The Human SLC25A33 and SLC25A36 are two Mitochondrial Pyrimidine Nucleotide Transporters

The human SLC25A33 and SLC25A36 are two members of solute carrier family 25 (SLC25) that transports a variety of metabolites across the inner mitochondrial membrane, connecting cytosolic and matrix functions. These two proteins are the close relatives of the Saccharomyces cerevisiae Rim2p, the mitochondrial carrier for pyrimidine nucleotides (1), whose absence by deletion of RIM2 gene causes total loss of mtDNA and lack of growth on non-fermentative carbon sources (2). This study reports the identification and in-depth functional characterization of SLC25A33 and of SLC25A36. The SLC25A33 and SLC25A36 genes were overexpressed in Escherichia coli and the recombinant proteins were purified and reconstituted in liposomes. Recombinant SLC25A33 transports uracil, thymine, and cytosine (deoxy)-nucleoside di- and tri-phosphates by an antiport mechanism and SLC25A36 transports cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Furthermore, subcellular localization demonstrated that SLC25A36 is a mitochondrial protein as previously shown for SLC25A33 (3). These results indicate that SLC25A33 and SLC25A36 are mitochondrial transporters for pyrimidine ribo- and deoxynucleotides. These nucleotides are essential for the synthesis of DNA and the various types of RNA including the RNA primers necessary for the initiation of DNA replication and repair, as well as the products of their breakdown. Furthermore, in vivo, the expression of SLC25A33 or SLC25A36 in RIM2 haploid strains restores the phenotype of S. cerevisiae by rescuing mtDNA, mitochondrial respiration, mitochondrial membrane potential and growth on glycerol and other respiratory substrates. These findings strongly support SLC25A33 and SLC25A36 controlling the uptake of pyrimidine (deoxy)nucleotides into mitochondria.