(Received for publication, May 9, 1996, and in revised form, October 30, 1996)
From the Division of Microbial and Molecular Ecology, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel
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 [14C]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.
Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals, and microorganisms. They are involved in cell energetics and play primary roles in the regulation of intracellular pH, cellular Na+ content, and cell volume (reviewed in Refs. 1-4).
Escherichia coli has two antiporters, NhaA (5) and NhaB (6), that specifically exchange Na+ or Li+ for H+ (4). nhaA is indispensable for adaptation to high salinity, for challenging Li+ toxicity, and for growth at alkaline pH (in the presence of Na+) (7). Accordingly, expression of nhaA which is dependent on NhaR, a positive regulator, is induced by Na+ in a pH-dependent manner (8-10). nhaB by itself confers a limited sodium tolerance to the cells but becomes essential when the lack of NhaA activity limits growth (11).
Both the NhaA and NhaB are electrogenic antiporters that have been purified to homogeneity and reconstituted in a functional form in proteoliposomes (12-14). With the purified antiporters it was found that the H+/Na+ stoichiometry of NhaA is 2H+/Na+ and that of NhaB is 3H+/2Na+; NhaB but not NhaA is sensitive to amiloride derivatives, and the rate of NhaA but not of NhaB is drastically dependent on pH, changing its Vmax over 3 orders of magnitude from pH 7 to pH 8 (12).
None of the eight histidines of NhaA were found essential for the Na+/H+ antiporter activity of NhaA (15). However, replacement of histidine 225 (His-225, previously mistakenly numbered His-226) by Arg (H225R) suggested that His-225 has an important role in the pH sensitivity of the antiporter (15). Whereas the activation of the wild-type NhaA occurs between pH 7.5 and pH 8, that of H225R antiporter occurs between pH 6.5 and pH 7.5. In addition, while the wild-type antiporter remains almost fully active, at least up to pH 8.5, H225R is reversibly inactivated above pH 7.5, retaining only 10-20% of the maximal activity at pH 8.5 (15). Furthermore, we have recently shown that replacement of His-225 with either cysteine (H225C) or serine (H225S) but not alanine (H225A) yielded an antiporter with a wild-type pH-sensitive phenotype, implying that polarity and/or hydrogen bonding, the common properties shared by His, Cys, and Ser, are essential at position 225 for pH regulation of NhaA (16).
In the present work we found that the three cysteine residues of NhaA are not essential in the mechanism of the antiporter, confirming previous findings on the lack of inhibition by SH reagents (17). The functional antiporter devoid of cysteinyl residues has provided the basis to design mutants of NhaA in which a suggested important residue such as His-225 is replaced with cysteinyl residue which can then be reacted specifically with sulfhydryl reagents. Analysis of these site-directed chemical modifications can bear upon both the importance as well as the topology of the original residue (Cys scanning). For this purpose, 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 NEM,1 C-less H225C was inhibited by this reagent. Furthermore, H225C was alkylated to a much higher level than any of the native cysteinyl residues of NhaA suggesting that H225C resides in the protein in a domain which is much more exposed to NEM than the native cysteinyl residues of NhaA. However, since NEM is membrane-permeable, it was impossible to deduce from these experiments the membrane topology of H225C with respect to neither the periplasmic nor the cytoplasmic face of the membrane.
Various SH reagents have been suggested to be membrane-impermeant and react only with residues exposed to the medium containing the reagents. Thus periplasmic exposed residues are expected to be affected only in intact cells or RSO membrane vesicles and cytoplasmic facing residues only in ISO membrane vesicles. On the other hand, membrane-permeant reagents are expected to react irrespective of the membrane orientation. On the basis of these differences various SH reagents were exploited to assay the cross-membrane topology of native or strategically placed Cys residues in membrane-embedded proteins. This approach was of value to the structural work of bacteriorhodopsin (18). A similar approach is now being used to probe functional regions with many membrane proteins including the acetylcholine receptor ion channel (19, 20) as well as LacY, the H+/lactose symporter (21), and UhpT, the anion exchange protein (22) both in E. coli.
