Unexpected oligomeric structure of the FocA formate channel of Escherichia coli : a paradigm for the formate–nitrite transporter family of integral membrane proteins

Authors


  • Editor: Jeff Cole

Correspondence: R. Gary Sawers, Institute for Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle (Saale), Germany. Tel.: +49 345 552 6350; fax: +49 345 552 7010; email: gary.sawers@mikrobiologie.uni-halle.de

Abstract

FocA is a predicted formate channel with a deduced mass of 31 kDa that catalyzes the bidirectional movement of formate across the cytoplasmic membrane of Escherichia coli and is the archetype of the formate–nitrite transporter (FNT) family. Overproduced FocA variants with either an N- or a C-terminal Strep-tag increased formate import into anaerobic E. coli cells as determined by the enhanced activity of a single-copy formate-dependent fdhFlacZ fusion. Using anti-FocA antibodies, we could show that both FocA variants were integrated into the cytoplasmic membrane. Circular dichroism spectroscopy of purified FocAStrep–N revealed a high α-helical content of 56% consistent with the predicted six transmembrane helices present in the protein. Analysis of the oligomeric state by blue-native polyacrylamide gel electrophoresis revealed FocA to have an unexpected pentameric quaternary structure. This study reports the first isolation of an FNT family member.

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