Function Unknown Number 26 (Fun26) Protein from Saccharomyces Cerevisiae is a Nucleoside Selective Integral Membrane Transporter

Abstract

Nucleoside transporters are polytopic integral membrane proteins (IMPs) that modulate a wide array of eukaryotic physiology by regulating plasmalemmal flux of purine and pyrimidine nucleosides. These transporters exhibit broad substrate selectivity and specificity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. In addition, nucleoside transporters serve as molecular targets for therapeutics designed to inhibit nucleoside flux across membranes (e.g., adenosine reuptake inhibitors) and are the primary means by which nucleoside analog therapeutics (e.g., gemcitabine) are able to enter eukaryotic cells. The current studies are focused on developing functional assays for purified eukaryotic nucleoside transporters as a foundation for defining the molecular basis for how therapeutics interact with this class of integral membrane proteins.Function Unknown Number 26 (FUN26) is a yeast ortholog of the human equilibrative nucleoside transporter (ENT) family. FUN26 was expressed and purified to homogeneity and incorporated into proteoliposomes for functional analysis. Our results demonstrate that FUN26 is: 1) functional in purified form using a defined proteoliposome system, 2) a nucleoside selective bidirectional transporter, 3) selective for purine over pyrimidine nucleosides, 4) sensitive to transport inhibition by known ENT chemotherapeutic substrates, and 5) sensitive to changes in the 2' position of nucleoside substrates. This study provides the first demonstration of functional activity of a purified eukaryotic equilibrative nucleoside transporter in a defined proteoliposome system.