In the present work, we have used the same approach to probe the reactivity of C-less H225C to various SH reagents, both in intact cells as well as in membrane vesicles of known orientation. The differential reactivity of the membrane-impermeant reagents with C-less H225C in the different experimental systems showed that H225C is exposed to the periplasmic face of the membrane.
EP432 is an
E. coli K12 derivative, which is melBLid,
nhaA1::kan,
nhaB1::cat,
lacZY,
thr1 (11). TA16 is
nhaA+nhaB+lacIQ
and otherwise isogenic to EP432 (12). DH5
(U.S. Biochemical Corp.)
was used as a host for construction of plasmids. Cells were grown
either in L broth (LB) or in modified L broth (LBK) in which NaCl was
replaced by KCl ((7) 87 mM, pH 7.5). Where indicated, the
medium was buffered with 50 mM BTP, and pH was titrated
with HCl. Cells were also grown in minimal medium A without sodium citrate (23). Thiamine (2.5 µg/ml) was added to all minimal media.
When required, threonine (0.1 mg/ml) was added. For plates, 1.6% agar
was used. Antibiotics were 100 µg/ml ampicillin and/or 50 µg/ml
kanamycin and/or 12 µg/ml chloramphenicol. Resistance to
Li+ and Na+ was tested as described previously
(15).
All plasmids are pBR322 derivatives. pGM36 carries wild-type nhaA (24). Plasmids encoding His-tagged antiporters are pET20b (Novagen) derivatives as described below. pEP3T is a plasmid overproducing nhaA (12).
Site-directed Mutagenesis of Cysteines of NhaA and Construction of C-less NhaA and C-less H225C NhaASite-directed Cys
replacements by serine, C200S, C308S, and C335S (Fig. 1), were obtained
following a polymerase chain reaction-based protocol (25). DNA of pGM36
was utilized as the template. The end primers and the mutagenic primers
are described in Table I. In all cases, the resulting mutagenized DNA
(2050 bp) was digested with BglII-MluI, yielding
a fragment of 682 bp containing each of the mutations (Fig. 1), which
was ligated to the 6.2-kb BglII-MluI fragment of
pGM36, to yield plasmid pC201S, pC309S, and pC335S. In each case, the
entire fragment originated by polymerase chain reaction and placed in
the recombinant plasmid was sequenced through the ligation junctions to
verify the accuracy of mutagenesis (Table I). This routine ensured that
the entire nhaA gene did not harbor mutations with the
exception of the changes described.
|
For construction of C-less NhaA, pC200S and pC335S were digested with BamHI and the BamHI-BamHI fragments; 1.9 kb of the former and 5 kb of the latter were ligated to yield plasmid pC201S-C335S. This plasmid was then used as a template to introduce C308S as above, and pC-less nhaA (C200S, C335S and C308S) was obtained. The last mutation introduced a PvuII site.
For construction of C-less H225C, we used DNA of pC-less as a template and the end and mutagenic primers as described in Table I. The BglII-MluI fragment containing the mutation was cloned instead of BglII-MluI of pGM36.
Construction of His-tagged NhaA DerivativesTo construct a
plasmid overproducing His-tagged NhaA we used pEP3T (12) and amplified
by polymerase chain reaction a fragment (1.5 kb) using two mutagenic
end primers (CTAGTCTAGAGGATCCGGAGCTTAT) and
(TTTTCCTTTTGCGGCCGCAACTGATGGACGCAAACGA) introducing
XbaI in the 5 and NotI in the 3
ends. The
fragment was cloned into TA vector (Invitrogen, San Diego, CA), and the
XbaI-NotI fragment of the recombinant plasmid was
cloned into XbaI-NotI fragment (3.6 kb) of
modified pET20b (Novagen, Madison, WI). The latter plasmid contains
between NotI and XhoI sequences encoding two factor Xa cleavage sites in frame with the sequences encoding the 6 histidines. The resulting recombinant plasmid, designated pYG10 (Fig.
