Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue Str. 9, D-60438 Frankfurt am Main, Germany.
Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue Str. 9, D-60438 Frankfurt am Main, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
J Mol Biol. 2022 Jan 30;434(2):167393. doi: 10.1016/j.jmb.2021.167393. Epub 2021 Dec 9.
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.
SLC23 家族成员是碱基或抗坏血酸的转运体。哺乳动物 SLC23 抗坏血酸转运体是钠离子偶联的，而非哺乳动物碱基转运体已被提议，但尚未正式证明是质子偶联的协同转运体。这种分配完全基于使用质子载体的体内转运测定。在这里，通过建立该蛋白家族的第一个体外转运测定，我们证明了 SLC23 碱基转运体的代表性成员作为单转运体起作用。我们通过鉴定将尿嘧啶转化为 UMP 的酶尿嘧啶磷酸核糖基转移酶在体内驱动尿嘧啶摄取的关键作用来解释这些相互矛盾的分配。对尿嘧啶磷酸核糖基转移酶的详细表征表明，质子载体存在时，完整细胞中尿嘧啶摄取的急剧减少是由酸化诱导的酶失活引起的。因此，SLC23 家族由单转运体和协同转运体组成，与结构上相关的 SLC4 和 SLC26 家族一致，这两个家族以前已经证明可以同时容纳两种转运模式。
