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Plant Cell, Vol. 13, 953-964, April 2001, Copyright © 2001, American Society of Plant Physiologists
Functional Characterization of a Maize Purine Transporter by Expression in Aspergillus nidulans
Eleftheria Argyroua,
Vicky Sophianopouloua,
Neil Schultesb, and
George Diallinasc
a National Center for Scientific Research "Demokritos, " Institute of Biology, 153 10 Aghia Paraskevi, Attiki, Greece
b Department of Biochemistry and Genetics, Connecticut Agricultural Experiment Station, New Haven, Connecticut 06511
c Microbiology Laboratory, Department of Biology, University of Athens, Athens 157 81, Greece
Correspondence to:
George Diallinas, diallina{at}biology.db.uoa.gr (E-mail), 301-7274136 (fax)
We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport–deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (Km = 30 ± 2.5 µM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.
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