1), encodes for NhaA fused in frame in its C terminus to the two factor
Xa cleavage sites followed by 6 histidines. To construct His-tagged
derivatives of the C-less NhaA and C-less H225C the
NheI-MluI (880 bp) fragment of pC-less or
pC-less-H225C was cloned into NheI-MluI (4.2 kb)
of pYG10 yielding plasmid His-tag C-less and His-tag C-less-H225C,
respectively. All constructs were verified by sequencing using the
Sequenase kit (Version 2.0, U. S. Biochemical Corp.).
Assays of Na+/H+
antiport activity were conducted on everted membrane vesicles (26). The
assay of antiport activity was based upon the measurement of
Na+ (or Li+) induced changes in the pH as
described (5, 27). NhaA in everted membrane vesicles (1 mg) was
quantitated by Western analysis as described previously (15). Protein
was determined according to Bradford (28).
The procedure was performed at 4 °C. Everted membrane vesicles (100-300 µg protein) were suspended in 1.15 ml containing 60 mM choline chloride, 4.5 mM Tris-Cl, pH.8, 110 mM sucrose, 20% glycerol, 100 mM MOPS, pH 7, and 1% DM. The suspension was gently mixed for 20 min and then centrifuged (Beckman model TLA100, 245,000 × g, 20 min). The supernatant was mixed with 90 µl of prewashed resin (Ni2+-NTA-agarose, Qiagen, Hilden, Germany). Prewashing was performed by cycles of resuspension and centrifugation (20,000 × g, 2 min); the first prewash was in 500 µl of H20, and the second was in binding buffer (modified Qiagen protocol: 5 mM imidazole, pH 7.9, 500 mM NaCl, 20 mM Tris-Cl, pH 7.9, 0.1% DM). For loading of the resin with His-tagged NhaA the mixture was incubated for 1 h with gentle mixing and then centrifuged.
The loaded resin was resuspended in 500 µl of binding buffer, mixed for 10 min, centrifuged, and washed again in wash buffer (30 mM imidazole, 500 mM NaCl, 20 mM Tris-Cl, pH 7.9, 0.02% DM, 10% glycerol). For elution the washed loaded resin was resuspended in 90 µl of elution buffer (300 mM imidazole, 500 mM NaCl, 20 mM Tris-Cl, pH 7.9, 0.02% DM, 10% glycerol) and incubated for 10 min as above. The supernatant devoid of the resin was collected by two consecutive centrifugations. The proteins in the supernatant were precipitated in 10% trichloroacetic acid for 30 min and collected by centrifugation (20,000 × g, 15 min), resuspended in sample buffer, and resolved by SDS-PAGE (29).
Treatment with NEMEverted membrane vesicles were treated
with NEM in 50 µl of reaction mixture containing, in final
concentrations, membrane vesicles (100 µg protein), 100 mM potassium phosphate (at the indicated pH), 10 mM MgS04, and 1 mM NEM. The
reaction (28 °C, 20 min) was stopped by addition of 20 mM dithiothreitol. Antiporter activity was assayed as
described above using K-ATP instead of D-lactate to
establish pH since NEM inactivates electron transport. If not
otherwise indicated for treatment with [14C]NEM, membrane
vesicles (100-300 µg of protein) were resuspended in 500 µl of
solution, pH 7.5, and incubated as above in the presence of 25 µM [14C]NEM (DuPont NEN, 40 mCi/mmol). Solubilization and purification of His-tagged NhaA protein
was as above.
NhaA, the Na+/H+ antiporter of E. coli, has three cysteinyl residues (Fig. 1). To study the role of the cysteinyl residues in the activity of the antiporter, we replaced each cysteine of NhaA separately to obtain mutations encoding serine instead of cysteine 200 (C200S), 308 (C308S), or 335 (C335S). In addition, both a cysteine-less (C-less) nhaA was constructed with all cysteine residues replaced by serines, as well as C-less with a Cys replacement of His-225 (C-less H225C).
