Functional characterization of a maize purine transporter by expression in Aspergillus nidulans.
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 (K(m) = 30 +/- 2.5 microM), 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.
National Center for Scientific Research Demokritos, Institute of Biology, 153 10 Aghia Paraskevi, Attiki, Greece.
E
E
Argyrou
V
V
Sophianopoulou
N
N
Schultes
G
G
Diallinas
D001205:Ascorbic Acid / Q000378:metabolism; D001233:Aspergillus nidulans / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001692:Biological Transport; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011687:Purines / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism; D003313:Zea mays / Q000235:genetics*
C099053:LPE1 protein, Zea mays; D026901:Membrane Transport Proteins; D010940:Plant Proteins; D011687:Purines; D019820:Xanthine; D014527:Uric Acid; D001205:Ascorbic Acid