Reconstitution of the phosphoglycerate transport protein of Salmonella typhimurium.
Operation of the phosphoglycerate transport protein (PgtP) of Salmonella typhimurium has been studied in proteoliposomes by using a technique in which membrane protein is solubilized and reconstituted directly from small volumes of cell cultures. When protein from induced cells was reconstituted into phosphate (Pi)-loaded proteoliposomes, it was possible to demonstrate a PgtP-mediated exchange of internal and external phosphate. For this homologous Pi:Pi antiport, kinetic analysis indicated a Michaelis constant (Kt) of 1 mM and a maximal velocity of 26 nmol/min mg of protein; arsenate inhibited with a Ki of 1.3 mM, suggesting that PgtP did not discriminate between these two inorganic substrates. Pi-loaded proteoliposomes also accumulated 3-phosphoglycerate and phosphoenolpyruvate, establishing for each of them a concentration gradient (in/out) of about 100-fold; phosphoenolpyruvate (Ki = 70 microM) rather than 3-phosphoglycerate (Kt = 700, Ki = 900 microM) was the preferred substrate for these conditions. We also concluded that such heterologous exchange was a neutral event, since its rate and extent were unaffected by the presence of a protonophore and unresponsive to the imposition of a membrane potential (positive or negative inside). In quantitative work, we found a stoichiometry of 1:1 for the exchange of Pi and 3-phosphoglycerate, and given an electroneutral exchange, this finding is most easily understood as the overall exchange of divalent Pi against divalent phosphoglycerate. These experiments establish that PgtP functions as an anion exchange protein and that it shares important mechanistic features with the Pi-linked antiporters, GlpT and UhpT, responsible for transport of glycerol 3-phosphate and hexose 6-phosphates into Escherichia coli.
Varadhachary
A
A
Department of Physiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Maloney
P C
PC
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D005988:Glyceric Acids / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials; D010710:Phosphates / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D005988:Glyceric Acids; D010710:Phosphates; C008885:2-phosphoglycerate; C005156:3-phosphoglycerate