Growth Phenotype and NhaA Expression of the Cysteine Replacement MutantsTo study the growth phenotype of the mutants, each mutated plasmid was introduced into a strain lacking both antiporters NhaA and NhaB (EP432) (11). This strain does not have Na+/H+ activity and thus is highly sensitive to Na+ but regains antiporter activity and becomes resistant to the ion upon transformation with multicopy plasmid bearing wild-type nhaA. It was therefore found most suitable to explore plasmidic antiporter mutations with respect to the capacity to confer Na+ resistance and antiporter activity (30). The results of the growth experiments show that at pH 7.5 in the presence of up to 0.7 M NaCl, the growth rate of all cysteine mutants was very similar to that of the wild-type (75-85 min doubling time). Also at pH 8.5 in the presence of 0.6 M NaCl, all mutants grew, but C200S, C-less, and the C-less H225C mutants were slightly slower (doubling time of 95, 90, and 95 min, respectively) compared with the wild-type and the other cysteine mutants (doubling time of 75 min). There was no significant difference in colony formation of the various strains on agar plates.
The level of the NhaA-Cys mutated proteins in the membranes was determined immunologically using polyclonal antibody produced against the C terminus of NhaA (15). The mutants C308S, C-less, and C-less-H225C were expressed at a level similar to that of the wild-type protein. The level of C335S and C200S in the membrane was 60 and 30% that of the wild-type, respectively.
Na+/H+ Exchange Activity in Everted Membrane VesiclesTo determine whether any of the mutations
affect antiporter activity, we isolated everted membrane vesicles from
EP432 transformed with each of the mutant plasmids and assayed the
specific Na+/H+ antiporter activity of these
membranes as a function of pH. Again this host proves very useful since
it has no background Na+/H+ antiporter activity
when transformed with the vector plasmid (pBR322, Fig.
2A). The data obtained from the mutants at pH
8.5 are shown in Fig. 2A, and the pH profile of the
Na+/H+ antiporter activity throughout the pH
range from 7 to 8.5 is summarized in Fig. 2B. For
comparison, the Na+/H+ antiporter activity
versus pH of wild-type plasmid transformants is also
shown.
The Na+/H+ antiporter activity of the C308S mutant is very similar to that of the wild-type NhaA both in maximal activity and pH sensitivity. Below pH 8 C335S and C200S show similar activity which is between 40 and 50% that of the wild-type. Above pH 8, whereas C335S reaches the steady state level of the wild-type, C200S remains at 60%.
The activity of the C-less mutant of NhaA is very similar to C200S and reaches a steady state level of about 60% of the wild-type at alkaline pH (Fig. 2, A and B). Thus, it is apparent that replacement of all three Cys residues, whether each separately or all together, in the same antiporter molecule does not markedly impair the antiporter activity, implying that neither of the cysteinyl residues of NhaA are mandatory to the Na+/H+ antiporter activity of NhaA.
It is obvious that high resolution structure of the transporter is required to determine the role of any residue in the mechanism. Nevertheless, it has recently become apparent (31, 32) that functional Cys replacements in transporters can be very useful since they allow site-specific labeling of the protein, at the reactive SH groups, with probes that can examine various aspects of structure and function relationship of the protein (Cys scanning). A principal difficulty with this general approach, however, is the complexity resulting from the presence of multiple cysteinyl residues in most proteins, three in the case of the NhaA antiporter. Thus, in addition to the important conclusion reached in this paper that cysteinyl residues do not play a critical role in the mechanism of the Na+/H+ antiport, the construction of a functional antiporter, devoid of cysteinyl residues, provides the basis for Cys-scanning analysis of NhaA.
Since His-225 has been shown to play an important role in the pH
response of NhaA, we have replaced His-225 of the C-less antiporter
with Cys. The Na+/H+ antiporter activity of
Cless H225C at pH 8.5 in everted membrane vesicles was about 60% that
of the C-less antiporter and 30-40% that of the wild-type (Figs.
3 and 2B). Most interestingly, the pH profile
of C-less H225C was similar to the wild-type or the C-less NhaA (Fig.
2B). Taken together these results show that C-less H225C
NhaA antiporter although at a decreased level is still functional and
exhibits a pH dependence similar to that of wild-type and the C-less
antiporter. These results are consistent with our previous data (16)
showing that an antiporter harboring an H225C mutation in otherwise
wild-type antiporter is similar to the latter both in activity and pH
sensitivity. The C-less H225C mutant thus affords the possibility to
test the effect of SH reagents at position 225 of NhaA.
