Translocation of metal phosphate via the phosphate inorganic transport system of Escherichia coli

Abstract

Pi transport via the phosphate inorganic transport system (Pit) of Escherichia coli was studied in natural and artificial membranes. Pi uptake via Pit is dependent on the presence of divalent cations, like Mg2+, Ca2+, Co2+, or Mn2+, which form a soluble, neutral metal phosphate (MeHPO4) complex. Pi-dependent uptake of Mg2+ and Ca2+, equimolar cotransport of Pi and Ca2+, and inhibition by Mg2+ of Ca2+ uptake in the presence of Pi, but not of Pi uptake in the presence of Ca2+, indicate that a metal phosphate complex is the transported solute. Metal phosphate is transported in symport with H+ with a mechanistic stoichiometry of 1. Pit mediates efflux and homologous exchange of metal phosphate, but not heterologous metal phosphate exchange with Pi, glycerol-3P, or glucose-6P. The metal phosphate efflux rate increased with pH, whereas the rate of metal phosphate exchange was essentially pH independent. Metal phosphate uptake was inhibited at low internal pH. Efflux was inhibited by a proton motive force (interior negative and alkaline), whereas exchange was inhibited by the membrane potential only. These results have been evaluated in terms of ordered binding and dissociation of metal phosphate and proton on the outer and inner surface of the cytoplasmic membrane.