Eudes, Aymerick , Kunji, Edmund , Noiriel, Alexandre , Klaus, Sebastian , Vickers, Tim , Beverley, Stephen , Gregory, Jesse , Hanson, Andrew .
Functional Analysis of Plant and Cyanobacterial Members of the Folate-Biopterin Transporter Family.
The Arabidopsis (Arabidopsis thaliana) At2g32040 protein and the orthologous Synechocystis Slr0642 protein belong to the folate-biopterin transporter (FBT) family and transport folates when expressed in Escherichia coli. Nothing is known about the native structure or transport mechanism of any FBT protein. First, to improve an E. coli folate synthesis (pabA) mutant as a host for recombinant FBT studies, we inactivated the cryptic folate transporter gene abgT. This eliminated low-frequency, spontaneous acquisition of the ability to grow on folate media. Guided by a topology model, we then used site-directed mutagenesis of Slr0642 to identify transport-critical residues by replacing them with Cys or Ala. Mutating polar residues in predicted transmembrane domains abolished growth on folate media but not Slr0642 protein accumulation in the membrane fraction, implicating these residues in the transport mechanism. All residues are conserved or conservatively replaced in At2g32040 but not in the eight other Arabidopsis FBT proteins, implying that the latter cannot transport folate. Consistent with this inference, other Arabidopsis FBT genes neither allowed growth of the pabA abgT mutant on folate, nor complemented folate or pterin transporter mutations in Leishmania major. When Arabidopsis cDNA libraries were screened using the double mutant, At2g32040 was the only FBT gene recovered. These data provide the first insights into the function of an FBT carrier and indicate that At2g32040 is the only Arabidopsis FBT protein that transports folates.
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1 - University of Florida, Horticultural Sciences Department, P.O. Box 110690, Gainesville, FL, 32611-0690, USA
2 - Medical Research Council,, Dunn Human Nutrition Unit, Hills Road, Cambridge, CB2 2XY, United Kingdom
3 - Washington University School of Medicine,, Department of Molecular Microbiology,, St. Louis, Missouri, 63110, USA
4 - University of Florida,, Food Science and Human Nutrition Department,, Gainesville, Florida, 32611, USA
5 - University of Florida, Horticultural Sciences Department, P.O. Box 110690, Gainesville, Florida, 32611, USA
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM