Characterization of a binding protein-dependent glutamate transport system of Rhodobacter sphaeroides

Abstract

The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (K(t) of 1.2 μM), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH. From the shock fluid, an L-glutamate-binding protein was isolated and purified. The protein binds L-glutamate (apparent K(d) of 1.3 μM) and L-glutamine (K(i) of 15 μM) with high affinity. The expression level of this binding protein is maximal at limiting concentrations of glutamine in the growth medium. The glutamate-binding protein restores the uptake of L-glutamate in spheroplasts. L-Aspartate is a strong competitive inhibitor of L-glutamate uptake (K(i) of 3 μM) but competes only poorly with L-glutamate for binding to the binding protein (K(i) of >200 μM). The uptake of L-aspartate in R. sphaeroides also involves a binding protein which is distinct from the L-glutamate-binding protein. These data suggest that in R. sphaeroides, the L-glutamate- and L-aspartate- binding proteins interact with the same membrane transporter.