Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals

Abstract

The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na+ and H+, but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes.

Molecular Basis of Nucleobase Transport Systems in Mammals

“…Dipyridamole, a potent inhibitor of both ENT1 and ENT2, has no effect on cellular [ Human ENBT1/SLC43A3 is expressed abundantly in the liver, lung, and heart, and ubiquitously in the pancreas, testis, thymus, placenta, and kidney. 10,48) Immunohistochemical studies showed that ENBT1/SLC43A3 is localized on the sinusoidal membrane of human hepatocytes 10) and on the basolateral membrane of epithelial cells of the proximal convoluted tubule in the human kidney. 48) Consistent with those results, GFPtagged ENBT1/SLC43A3 is localized on the basolateral membrane of polarized MDCKII cells.…”

“…48) This issue was resolved by a study attempting to identify the cDNA of a purine nucleobase transporter using comprehensive functional screening, in which human SLC43A3 was found to stimulate the accumulation of [ 3 H] adenine in HEK293 cells. 10) Currently, SLC43A3 is considered to be a molecular entity of the facilitative purineselective nucleobase transport system involved in the uptake of adenine and hypoxanthine in many types of cells. ] adenine uptake occurs when ENBT1/SLC43A3 is introduced in mouse fibroblast-derived A9 cells that lack the capability to salvage purine nucleobases.…”

“…Review basis of purine nucleobase transport systems, which involve a concentrative transporter (sodium-dependent nucleobase transporter 1; SNBT1 7) ) and three facilitative transporters (ENT1, 8) ENT2, 9) and ENBT1/SLC43A3 10) ). Their substrate specificities as nucleobase transporters are summarized in Table 1. 3.…”

“…Although the existence of purine nucleobase transport systems has been previously reported in a variety of mammalian cells and tissues, 5,6) the underlying molecular mechanisms remain to be elucidated. Recently, several solute carrier (SLC) transporters such as sodium-dependent nucleobase transporter 1 (SNBT1)/ SLC23A4, 7) equilibrative nucleoside transporter 1 (ENT1)/ SLC29A1, 8) ENT2/SLC29A, 9) and equilibrative nucleobase transporter 1 (ENBT1)/SLC43A3 10) have been reported to mediate the transport of purine nucleobases, and their functions have been characterized. Specifically, ENBT1/SLC43A3 is a novel transporter that has been more recently identified as a selective purine nucleobase transporter.…”

“…Such SNBT1-like transport systems are present in LLC-PK1 [15][16][17] and OK cells 18) hENT1/ SLC29A1 8) hENT2/ SLC29A2 8,9) rENT2/ Slc29a2 9) hENBT1/ SLC43A3 10,56) derived from the renal proximal tubules of the pig and opossum, respectively. These transport systems recognize purine and pyrimidine nucleobases, with the exception of adenine, with Na + /substrate stoichiometry of 1 : 1.…”