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Dr. George Mourad
Department of Biology
Indiana University - Purdue University Fort Wayne
Nucleobase biochemistry plays a central role in serving various needs of plants during their growth and development.The synthesis of nucleic acids, carbohydrate metabolism, and overall plant growth, development, and reproduction depends on a continuous flow of purines and pyrimidines attributed to a balance of different pathways like de novo synthesis, salvage pathways, and catabolic pathways.This intricate nucleobase biochemistry requires intra- and inter-cellular movement of nucleobases facilitated by different families of membrane transporter proteins.The genome of Zea mays contains three genes that encode for putative azagunainelike transporters (AZG) with high similarity to a previously characterized AZG transporter of Arabidopsis thaliana in our lab.The goal of this research is to identify the substrate specificity and binding properties of the three putative members of AZG-like family of Zea mays; ZmAZG1, ZmAZG2 and ZmAZG3.The genes encoding for ZmAZG1, ZmAZG2, and ZmAZG3 were independently PCR-cloned then spliced into a yeast expression vector, and the recombinant plasmids transformed into yeast cells lacking their native nucleobase transporters.The transgenic yeast cells were assayed for their ability to uptake a panel of radiolabeled nucleobases to reveal the transport profiles of the heterologously expressed ZmAZG transporters.The results show that ZmAZG1 facilitates transport of uracil whereas, ZmAZG3 transports adenine, guanine, cytosine and hypoxanthine.The kinetic properties of AZG-like transporters were further revealed using heterologous competition between radiolabeled [3H]- hypoxanthine as a substrate and an array of non-radioactive cold nucleobase competitors.For ZmAZG3, the nucleobase adenine competitively inhibited [3H]-hypoxanthine transport, whereas cytosine and xanthine were respectively found to be weak and poor competitive inhibitors.As for ZmAZG2, the nucleobases adenine and cytosine competitively inhibited [3H]-hypoxanthine transport, while xanthine and uric acid were respectively found to be weak and poor inhibitors.Moreover, both ZmAZG2 and ZmAZG3 continually transported [3H]-hypoxanthine over a period of two hours without reaching a saturation point suggesting their pivotal role in hypoxanthine transport.These findings shed light on the significant role the AZG-like transporters play in maize growth and development.
Buda, Geeta; Mourad, George; and Test, Opus, "Heterologous complementation in yeast reveals the substrate specificity of the three-member azaguanine-like family of transporters of Zea mays, ZmAZG1, ZmAZG2, and ZmAZG3" (2015). 2015 IPFW Student Research and Creative Endeavor Symposium. 10.