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

Liaoyuan A Hu,Steven C King

doi:10.1074/jbc.273.32.20162

Liaoyuan A Hu, Steven C King

Open Access

https://doi.org/10.1074/jbc.273.32.20162

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Abstract

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-forming domain that is found to be evolutionarily conserved within the APC (amine-polyamine-choline) transporter superfamily. Upon the polar surface of the CAR, all known gab permeases display a "signature cysteine" not found in other members of the APC superfamily, suggesting that discrete features within the CAR might play a role in imparting specificity (kcat/Km) to the translocation reaction. Here we show that among 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 (equilibrium 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 structurally diverse array (kojic amine, 5-aminovaleric acid, GABA, nipecotic acid, and cis-4-aminocrotonic acid) of competitive ligands. These results raise the possibility that other GABA transporters might rely analogously upon conserved cysteine residues positioned within the amphipathic helix 8 and loop 8-9 regions.

Highlights

The Escherichia coli gab permease (GabP) is an archetypal APC1 superfamily transporter that catalyzes 4-aminobutyrate (GABA) translocation across the plasma membrane
To investigate the possibility that discrete elements within the consensus amphipathic region” (CAR) have a significant role in determining transport specificity, we have focused on Cys-300 of the E. coli GabP, a “signature cysteine” found in the CAR of GABA transporters but not other members of the APC superfamily
Amino acid sequence alignments show that the CAR of all known gab permeases contains a signature cysteine that is not found at this position in the CAR of other APC superfamily members (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Materials—[3H]GABA (31.6 Ci/mmol) were from NEN Life Science Products. Plasmid pSE380 was from Invitrogen (San Diego, CA), and pBluescript II KS(Ϫ) was from Stratagene (La Jolla, CA). Site-directed Mutagenesis—Mutagenesis was performed using the method of Kunkel [6] as implemented commercially by Bio-Rad. Singlestranded phagemid DNA (from pSCK-GP8 or pSCK-GP9) containing uracil was produced from the E. coli strain, CJ236 (obtained from Bio-Rad), infected with the kanamycin-resistant helper phage, VCSM13 (obtained from Stratagene). Cells grown overnight (16 h) were diluted 100-fold into fresh medium containing 1 mM IPTG (to induce gabP expression from the plasmid) (for SK35) or diluted into medium supplemented with 0.5% glucose, 0.2% ammonium sulfate (to cause catabolite repression of chromosomal genes in CC118), and 1 mM IPTG. Transport reactions were initiated by adding 90 ␮l of washed cells with rapid vortex mixing to 10 ␮l of solution containing 100 ␮M [3H]GABA (3 ␮Ci/ml). Uptake was rapidly quenched by adding 1 ml of a “stop solu-

The GabP Signature Cysteine

This work

RESULTS

DISCUSSION