FUNCTIONAL-SIGNIFICANCE OF THE SIGNATURE CYSTEINE IN HELIX-8 OF THE ESCHERICHIA-COLI 4-AMINOBUTYRATE TRANSPORTER FROM THE AMINE-POLYAMINE-CHOLINE SUPERFAMILY - RESTORATION OF CYS-300 TO THE CYS-LESS GABP

gab permease (GabP) is the exclusive mediator of 4-aminobutyrate (GABA ) transport across the Escherichia coli plasma membrane. Helix 8 and a portion of the adjoining cytoplasmic region (loop 8-9) constitute the GabP ''consensus amphipathic region'' (CAR), a potential channel-form ing domain that is found to be evolutionarily conserved within the APC (amine-polyamine-choline) transporter superfamily, Upon the polar sur face of the CAR, all known gab permeases display a ''signature cystein e'' not found in other members of the APC superfamily, suggesting that discrete features within the CAR might play a role in imparting speci ficity (k(cat)/K-m) to the translocation reaction. Here we show that a mong the five cysteine residues in the E. coli GabP, only Cys-300, the signature cysteine, can restore wild type properties to the Cys-less GabP mutant. We conclude (i) from partial reaction studies (equilibriu m exchange, counterflow) that rapid translocation of the GABA binding site from one side of the membrane to the other is greatly facilitated by Cys-300 and (ii) from pharmacological studies that loss of Cys-300 has little effect on the affinity that GabP exhibits for a structural ly diverse array (kojic amine, 5-aminovaleric acid, GABA, nipecotic ac id, and cis-4-aminocrotonic acid) of competitive ligands. These result s raise the possibility that other GABA transporters might rely analog ously upon conserved cysteine residues positioned within the amphipath ic helix 8 and loop 8-9 regions.