Genetic and Biochemical Characterization Reveals the Substrate Specificity of the Three-Member Azaguanine-like Transporter Family of Zea mays

Geeta Buda, Purdue University

Abstract

Nucleobases, purines and pyrimidines, along with their precursors are central to nucleic acid biochemistry, a process essential for all forms of life. Not only do they play a role in metabolism of phospholipids, carbohydrates and glycoproteins, they have a pronounced role in synthesis of secondary metabolites in addition to serving as energy source. In maize (Zea mays), ZmAZG (azaguanine-like transporter family), one of the several nucleobase transporters, plays a major role in ensuring the movement of nucleobases between and within cells and between the organelles and cytoplasm. With an aim to characterize these transporters in Zea mays, this research was conducted. The study focused on identifying the substrate specificity and binding properties of the three putative members of the AZG-like family of Zea mays; ZmAZG1, ZmAZG2 and ZmAZG3. Transgenic yeast cells, deficient in their native nucleobase transporter, 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 showed that ZmAZG1 facilitates the transport of uracil, xanthine and hypoxanthine whereas ZmAZG2 and ZMAZG3 transport adenine, guanine, cytosine, hypoxanthine and xanthine. The kinetic properties of AZG-like transporters were further revealed using homologous competition between radiolabeled nucleobase and varying concentrations of same cold competitor, heterologous competition of radiolabeled nucleobase against a concentration series of various cold nucleobases, and heterologous competition between radiolabeled hypoxanthine as a substrate and an array of non-radioactive cold nucleobase competitors. Effects of protonophores and Na+ pump inhibitors on the function of the ZmAZG transporters revealed that they are proton-driven symporters. Moreover, all three ZmAZG transporters continually transported [3H]-hypoxanthine over a period of two hours without reaching a saturation point suggesting their pivotal role in hypoxanthine transport. Additionally, ZmAZG2 complemented the missing azaguanine-like transporter function in a transgenic homozygous double knock-out Arabidopsis mutant Atazg1-1/Atazg1-1, Atazg2-1/Atazg2-1 providing in planta confirmation of being a proton-driven symporter. These findings shed light on the significant role the AZG-like transporters play in maize growth and development.

Degree

M.S.

Advisors

Mourad, Purdue University.

Subject Area

Biology|Genetics|Biochemistry

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