HISTIDINE-225, A RESIDUE OF THE NHAA-NA+ H+ ANTIPORTER OF ESCHERICHIA-COLI IS EXPOSED AND FACES THE CELL EXTERIOR/

Cysteine residues were found nonessential in the mechanism of the NhaA antiporter activity of Escherichia coli. The functional C-less NhaA h as provided the groundwork to study further histidine 225 of NhaA whic h has previously been suggested to play an important role in the activ ation of NhaA at alkaline pH (Rimon, A., Gerchman, Y., Olami, Y., Schu ldiner, S. and Padan, E. (1995) J. Biol. Chem. 270, 26813-26817). C-le ss H225C was constructed and shown to possess an antiporter activity 6 0% of that of C-less antiporter and a pH profile similar to that of bo th the C-less or wild-type antiporters. Remarkably, whereas neither th e wild-type nor the C-less antiporters were affected by N-ethylmaleimi de, C-less H225C was inhibited by this reagent. To determine the degre e of alkylation of the antiporter protein by N-ethylmaleimide, antipor ter 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 [C-14]N-ethylmaleimide, affinity pu rification of the His-tagged antiporter, and determination of the radi oactivity of the purified protein. This assay showed that H225C is alk ylated to a much higher level than any of the native cysteinyl residue s of NhaA reaching saturation at alkyl/NhaA stoichiometry of 1. The wi ld-type derivative showed at least 10-fold less alkylation even at hig her concentrations, suggesting that H225C resides in a domain that is much more exposed to N-ethylmaleimide than the native cysteinyl residu es of NhaA. Since H225C residues both in right-side out and in side-ou t membrane vesicles were quantitatively alkylated by N-ethylmaleimide this assay was used to determine the accessibility of H225C to other S H reagents by titrating the H225C left free to react with N-ethylmalei mide, 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 reag ent, the assay was used to determine the membrane topology of H225C.As expected for a membrane-permeant probe, p-chloromercuribenzoate react ed with H225C as efficiently as N-ethylmaleimide in both membrane orie ntations. Similar results were obtained with methanethiosulfonate ethy lammonium supporting the recent observations that this probe is membra ne-permeant. On the other hand, both membrane-impermeant reagents p-ch loromercuribenzosulfonate and methanethiosulfonate ethyl-trimethyl amm onium bromide reacted with H225C 10-fold more in right-side out than i n inside-out vesicles, and p-chloromercuribenzosulfonate also blocked completely the H225C in intact cells. These results strongly suggest t hat H225C is exposed at the periplasmic face of the membrane.