While NEM Has No Effect on Wild-type or C-less NhaA It Inactivates Both H225C and C-less H225C
In accordance with previous results (17), the Na+/H+ antiporter activity of the wild-type NhaA protein, like the C-less antiporter, was not affected by treatment of the membranes with NEM (up to 2 mM) (Fig. 3). However, NEM (at 0.5 mM) inhibited, significantly, the C-less H225C antiporter (Fig. 3A). The most significant effect was obtained when the NEM treatment was conducted at pH 7.5 (Fig. 3B). The maximal inhibition (60-70%) was not increased with further increase of the NEM concentration. The presence of Na+ had no effect on the degree of NEM inhibition. Similar results (not shown) were obtained with NhaA mutant which harbors H225C in an otherwise wild-type protein (H225C). Interestingly, the residual activity of the NEM-inactivated C-less H225C is not sensitive to pH (not shown). Taken together these results emphasize again the importance of His-225 in the unique response of NhaA to pH (15, 16).
H225C Is Exposed to Alkylation by [14C]NEM Much More Readily Than the Native Cysteinyl Residues of NhaAThe observation that NEM significantly affects the NhaA antiporter, harboring instead of histidyl a cysteinyl residue at position 225, but has no effect on a wild-type antiporter with its three cysteines (C200, C308, C335), raised two alternatives: a modification of the cysteinyl residues by NEM has no inhibitory effects on the antiporter activity, and the Cys residues, which are in putative transmembrane segment (15), are not accessible to NEM.
To test whether the NhaA cysteinyl residues are NEM-accessible, we
constructed His-tagged NhaA (Fig. 1) (and in addition His-tagged C-less
and His-tagged C-less H225C). Plasmidic His-tagged NhaA was as
effective as plasmidic wild-type antiporter in conferring Na+ resistance upon nhaA
nhaB
and in promoting Na+/H+ antiporter activity in
isolated membrane vesicles (not shown). The pH sensitivity of the
His-tagged NhaA was also similar to that of the wild-type antiporter.
The His-tagged NhaA readily bound onto the Ni+2 resin (Fig.
4A). Out of the many membrane proteins (Fig.
4A, lane 2) exposed to the resin many did not bind (Fig.
4A, lane 3) or were eluted by the washes at low imidazole
concentrations (
30 mM, Fig. 4A, lanes 4 and
5). At 300 mM imidazole the His-tagged NhaA was
eluted as a prominent band at 30 kDa (Fig. 4, lane 6). As
expected from its longer C terminus His-tagged NhaA was slightly heavier than the native NhaA (Fig. 4A, lane 7). The two
additional very weak bands of 51 and 83 kDa shown are most probably
aggregates of the antiporter, a property noted before for this protein
(12).
This rapid isolation of the His-tagged NhaA formed the basis for an assay designed to probe in situ the reactivity of the NhaA Cys residues to NEM as well as to other SH reagents. In this assay everted membrane vesicles were treated with [14C]NEM, and the His-tagged NhaA derivatives were isolated on the Ni+2 resin and subjected to autoradiography (Fig. 4B) to estimate the number of NEM titratable molecules per purified NhaA protein.
These results show that when the [14C]NEM treatment was conducted at a concentration of 25 µM, His-tagged C-less H225C (Fig. 4B, lane 1) was labeled to a level at least 10-fold higher than the His-tagged wild-type antiporter (Fig. 4B, lane 3), in spite of the three cysteines of the latter. As expected there was no labeling of the His-tagged C-less NhaA (Fig. 4B, lane 2).
The concentration dependence of the alkylation by
[14C]NEM was determined with His-tagged C-less H225C
(Fig. 5). The results show that at 0.5 mM
NEM saturation of the alkylation reaction was reached. To estimate the
maximal number of [14C]NEM molecules bound per purified
His-tagged C-less H225C NhaA molecule, the amount of the radioactivity
bound to the antiporter at 0.5 mM NEM was also determined
directly by scintillation counting of the purified antiporter fraction.
