FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity, High Affinity, Nucleoside and Nucleobase Transporter

Background: FUN26 is a nucleoside transporter expressed in yeast vacuoles. Results: Proteoliposome studies of purified FUN26 reveal broad nucleoside and nucleobase uptake that is sensitive to C(2)-ribose modifications. Conclusion: FUN26 is a high affinity and broad selectivity nucleoside and nucleobase transporter. Significance: FUN26 has a unique substrate transport profile relative to other ENTs and retains activity following detergent solubilization and purification. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2)- and C(5)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [H-3]Adenine nucleobase transport efficiency is increased approximate to 4-fold relative to nucleosides tested with no observed [H-3]adenosine or [H-3]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.