Heterologous Complementation in Yeast Reveals the Solute Specificity of the Nucleobase Cation Symporter 1 proteins of the plants Zea mays and Setaria viridis

Nucleobases and their precursors are vital organic molecules in al living cels due to their role in nucleic acid biochemistry, sugar metabolism, as wel as serving as energy molecules. In plants, the salvage and catabolism of nucleobases requires an intricate membrane transport mechanism that ensures their movement between cels and also within cels between the organeles and cytoplasm. One of several nucleobase transporter membrane families that have been identified is the Nucleobase Cation Symporter 1 (NCS1). In this report the specific nucleobase substrates transported by the sole NCS1 protein of corn (Zea mays), ZmNCS1, and by its relative grass Setaria viridis, SvNCS1, are identified. The genes encoding ZmNCS1 and SvNCS were PCR-cloned then spliced into a yeast expression vector. The yeast vectors containing the plant genes were transformed into yeast cels lack ing their native NCS1. Such yeast cels were then used to identify the substrate specificity of the expressed plant NCS1 proteins using two approaches. First, by growing on media containing toxic structural analogs of various nucleobases. Second, by assaying for the uptake of a panel of radiolabeled nucleobases from the growth medium into the cels. In addition, heterologous competition between radiolabeled substrates and an aray of non-radioac tive nucleobases revealed the kinetic properties of uptake for both plant NCS1 proteins. The results revealed that ZmNCS1 and SvNCS1 proteins have novel functions, in that they are both strict transporters of the purines adenine and guanine. In addition, SvNCS1 transports hypoxanthine, a unique function when compared to other known plant NCS1s. Nucleobases are nitrogen-rich compounds vital to al organisms. They are involved in DNA, metabolism, and serve as energy molecules for cels. There are two classifications of nucleobases; purines and pyrimidines, both of which are essential to these celular functions. Purine Pyrimidine The goal of this report was to determine the substrate specificity of the two NCS1 proteins in corn (ZmNCS1) and green foxtail (SvNCS1). Figure 1: Amino Acid Alignment of Plant NCS1s Cels gain access to nucleobases through two primary methods. First, cels can synthesize new nucleobases. Second, cels have evolved the ability to break down complex molecules, like DNA, and salvage the nucleobases within. This process is much more energy-eficient, and thus vital to a cel. After a nucleobase has been salvaged, it must be transported both within a cel, and between other cels. There are several families of transport proteins that serve this purpose, with one such family being the Nucleobase Cation Symporter 1 (NCS1) family. Though similar in function, not al NCS1 proteins transport the same nucleobases. The gene for the putative NCS1 transporters in corn (Zea mays) and green foxtail (Setaria viridis) has been isolated, but not fuly characterized. Introduction The functions of both NCS1 transporters were determined using two primary methods. First, the genes were isolated and spliced into an expression vector. This expression vector was then transformed into yeast cels (Saccharomyces cerevisiae) deficient in their native nucleobase transporters. Second, three types of experiments were caried out to determine the function of the transporters. In one procedure, yeast cels expressing the plant NCS1s were plated on media containing toxic mimics of various nucleobases. If cels were kiled on said media, the toxic mimics were transported into the cel due to the relevant plant NCS1. In a second method, cels were exposed to radiolabeled nucleobases. After five minutes, the cels were filtered and their radioactivity measured using a scintilation counter. The levels of radioactivity were used to determine the amount of uptake by the yeast cels. The final method was a competition study done to determine whether certain nucleobase could inhibit the uptake of radiolabeled nucleobases. Approaches Figure 2: Transport of 8-Azaadenine Transport of Toxic Purine and Pyrimidine Analogs A B A B Figure 3: Transport of [3H] Guanine Figure 4: Transport of [3H] Uracil Transport of Radiolabeled Purines and Pyrimidines Competition Study Between Potential Substrates Figure 5: Competition Between Potential Substrates Figure 6: Summary of Substrate Specificity