CHARACTERIZATION OF A BINDING PROTEIN-DEPENDENT GLUTAMATE TRANSPORT-SYSTEM OF RHODOBACTER-SPHAEROIDES

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 mu M), 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 mu M) and L-glutamine (K-i of 15 mu M) 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 mu M) but competes only poorly with L-glutamate for binding to the binding protein (K-i of >200 mu M). 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.