Functional characterization of the sole Nucleobase Cation Symporter 1 (PgNCS1) of the gymnosperm Picea glauca

Mahanish J Thapa, Purdue University

Abstract

Purines, pyrimidines, their precursors, and derivatives play a vital role in plant metabolism including nucleic acids, carbohydrates, phospholipids, and glycoproteins. In addition, nucleobases serve as energy source. This role of nucleobases requires the action of nucleobase membrane transporters to move nucleobases within cells, between organelles and the cytoplasm, as well as between cells. Nucleobase Cation Symporter 1 (NCS1) is one of several classes of nucleobase transporters that have been identified and characterized in plants. We found that the genome of the white spruce Picea glauca encodes a single putative NCS1 (PgNCS1). The goal of this research is to identify the solute specificity and binding affinity of PgNCS1. To achieve this goal, PgNCS1 was cloned and heterologously expressed in yeast strains lacking their native NCS1 transporters. The kinetic parameters of PgNCS1 for specific solutes were also determined using radiolabel nucleobase uptake experiments. Our results show that PgNCS1 transports a wide range of solutes including adenine, guanine, uracil, xanthine, and hypoxanthine. Even though PgNCS1 doesn’t transport cytosine, 5-Flurocytosine, uric acid or caffeine, all such compounds bind to PgNCS1 and act as efficient competitive inhibitors. PgNCS1 displays high affinity for guanine (1.71 µM) and adenine (3.87 µM), hypoxanthine (1.83 µM) and somewhat lesser affinity for cytosine (4.95 µM) and xanthine (5.36 µM). Arabidopsis plants deficient in their native AtNCS1, yet genetically-engineered with PgNCS1, showed significant uptake of radiolabeled adenine furnishing an in planta confirmation of the nature of PgNCS1 as a nucleobase transporter.

Degree

M.S.

Advisors

Mourad, Purdue University.

Subject Area

Biology|Molecular biology|Genetics

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