Unexpected oligomeric structure of the FocA formate channel of Escherichia coli : a paradigm for the formate-nitrite transporter family of integral membrane proteins.
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 fdhF::lacZ fusion. Using anti-FocA antibodies, we could show that both FocA variants were integrated into the cytoplasmic membrane. Circular dichroism spectroscopy of purified FocA(Strep-N) revealed a high alpha-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.
Falke
Dörte
D
Institute for Biology/Microbiology, Martin-Luther University Halle-Wittenberg, Halle, Germany.
Schulz
Kristin
K
Doberenz
Claudia
C
Beyer
Lydia
L
Lilie
Hauke
H
Thiemer
Barbara
B
Sawers
Robert Gary
RG
D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification; D005561:Formates / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D009573:Nitrites / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D029968:Escherichia coli Proteins; C088103:FocA protein, E coli; D005561:Formates; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009573:Nitrites; C030544:formic acid