A NEM/NhaA mol ratio of 1.02 was calculated from the amount of
radioactivity, specific activity of [14C]NEM, and the
amount of the pure protein in the fraction, suggesting a NEM/NhaA
stoichiometry of 1. A similar stoichiometry was found with RSO membrane
vesicles. At this NEM concentration maximal inhibition (at pH 7.5) of
the Na+/H+ antiporter activity was observed in
everted membrane vesicles isolated from C-less H225C cells (Fig.
3).
The effect of NEM concentration on the alkylation of the native cysteines of the wild-type NhaA was also determined (Fig. 5). It was observed that up to 0.5 mM the alkylation was very low but increased with increasing concentrations. However, even at 2 mM [14C]NEM there was no saturation of the reaction. It is concluded that H225C is much more exposed to NEM than the native NhaA cysteines.
The fact that H225C is much more exposed to NEM as compared with the
native Cys residues of NhaA is intriguing. Since H225C in right-side
out membranes has been found as reactive to NEM as in everted membrane
vesicles, it is suggested that neither membrane permeability nor the
topology of H225C with respect to the face of the membrane is the cause
for the preferential reactivity of H225C. Since the native
cysteines are located in putative helices while H225C is supposed
to reside at the membrane surface (15), a tempting explanation is that
His-225 is exposed to the aqueous phase of the membrane, a property
which readily exposes it to NEM. On the other hand, the residues
embedded in the transmembrane domains of the protein are masked from
the reagent. Permeation of NEM into the protein thus appears the main
factor determining the NEM reactivity of the cysteinyl residues. Hence
NEM reactivity may bear upon different domains of membrane proteins.
Probing Cys replacements of the TetB protein has recently led to
similar conclusions.2
The cross-membrane topology of H225C was investigated by probing in situ the accessibility of H225C to membrane-impermeant SH reagents both in oriented membrane preparation, ISO, or RSO as well as intact cells. Accessibility to the probe implied that H225C is located at or near the face of the membrane exposed to the side of application of the impermeant probe. Inaccessibility of the H225C residue to the impermeant probe but accessibility to a permeant probe in membranes of the reversed orientation served as powerful internal controls in these experiments.
In the protocol devised to determine the reactivity of His-tag C-less H225C to SH reagents, cells or membrane vesicles were first treated with the membrane-impermeant reagent, washed, and subsequently exhaustively alkylated by [14C]NEM. The level of the alkylation was determined by autoradiography of the affinity purified antiporter (Ni+2 resin) separated on SDS-PAGE. As shown above (Fig. 5), in untreated membranes the alkyl/NhaA stoichiometry was close to one whether right-side out or everted membrane vesicles were used. In addition, C-less NhaA was not labeled under the conditions employed (Fig. 4B). Both these controls implied that alkylation by [14C]NEM can be used to titrate the number of reactive H225C residues in NhaA. Therefore the difference between the number of NEM alkylatable H225C before and after exposure to the -SH reagent of selective permeability allowed us to estimate the reactivity of H225C to this reagent.
Small, charged and highly water-soluble sulfhydryl-specific reagents derived from methanethiosulfonate (MTS) have previously been used to probe the cross-membrane topology of cysteines strategically placed in the acetylcholine receptor (19, 20). These include the positively charged MTSEA and MTSET, both considered membrane-impermeant (20, 33).
A typical experiment is shown in Fig. 6A
using MTSEA. Surprisingly, treatment with MTSEA for 1 min whether
applied to right-side out or inside-out membrane vesicles reduced
completely the number of NEM-titratable H225C residues. These results
could be interpreted in two ways, either MTSEA is permeant in the
E. coli membrane and modifies H225C from both sides of the
membrane or H225C is accessible from both sides of the membrane.
However, we cannot exclude the existence of a carrier-mediated
transport of MTSEA across the membrane. Furthermore, since MTSEA is a
weak base with a pK of around
8.5,3 it is possible that it equilibrates
across the membrane in its undissociated form similar to other weak
amines of similar pK (35, 36). We therefore probed H225C
with additional SH reagents suggested to be impermeant to
membranes.
