Functional Characterization of Two Putative Nucleobase Transporters in Arabidopsis Using Heterologous Complementation in Yeast

Miller, Sara E. M.S., Purdue University, August 2012. Functional Characterization of Two Putative Nucleobase Transporters in Arabidopsis Using Heterologous Complementation in Yeast. Major Professor: George S. Mourad. To identify the substrate profile function for AtNAT5, one of the twelve members of the NAT/NCS2 gene family putatively identified to transport xanthine and/or uric acid, and to identify other functional aspects of transport for the already characterized AtNCS1 adenine‐ guanine‐uracil transporter (Mourad et al., 2012) we used heterologous complementation techniques in S. cerevisiae mutant strains deficient in nucleobase transport. The ability of transformed yeast cells expressing the AtNAT5 locus to grow in the presence of a panel of nucleobase toxic analogs revealed that the AtNAT5 protein is unable to transport adenine, guanine, cytosine, or uracil as expected by its previously defined putative function. Using uptake based assays, the ability of AtNAT5 to uptake [H]‐nucleobases from the growth medium confirmed that using this particular methodology AtNAT5 does not transport xanthine. Heterologous complementation in yeast deficient in native ScNCS1 transporters provided additional evidence of transporter function for the previously characterized AtNCS1 transporter (Mourad et al., 2012). When AtNCS1 transformed strains were grown on media containing the toxic analogs 5‐bromo‐2‐deoxyuridine and pyrithiamine, allantoin as the sole nitrogen source, and media containing various concentrations of pyridoxine (Vitamin B6), AtNCS1 did not function to transport toxic analogs of uridine or thiamine, nor allantoin or pyridoxine.