Abstract
Equilibrative nucleoside transporters (ENTs) are polytopic membrane transporters responsible for the translocation of nucleosides, nucleobases—to a lesser extent—and nucleoside analog therapeutics across cellular membranes. ENTs function in a diffusion controlled bidirectional manner and are thought to utilize an alternating access transport mechanism. However, a detailed understanding of ENT function at the molecular level has remained elusive. ScENT1 (formerly known as Function Unknown Now 26 or FUN26) is the only known ENT ortholog endogenously expressed in S. cerevisiae, and a proteoliposome assay system was used to study homogenously overexpressed and purified ScENT1 (wildtype relative to L390A and F249I mutants). L390 and F249 are highly conserved residues and were found to alter transporter function. L390A produced a reduction of mean transport activity while F249I increased mean substrate translocation relative to wildtype protein. However, both mutations resulted in transport of UTP—a novel gain of function for any ENT. These residues were then mapped onto an ab initio model of FUN26 which suggests they function in substrate translocation (L390) or cytoplasmic gating (F249). Furthermore, wildtype, L390A, and F249I were found to be sensitive to the presence of alcohols. Ethanol attenuated ScENT1-mediated transport of uridine by ~50%. These findings further demonstrate functional similarities between ScENT1 and human ENT isoforms and support identification of FUN26 as ScENT1, the first ENT isoform in S. cerevisiae.
Highlights
Nucleoside transporters are fundamental contributors to nucleoside physiology, pathophysiology, and the beneficial exploitation of nucleoside analog therapeutics
Data obtained in the present study demonstrates that (1) the L390A and F249I mutations are capable of modulating substrate influx and altering the overall substrate transport profile; (2) residues L390, and G463, appear to contribute to the substrate translocation pore while G216 contributes to structural stability of the protein and F249 may regulate cytoplasmic gating; (3) L390A and F249I ScENT1 mutants are capable of low levels of [3 H]-UTP transport—the first demonstration of Equilibrative nucleoside transporters (ENTs)-mediated nucleotide transport; (4) ScENT1-mediated uridine transport is attenuated in the presence of ethanol; and (5) the overall substrate transport profile of ScENT1 is altered by alcohols
ScENT1 is the S. cerevisiae ortholog to hENT1-3 and mutagenesis studies have analyzed the function of residues conserved throughout various members of the ENT
Summary
Nucleoside transporters are fundamental contributors to nucleoside physiology, pathophysiology, and the beneficial exploitation of nucleoside analog therapeutics. Nucleosides serve as metabolic precursors in de novo nucleic acid synthesis, metabolic precursors of energy metabolism (e.g., ATP and GTP), and as ligands for purinergic receptors (e.g., adenosine and inosine) [1,2]. Nucleoside and nucleobase analogs represent important classes of antineoplastic agents and antiviral therapeutics [3]. Activity of many of these hydrophilic compounds relies upon their entry into intracellular metabolic pathways to exert their effectiveness. Transport through cellular membranes is an essential component of therapeutic efficacy for nucleoside and nucleobase derived therapeutics.
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