Biochemical and kinetic analysis of Arabidopsis nucleobase transport
Abstract
Due to its relevance in plant growth, reproduction and development, inter- and intracellular transport of nucleobases remains to be an important area of basic research. In this work, two nucleobase transporters of Arabidopsis thaliana from two distinct families were partially characterized with the goal of determining their solute transport specificity. The first transporter studied is nucleobase ascorbate transporter 2 (AtNAT2 ), one of a 12-member putative gene family predicted to function as a nucleobase-ascorbate transporters. The coding sequence of AtNAT2 was cloned into a yeast expression vector driven by a yeast constitutive promoter. Yeast expressing AtNAT2 were tested through growth studies utilizing toxic purine/pyrimidine analogues as well as through radiolabel uptake studies. We found evidence that AtNAT2 does not transport adenine, guanine, cytosine, uracil and xanthine. The second transporter studied was the nucleobase cation sympoter 1 of A. thaliana ( AtNCS1). Here kinetic analysis of the recently functionally characterized A. thaliana nucleobase cation symporter 1 (AtNCS1) was undertaken through radiolabeled uptake assays utilizing yeast strains expressing the AtNCS1 gene. Substrate saturation kinetics was approached by gradually increasing the substrate concentration, and the apparent Km and Vmax values were determined.
Degree
M.S.
Advisors
Mourad, Purdue University.
Subject Area
Genetics
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