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

TitleIdentification of transport-critical residues in a folate transporter from the folate-biopterin transporter (FBT) family.
Publication TypeJournal Article
Year of Publication2010
AuthorsEudes, A, Kunji, ERS, Noiriel, A, Klaus, SMJ, Vickers, TJ, Beverley, SM, Gregory, JF, Hanson, AD
JournalJ Biol Chem
Volume285
Issue4
Pagination2867-75
Date Published2010 Jan 22
ISSN1083-351X
KeywordsAmino Acid Sequence, Arabidopsis, Arabidopsis Proteins, Dicarboxylic Acid Transporters, Escherichia coli, Folic Acid, Gene Expression, Gene Library, Genetic Vectors, Leishmania donovani, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Structure, Secondary, Synechocystis, Tetrahydrofolates
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.

DOI10.1074/jbc.M109.063651
Alternate JournalJ. Biol. Chem.
Citation Key10.1074/jbc.M109.063651
PubMed ID19923217
PubMed Central IDPMC2807340
Grant ListR01 AI021903 / AI / NIAID NIH HHS / United States
R01 AI031078 / AI / NIAID NIH HHS / United States
R37 AI021903 / AI / NIAID NIH HHS / United States
MC_U105663139 / / Medical Research Council / United Kingdom
R01 GM071382 / GM / NIGMS NIH HHS / United States
AI21903 / AI / NIAID NIH HHS / United States