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

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.


Preparation of plasmids
The cDNA of human ENBT1 was cloned from the human lung total RNA (Clontech, Mountain View, CA) by RT-PCR. In brief, an RT reaction was performed using 3 μg of the total RNA, an oligo(dT) primer and ReverTra Ace (Toyobo, Osaka, Japan). The cDNA of ENBT1 was isolated from the obtained cDNA mixture by PCR using KOD FX DNA polymerase (Toyobo) and the following primers: forward primer, 5'-ATT TTC CAA GTG CTC AAA CGC -3'; reverse primer, 5'-CTG CCA AGG CTA AGT GCA AGG -3'. These primers were designed based on the sequence in GenBank (accession no. NM_017611). PCR was performed using the following conditions: 94°C for 2 min; 33 cycles of (i) 94°C for 20 s, (ii) 56°C for 20 s and (iii) 72°C for 1.5 min. The second PCR was performed using the PCR product as a template and a forward primer containing an EcoRI restriction site (underlined), 5'-AGG AAT TCT GCT CAT GGC GG GCC A -3', and a reverse primer containing an XbaI restriction site (underlined), 5'-GCT CTA GAA CTA TGC AAT TGC AGA -3'. Then the amplified product was introduced at the EcoRI and XbaI sites into a mammalian expression vector, pCI-neo (Promega, Madison, WI). The sequence of the final product was determined with an automated sequencer (ABI PRISM 3100; Applied Biosystems, Foster City, CA).
To generate ENBT1 fused with green fluorescent protein (GFP-ENBT1), the cDNA fragments of ENBT1 were prepared by digestion of the pCI-neo-based plasmids carrying ENBT1 cDNA with EcoRI and XbaI, and then introduced into pEGFP-C1 vector (Clontech).
The cDNAs of the APRT and HPRT1 of human were similarly cloned from the human liver total RNA (Clontech) by RT-PCR, using PCR primers designed on the basis of the sequences in GenBank (accession no. NM_000485 and NM_000194, respectively). For the cloning of APRT, the conditions of PCR were as follows: 94°C for 2 min; 33 cycles of (i) 98°C for 20 s and (ii) 68°C for 20 s. The primers for the first PCR were as follows: forward primer, 5'-CTG CCG CTG GCT CTT CGC ACG -3'; reverse primer, 5'-GCA GCC GGT GCC CCT GGT CACT -3'. The second PCR was performed using a forward primer containing an EcoRI restriction site (underlined), 5'-AT G AAT TCA GCC ATG GCC GAC TCC -3', and a reverse primer containing an XbaI restriction site (underlined), 5'-GAC TCT AGA GAG GCC CTG TGG TCA -3'. For the cloning of HPRT1, the conditions of PCR were as follows: 94°C for 2 min; 33 cycles of (i) 98°C for 20 s, (ii) 57°C for 30 s and (iii) 68°C for 1 min. The primers for the first PCR were as follows: forward primer, 5'-CCT CCT CCT GAG CAG TCA GC -3'; reverse primer, 5'-TTT AGG AAT GCA GCA ACT GAC A -3'. The second PCR was performed using a forward primer containing an EcoRI restriction site (underlined), 5'-AGT GAA TTC CGT TAT GGC GAC CCG CA -3', and a reverse primer containing an XbaI restriction site (underlined), 5'-GCC TCT AGA ACA TTG ATA ATT TTA C -3'.

Western blot analysis
HeLa cells treated for silencing ENBT1 or APRT were washed twice with ice-cold PBS, scraped off, and pelleted by centrifugation at 800 × g for 3 min at 4°C. The cell pellet was homogenized by sonication in an ice cold buffer (pH 7.4) containing 250 mM sucrose, 150 mM NaCl, 20 mM Tris-HCl and supplemented with protease inhibitor cocktail (Sigma-Aldrich), and then centrifuged at 2,000 × g for 10 min at 4°C. The supernatant was recentrifuged at 15,000 × g for 30 min at 4°C, and the resultant pellet was used as the crude membrane fraction sample. The sample (30 μg) was separated on the 10% SDS-polyacrylamide gel by electrophoresis and transferred to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked with 5% skim milk in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 (TBS-T) and then probed with primary antibody/anti-human SLC43A3 (ENBT1) rabbit-polyclonal antibody (Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden) and anti-human APRT purified rabbit polyclonal IgG (GeneTex Inc., Irvine, CA) at a dilution of 1:200 and 1:1,000, respectively, for overnight at 4°C.
After washing three times with TBS-T, the membrane was incubated with secondary antibody/peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a dilution of 1:10,000 for 1 h at room temperature. Then, the expression levels of ENBT1 and APRT were determined by enhanced chemiluminescence using Immobion Western (Millipore, Billerica, MA), according to the manufacturer's instructions.