32, imports folate into mitochondria (Urano et al 2014; Lawrence et al 2011). SLC25A32 appears to be essential for glycine synthesis inside mitochondria as folate is required for the conversion of serine to glycine by mitochondrial serine hydroxymethyltransferase (Locasale 2013; Giardina et al 2015; Saint-Marc et al 2015).…”

The peroxisomal NAD+ carrier of Arabidopsis thaliana transports coenzyme A and its derivatives

“…Furthermore, the great majority of amino acids which have been found essential for function particularly by site-directed mutagenesis studies (Heidkämper et al 1996;Briggs et al 1999;Echtay et al 2001;Stipani et al 2001;Indiveri et al 2002;Tonazzi et al 2005;De Lucas et al 2008;Cappello et al 2006Cappello et al , 2007Aluvila et al 2010;Miniero et al 2011;Giangregorio et al 2010;Lawrence et al 2011;Monné et al 2012;Tonazzi et al 2012) belong to the mitochondrial carrier signature motif or protrude into the carrier's internal cavity, i.e., into the substrate translocation path. The residues of triplets 23, 30, 33, 77 and 80 protruding into the carrier cavity differ between PXN (QVT, QWL, NVK, STK and GMQ, respectively) and SLC25A17 (MVT, DWQ, RNQ, VSK and SLQ, respectively).…”

“…With all these transporters, except yeast Ant1p and its homologs in Arabidopsis, PXN shares the distinct feature of having a tryptophan instead of an acidic residue in the mitochondrial carrier signature motif PX(D/E)XX(R/K) present in the second repeat of these proteins (Cappello et al 2007;Fig. 2 of Palmieri et al 2011;Lawrence et al 2011). Nevertheless, the substrate specificity of PXN is distinct from that of any other carrier present in the above-mentioned cluster, although some overlapping functions (i.e., substrates transported) occur, as well as from that of any other previously characterized members of the mitochondrial carrier family (Palmieri and Pierri 2010a;Palmieri et al 2011;Palmieri 2012).…”

Mammalian Mitochondrial and Cytosolic Folylpolyglutamate Synthetase Maintain the Subcellular Compartmentalization of Folates

“…A transporter is present in the inner mitochondrial membrane, the mitochondrial folate transporter (MFT, SLC25A32), which facilitates the entry of folates into the mitochondrial matrix (15,24,31). From the data of Fig.…”