Reference "Identification of transport-critical residues in a folate transporter from the folate-biopterin transporter (FBT) family"

Reference ID

54980

Title

Identification of transport-critical residues in a folate transporter from the folate-biopterin transporter (FBT) family

Source

J Biol Chem, 2010, vol. 285, pp. 2867-2875

Authors (8)

Eudes-A	Kunji-E-R
Noiriel-A	Klaus-S-M
Vickers-T-J	Beverley-S-M
Gregory-J-F-3rd	Hanson-A-D

Abstract

The Synechocystis Slr0642 protein and its plastidial Arabidopsis (Arabidopsis
thaliana) ortholog At2g32040 belong to the folate-biopterin transporter (FBT)
family within the major facilitator superfamily. Both proteins transport
folates when expressed in Escherichia coli. Because the structural requirements
for transport activity are not known for any FBT protein, we applied mutational
analysis to identify residues that are critical to transport and interpreted
the results using a comparative structural model based on E. coli lactose
permease. Folate transport was assessed via the growth of an E. coli pabA abgT
strain, which cannot synthesize or take up folates or p-aminobenzoylglutamate.
In total, 47 residues were replaced with Cys or Ala. Mutations at 22 positions
abolished folate uptake without affecting Slr0642 expression in membranes,
whereas other mutations had no effect. Residues important for function mostly
line the predicted central cavity and are concentrated in the core alpha-
helices H1, H4, H7, and H10. The essential residue locations are consistent
with a folate-binding site lying roughly equidistant from both faces of the
transporter. Arabidopsis has eight FBT proteins besides At2g32040, often
lacking conserved critical residues. When six of these proteins were expressed
in E. coli or in Leishmania folate or pterin transporter mutants, none showed
evidence of folate or pterin transport activity, and only At2g32040 was
isolated by functional screening of Arabidopsis cDNA libraries in E. coli. Such
negative data could reflect roles in transport of other substrates. These
studies provide the first insights into the native structure and catalytic
mechanism of FBT family carriers.