(Received for publication, May 31, 1995; and in revised form, August 16, 1995)
From the
We have previously shown that replacement of His-226 in the NhaA
Na/H
antiporter of Escherichia
coli to Arg (H226R) shifts the pH profile of the antiporter toward
acidic pH and as a result a
nhaA
nhaB strain
bearing this mutation is Na
sensitive at alkaline pH
(Gerchman, Y., Olami, Y., Rimon, A., Taglicht, D., Schuldiner, S. and
Padan, E.(1993) Proc. Natl. Acad. Sci. U. S. A. 90,
1212-1216).
In the present work the role of His-226 in the
response of NhaA to pH has been studied in detail. The Na sensitivity of the
nhaA
nhaB mutant
bearing the H226R-NhaA plasmid at alkaline pH provided a very powerful
tool to isolate revertants and suppressants of H226R growing on high
Na
at alkaline pH. With this approach cysteine (H226C)
and serine (H226S) replacements were found to efficiently replace
His-226 and yield an antiporter, which like the wild-type protein, is
activated by pH between pH 7 and 8. These results imply that polarity
and/or hydrogen bonding, the common properties shared by these amino
acid residues, are essential at position 226 for pH regulation of NhaA.
This suggestion was substantiated by site-directed mutagenesis of His-226 either to alanine (H226A) or aspartate (H226D). Whereas H226A-NhaA shows very low activity which is not activated by pH, H226D-NhaA is active and regulated by pH. The pH profile of H226D is shifted by half a pH unit toward alkaline pH, as opposed to the previously isolated mutant H226R which has a pH profile shift, to the same extent, but toward acidic pH. It is suggested that charge modifies the pH profile but is not essential for the pH regulation of NhaA.
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, play primary roles in signal transduction
and in regulation of intracellular pH, cell Na
content, and cell volume (for reviews, see (1, 2, 3, 4) ).
Escherichia
coli has two antiporters, NhaA (5) and NhaB(6) ,
which 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, 9, 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 which have been purified to
homogeneity and reconstituted in a functional form in
proteoliposomes(12, 13, 14) . The V of NhaA is highly dependent on pH, changing
more than 1000-fold over the pH range between 7 and 8(12) .
This pH dependence of NhaA is expressed also in isolated membrane
vesicles (15) and is unique to NhaA. The V
of NhaB is pH independent(7, 13) .
The steep
pH dependence of NhaA is expected from its proposed role in pH
homeostasis of the cytoplasm at alkaline pH(3, 16) .
It has been suggested that when the pH increases, the antiporter is
activated so that it can acidify the cytoplasm back to the
``resting pH'' in a self regulated
mechanism(4) . This pattern of a ``pH meter'' and
``titrator'' in the same molecule also exists in other
transporters involved in pH regulation which show very little sequence
similarity with NhaA: in the mammalian Na
/H
antiporter (NheI)(17) , and in the non-erythroid
Cl
/HCO
exchanger(18) . The pH at which half-maximal activity
(the set point) of the human protein (nhe1) is observed seems
to be regulated by hormones and environmental conditions such as
osmolarity (19) . In the eukaryotic systems none of the protein
residues involved in pH sensing are known.
In Escherichia coli His-226 has been shown to be involved in the pH sensitivity of
NhaA(20) . Site-directed mutagenesis of each of the eight
histidines of NhaA showed that none are essential for
Na/H
antiport activity of NhaA.
However, the replacement of His-226 by Arg (H226R) markedly changed the
pH dependence of the antiporter. A strain deleted of both antiporters
genes, nhaA and nhaB, transformed with multicopy
plasmid bearing wild-type nhaA, exhibits both Na
and Li
resistance, throughout the pH range of
6-8.5. In marked contrast, transformants of plasmid bearing
H226R-nhaA are resistant to Li
and
Na
at neutral pH, but they become sensitive to
Na
above pH 7.5. Analysis of the
Na
/H
antiporter activity of membrane
vesicles derived from H226R cells shows that the mutated protein is
activated by pH to the same extent as the wild type. However, whereas
the activation of the wild-type NhaA occurs between pH 7 and 8, that of
H226R antiporter occurs between pH 6.5 and 7.5. In addition, while the
wild-type antiporter remains almost fully active, at least up to pH
8.5, H226R is reversibly inactivated above pH 7.5, retaining only
10-20% of the maximal activity at pH 8.5 (20) .
