Abstract
Acetate is found ubiquitously in the natural environment and can be used as an exogenous carbon source by bacteria, fungi, and mammalian cells. A representative member of the acetate uptake transporter (AceTr) family named SatP (also yaaH) has been preliminarily identified as a succinate-acetate/proton symporter in Escherichia coli However, the molecular mechanism of acetate uptake by SatP still remains elusive. Here, we report the crystal structure of SatP from E. coli at 2.8 Å resolution, determined with a molecular replacement approach using a previously developed predicted model algorithm, which revealed a hexameric UreI-like channel structure. Structural analysis identified six transmembrane (TM) helices surrounding the central channel pore in each protomer and three conserved hydrophobic residues, FLY, located in the middle of the TM region for pore constriction. According to single-channel conductance recordings, performed with purified SatP reconstituted into lipid bilayer, three conserved polar residues in the TM1 facing to the periplasmic side are closely associated with acetate translocation activity. These analyses provide critical insights into the mechanism of acetate translocation in bacteria and a first glimpse of a structure of an AceTr family transporter.
Highlights
Acetate is found ubiquitously in the natural environment and can be used as an exogenous carbon source by bacteria, fungi, and mammalian cells
EcSatP, with a molecular mass of 20 kDa, was eluted at ϳ12 ml from size-exclusion chromatography, which suggested that ecSatP maintains more than 158 kDa within detergent micelles and indicated that ecSatP stabilizes at an oligomeric state (Fig. S1A)
We carried out an in vitro cross-link assay using purified ecSatP, and the result strongly implied that ecSatP was present in a hexameric state (Fig. S1B)
Summary
Acetate is found ubiquitously in the natural environment and can be used as an exogenous carbon source by bacteria, fungi, and mammalian cells. A representative member of the acetate uptake transporter (AceTr) family named SatP ( yaaH) has been preliminarily identified as a succinate–acetate/proton symporter in Escherichia coli. According to single-channel conductance recordings, performed with purified SatP reconstituted into lipid bilayer, three conserved polar residues in the TM1 facing to the periplasmic side are closely associated with acetate translocation activity. These analyses provide critical insights into the mechanism of acetate translocation in bacteria and a first glimpse of a structure of an AceTr family transporter. The unclear translocational mechanism of acetate and novel fold structure of the AceTr family may be highly conserved from bacteria to fungi based on their sequence similarity. The authors declare that they have no conflicts of interest with the contents of this article
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