Uniport, Not Proton-Symport, in a Non-Mammalian SLC23 Transporter

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.