Resolution of the Multiplicity of the Glutamate and Aspartate Transport Systems of Escherichia coZi*

Mutants of Escherichia coli D,W with altered levels of glutamate and aspartate transport were used to resolve the following five separate transport systems for these two amino acids: (a) a binding protein-dependent, sodium-independent, cysteate-inhibitable glutamate-aspartate system; (b) a binding protein-independent, sodium-independent, glutamate-aspartate system which is inhibitable by fi-hydroxyaspartate or cysteate; (c) a binding protein-independent, sodium-dependent, cu-methylglutamate-inhibitable glutamate-specific system (Miner, K. M., and Frank, L. (1974) J. Bacterial. 117, 1093-1098; Halpern, Y. S., Barash, H., Dover, S., and Druck, K. (1973) J. Bacterial. 114, 53-58); (d) a binding protein-independent aspartate-specific system (Kay, W. W. (197115. Biol. Chem. 246, 7373-7382); and (e) a dicarboxylic acid transport (dct) system which transports succinate, malate, fumarate, and aspartate (Kay, W. W., and Kornberg, H. L. (1971) Eur. J. Biochem. 18, 274-281). Mutants were generated which have specific alterations in the level of each system, and through the use of these mutants K,,S values were obtained for three of these systems as well as estimates for the contribution of four of these systems to the total glutamate and aspartate transport in E. coli D,W measured in the presence of 40 mu sodium ion and at substrate concentrations of 28 PM. The binding protein-dependent system has K,, values of 0.5 PM for glutamate and 0.5 JAM for aspartate, and it is responsible for 15% of the glutamate and 25% of the aspartate transport. The /3-hydroxyaspartate-inhibitable glutamate-aspartate transport system has K,, values of 5 ~IVI for glutamate and 4 PM for aspartate, and it is responsible for 60% of the total glutamate and 52% of the total aspartate transport. The sodium-dependent glutamate-specific transport system has a K,,! value of 1.5 PM for glutamate and accounts for 25% of the total glutamate transport. The aspartate-specific system is responsible for 23% of the total aspartate transport. The amount of aspartate transported through the dicarboxylic

The transport system elevated in this mutant does not correspond to the P-hydroxyaspartate-inhibitable glutamate-aspartate transport system since /3-hydroxyaspartate does not inhibit either the elevated glutamate or aspartate transport (Fig. 3) (Fig.   8). The same data plotted by the method of Cornish-Bowden (34) (data not shown) indicate that the inhibition is competitive.
Cysteate also inhibits aspartate binding (data not shown).
Glutamate binding was not significantly inhibited by a 300-fold excess of m-a-methylglutamate, an inhibitor of the sodium-dependent glutamate transport system (Fig. 11, or by a loo-fold excess of /3-hydroxyaspartate, an inhibitor of the /3-  . 3). In the absence of sodium, 90% of the glutamate uptake and 78% of the aspartate uptake is inhibitable by P-hydroxyaspartate (Table III). 0 The difference in aspartate transport between mutants HA9 (17 nmol/min/mg) and HA12 (5.6 nmol/min/mg) (Table II) is estimated to be the contribution of the P-hydroxyaspartate-inhibitable glutamate-aspartate system to the total aspartate transport.
The contribution of this system to the total glutamate transport is taken as the portion of the glutamate uptake in the wild type strain D,W inhibitable by P-hydroxyaspartate (12.6 nmollminimg) (Fig. 3B). h N.D. indicates value not determined. ' The glutamate-specific transport system accounts for 85% of the glutamate uptake in the presence of sodium ion in mutant HA12-MGl with the remaining uptake being through the glutamateaspartate binding protein-dependent system. The velocity contribution of the glutamate-specific system in this mutant was determined by subtracting the velocity of the glutamate-aspartate binding protein-dependent system (8 nmoliminlmg) from the total glutamate transport activity of mutant HAlB-MGl (53 nmol/min/mg, Table  III). The activity of the binding protein-dependent system is estimated as the portion of the glutamate transport (in the absence of sodium ion) inhibitable by aspartate (Fig. 6). J The glutamate-specific system velocity is estimated from the portion of the sodium-stimulated glutamate transport in mutant HA12 resistant to inhibition by aspartate or cysteate (5.3 nmol/min/ mg) (Fig. 5).
* The contribution of the aspartate-specific system to the total aspartate transport is taken as the amount of aspartate transport lost when this system is genetically removed in mutant HA9 (5 nmol/min/mg) (Table II).
( yaspartate-inhibitable glutamate-aspartate system. The combination of the inhibition data and the specific activities of transport in the mutants allows for the estimation of the contributions of four of the transport systems to the total glutamate and aspartate transport in the wild type strain DZW (Table VI). Under the conditions of these assays, the dct system does not appear to catalyze a significant percentage of the total aspartate transport since the genetic removal of this system (mutant HA12-ST511 does not result in a significant change in the level of aspartate transport (Table II). Under the assay conditions used, 60% of the glutamate transport is mediated through the p-hydroxyaspartate-inhibitable glutamate-aspartate transport system, 25% through the sodium-dependent glutamate-specific system, and 15% through the binding protein-dependent system. For aspartate, 52% of the uptake is mediated by the P-hydroxyaspartate-inhibitable glutamate-aspartate system, 23% by the aspartate-specific system, and 25% by the binding protein-dependent system.

DISCUSSION
The studies presented in this paper were undertaken to characterize the glutamate and aspartate transport systems of Escherichia coli and to assess the role of the glutamateaspartate binding protein in transport. Previous studies in our laboratory (10, 19) as well as those by Barash and Halpern (11) and Kahane et al. (18) have suggested that the glutamateaspartate binding protein is probably involved in the transport of glutamate and aspartate; however, the exact role of this protein had remained elusive. Our attempts to resolve this question have resulted in the work reported here where we have found that the binding protein appears to serve as the substrate recognition component for one of five separate transport systems which operate by at least three different mechanisms (i.e. sodium-independent membrane-bound, sodium-dependent membrane-bound, and binding protein-dependent). Earlier work resulted in the partial characterization of three of the five systems (2,3,5,7); however, in many cases the data were difficult to interpret due to the unresolved multiplicity of the transport systems which accumulate these two amino acids. The five separate transport systems for these two amino acids are: the membrane-bound aspartatespecific system (3), a previously uncharacterized binding protein-independent P-hydroxyaspartate-inhibitable system shared by glutamate and aspartate, a sodium-dependent, membrane-bound glutamate-specific system (7), a binding protein-dependent glutamate-aspartate system, and the dicarboxylic acid transport system (2, 3) which recognizes succinate, fumarate, malate, and aspartate ( Fig. 1).
Genetic removal of the aspartate-specific system (mutant HA9) results in the loss of a component of aspartate transport which is resistant to inhibition by glutamate or cysteate (Fig.  2) while glutamate transport is not altered (Table II). The system lost in this mutant appears to be the same aspartatespecific system previously described by Kay (3). In addition to aspartate, this system probably also recognizes P-hydroxyaspartate since mutant HA9 was isolated by virtue of its resistance to this analogue (Table I). Kay has also reported that P-hydroxyaspartate inhibits aspartate transport through the aspartate-specific system (3), although the ratio of inhibitor to substrate needed to demonstrate significant inhibition was much higher than the ratio used in this study (Fig. 3). Kay's studies indicate that in whole cells the aspartate-specific system has a K, for aspartate transport of 3.7 p,M and that this system is retained in membrane vesicles. Willis and Furlong (19) have also reported aspartate transport to be retained in membrane vesicles prepared from cultures of E. coli D,W grown on succinate as the sole carbon source.