Histidine 225, a Residue of the NhaA-Na+/H+ Antiporter of Escherichia coli Is Exposed and Faces the Cell Exterior

Abstract

Cysteine residues were found nonessential in the mechanism of the NhaA antiporter activity of Escherichia coli. The functional C-less NhaA has provided the groundwork to study further histidine 225 of NhaA which has previously been suggested to play an important role in the activation of NhaA at alkaline pH (Rimon, A., Gerchman, Y., Olami, Y., Schuldiner, S. and Padan, E. (1995) J. Biol. Chem. 270, 26813-26817). C-less H225C was constructed and shown to possess an antiporter activity 60% of that of C-less antiporter and a pH profile similar to that of both the C-less or wild-type antiporters. Remarkably, whereas neither the wild-type nor the C-less antiporters were affected by N-ethylmaleimide, C-less H225C was inhibited by this reagent. To determine the degree of alkylation of the antiporter protein by N-ethylmaleimide, antiporter derivatives tagged at their C termini with six histidines residues were constructed. Alkylation of C-less H225C was measured by labeling of everted membrane vesicles with [14 C]N-ethylmaleimide, affinity purification of the His-tagged antiporter, and determination of the radioactivity of the purified protein. This assay showed that H225C is alkylated to a much higher level than any of the native cysteinyl residues of NhaA reaching saturation at alkyl/ NhaA stoichiometry of 1. The wild-type derivative showed at least 10-fold less alkylation even at higher concentrations, suggesting that H225C resides in a domain that is much more exposed to N-ethylmaleimide than the native cysteinyl residues of NhaA.Since H225C residues both in right-side out and inside-out membrane vesicles were quantitatively alkylated by N-ethylmaleimide, this assay was used to determine the accessibility of H225C to other SH reagents by titrating the H225C left free to react with N-ethylmaleimide, following exposure of the membranes to the reagents. Furthermore, since membrane-impermeant probes can react with residues in membrane-embedded protein only if accessible to the medium containing the reagent, the assay was used to determine the membrane topology of H225C.As expected for a membrane-permeant probe, p-chloromercuribenzoate reacted with H225C as efficiently as N-ethylmaleimide in both membrane orientations. Similar results were obtained with methanethiosulfonate ethylammonium supporting the recent observations that this probe is membrane-permeant. On the other hand, both membrane-impermeant reagents p-chloromercuribenzosulfonate and methanethiosulfonate ethyl-trimethyl ammonium bromide reacted with H225C 10-fold more in right-side out than in inside-out vesicles, and p-chloromercuribenzosulfonate also blocked completely the H225C in intact cells. These results strongly suggest that H225C is exposed at the periplasmic face of the membrane.

The Aqueous Accessibility in the External Half of Transmembrane Domain I of the GABA Transporter GAT-1 Is Modulated by Its Ligands

“…However, even when sodium was replaced by choline, none of these cysteine mutants was impacted by MTSET (data not shown). It is possible that the cysteines at positions 53, 59, and 60 are inactivated by MTSEA reaching from the intracellular side, as it has been shown that this reagent can permeate through the membrane (30,31). However, because MTSEA is smaller than MTSET and MTSES, it is also possible that the aqueous accessibility pathway becomes more narrow toward the cytoplasm, so that the small MTSEA could reach deeper into the "crevice" than MTSET and MTSES.…”

“…3) and MTSES (data not shown). We have therefore used the smaller MTSEA, which is also somewhat membrane permeant (30,31), to see if it affects those cysteine mutants (Fig. 8).…”

The Monoclonal Antibody 1F6 Identifies a pH-dependent Conformational Change in the Hydrophilic NH2 Terminus of NhaA Na+/H+ Antiporter ofEscherichia coli

“…DNA of pGMAR100 was used as a DNA template with the forward mutagenic primer 5Ј-GTG AAA TGT CTC TGC AGA TTC TTT AGC-3Ј and the backward mutagenic primer 5Ј-GCT AAA GAA TCT GCA GAG ACA TTT CAC-3Ј. Isolation of Membrane Vesicles, Assay of Na ϩ /H ϩ Antiporter Activity-Assays of Na ϩ /H ϩ antiporter activity were conducted on everted membrane vesicles (22). The assay of antiport activity was based upon the measurement of Na ϩ -(or Li ϩ )-induced changes in the ⌬pH as described (5,22).…”

“…Resistance to Li ϩ and Na ϩ was tested as described previously (13). Plasmid pAXH, which encodes Xa-His-tagged NhaA, was constructed previously (22) and contains nhaA fused at its 5Ј end to the tac promoter for overexpression and at its 3Ј end to a piece of DNA encoding in tandem two factor Xa cleavage sites followed by six His residues for affinity purification of the protein on Ni 2ϩ -NTA column (22). pG338S-XH is a plasmid isogenic with pAXH but encodes NhaA with a Ser-338 instead of Gly (G338S) (15).…”

“…The transformed cells were grown in minimal medium to A 600 ϭ 0.6, induced with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside, grown for 2 h to A 600 ϭ 1-1.2, harvested, and used for preparation of membranes either after storage overnight at 4°C or after freezing at Ϫ20°C (11). His-tagged NhaA and the mutant His-tagged G338S were affinity purified on Ni 2ϩ -NTA-agarose column (Qiagen, Hilden, Germany) as described (22).…”