The results obtained by similar protocol conducted with MTSET are summarized in Fig. 6B. A clear difference in the number of NEM-titrable residues was observed between NhaA isolated from RSO- or ISO-treated membrane vesicles; only 4% of the NEM-titrable residues of H225C remained in RSO membrane vesicles following 20 min treatment as compared with 44% in treated ISO membranes. Further incubation had no significant additional effect. Hence after 20 min of treatment the number of NEM-titratable H225C in RSO as compared with ISO vesicles were 8-10-fold lower. This result suggests that H225C is much more exposed to modification by MTSET in RSO vesicles as compared with ISO vesicles.
PCMB and its sulfonic acid derivative, PCMBS, are organomercurials that share chemical specificity for the reaction with cysteines. They differ, however, in several properties. PCMB is lipid-soluble weak acid which when protonated (pK 4) has access to cysteine residues at both membrane surfaces. On the other hand, in PCMBS the strongly acidic sulfonic acid group (pK 1.5) is very soluble in water and is largely membrane-impermeant, which attacks those cysteines exposed to the medium. Therefore a comparison of the response to PCMB and PCMBS could, in principle, give important information concerning the location of reactive cysteines as shown most elegantly with UhpT transporter (22).
Fig. 7 shows the results obtained with PCMB and PCMBS
with both RSO and ISO membrane vesicles. In both membrane types PCMB reduced dramatically the titratable NEM residues as expected for membrane-permeable SH reagent (Fig. 7, lanes 2 and
6). On the other hand, only in RSO membrane vesicles PCMBS
had a pronounced effect, reducing by 10-15-fold the NEM-titratable
residues (compare lanes 8 to 4 in Fig. 7). In the
ISO vesicles only a 2-fold decrease was observed (Fig. 7, lane
4). These results show that H225C is significantly more exposed to
PCMBS in RSO than in ISO vesicles.
It is not clear why some reactivity with PCMBS was observed in ISO membrane vesicles. One possibility is that some heterogeneity exists in this membrane preparation with respect to orientation. This, however, is unlikely since these membrane preparations have been shown to be very homogenous consisting of mainly everted membrane vesicles (26, 36). Another alternative that can be considered is that the membranes are slightly permeable to PCMBS either originally or due to the experimental manipulation.
The very different reactivity of H225C to the hydrophilic SH reagents (MTSET and PCMBS) in RSO and ISO membrane vesicles and similar high accessibility to the hydrophobic SH reagents (MTSEA and PCMB) in both membrane types suggest that H225C is located at or near the periplasmic face of the membrane.
Given this conclusion it was expected that PCMBS would be able to react with H225C of NhaA even in intact cells. For this experiment intact cells were exposed to PCMBS and then after extensive washing of the reagent ISO membrane vesicles were isolated and exposed to [14C]NEM for titrating the free H225C residues. Indeed, in intact cells PCMBS blocked completely the H225C residues leaving no NEM-titratable residues in NhaA (Fig. 7, lanes 9-11).
Based on the hydropathic analysis of the predicted protein sequence of NhaA we have previously published a putative secondary structure model with 11 hydrophobic TMS connected with hydrophilic loops (12, 15). In this model H225C was predicted to be located in the loop connecting TMS VII and VIII at the edge of TMS 7. However, a careful study of the topology of NhaA using phoA fusions showed that NhaA consists of 12 TMS, and in this model H225C is located at the edge of TMS 8 facing the periplasmic face of the membrane (37). In line with this model the C terminus was found exposed to the cytoplasm. The results obtained in the present work showing directly that H225C is exposed at the periplasmic face of the membrane strengthens the new 12 TMS model of NhaA.
The periplasmic topology of H225C found in the present work supports the contention that the "pH sensor" of NhaA interacts indirectly with His-225 (30). In ISO membrane vesicles H225C faces the lumen of the vesicles. Since these membrane vesicles pump protons in, their intravesicular space becomes very acidic during respiration. This acid pH is expected to totally inhibit the antiporter (12). However, although H225C is exposed to this acidic space in ISO vesicles, the antiporter is active under these conditions. Hence, although we know that His-225 affects the pH sensitivity of NhaA, as yet we do not know which residue directly senses pH and how this signal is transduced by the NhaA protein.