In the
present work the role of His-226 in the response of NhaA to pH has been
studied in detail. Since both histidine and arginine potentially bear a
positive charge, we tested the effect of a negative charge or no
charge, by site-directed mutagenesis of His-226 to aspartate (H226D) or
alanine (H226A), respectively. Furthermore, the Na sensitivity of the
nhaA
nhaB mutant
bearing the H226R-NhaA plasmid at alkaline pH provided a very powerful
tool to isolate first-site revertants and suppressants of H226R growing
on high Na
at alkaline pH. Combining both approaches
we found that positively charged or polar residues, histidine,
cysteine, and serine, allow the increase in activity of NhaA at
alkaline pH. Aspartate shifts the pH profile toward alkaline pH.
Alanine, however, drastically reduces the activity of the antiporter at
any pH.
Random mutagenesis of H226R-nhaA was
conducted either in vitro, by chemical mutagenesis of pH226R
utilizing hydroxylamine(23) , or in vivo, by
proliferation of pH226R in mutD
strain(24, 25) . For the chemical mutagenesis, pH226R
(5 µg) was incubated in 1 ml with 50 mM sodium phosphate
(pH 6), 1 mM sodium EDTA, and 0.4 M hydroxylamine
hydrochloride (pH 6) for 3 h at 70 °C, the DNA purified on JetSorb
(Genomed) and transformed into EP432 by electroporation (Bio-Rad
version 1.0). The transformants were grown on LBK plates and the grown
colonies screened for Na resistance (at alkaline pH)
on LB containing 700 mM NaCl (pH 8.3).
To verify that the
resistance is conferred by a mutation in the plasmid, plasmids were
isolated from the Na-resistant colonies, retransformed
into EP432, and rescreened on the high Na
/high pH
plates.
To locate the mutation, the BglII-MluI
fragment of the plasmid was excised and cloned into a BglII-MluI fragment of the wild-type plasmid (pGM36)
as described above. If the Na resistance was retained,
both strands of the entire cloned fragment were sequenced.
For the in vivo random mutagenesis, pH226R was transformed into CC130
mutD5(24), grown for 1 h in LBK (20 µg/ml thymidine), and then for
an additional 12 h in the presence of ampicillin and kanamycin,
plasmids were isolated and transformed by electroporation into EP432.
The screen for mutants resistant to Na at alkaline pH
and the identification of the mutations were conducted as above.
To further explore the pH-sensitive domain of NhaA we used
the previously isolated mutant H226R, which shows a pH optimum of
activity shifted toward acidic pH and as a result is inactive at
alkaline pH (20) . When transformed into a strain lacking both
Na/H
antiporters nhaA and nhaB (EP432), this antiporter mutant, as opposed to the wild
type, renders the cells Na
sensitive at alkaline
pH(20) . Hence this sensitivity to Na
at
alkaline pH affords a very powerful tool to select for
Na
-resistant bacteria at alkaline pH created by
reversion of H226R and/or either first-site or second-site suppression
in the pH-sensitive domain of NhaA.
To increase the mutation
frequency in H226R-nhaA, pH226R was treated in vitro by hydroxylamine or propagated in a mutD host. The
randomly mutagenized plasmids were transformed into
nhaA
nhaB host, plated at pH 8.2 in the
presence of 700 mM Na
and the
Na
-resistant clones isolated.
To verify that the
mutations are plasmidic, the plasmids were isolated and shown to confer
Na resistance at alkaline pH upon retransformation
into
nhaA
nhaB. To locate the mutation, a
fragment (BglII-MluI), bearing codon 226, of the
mutated nhaA was cloned instead of an identical fragment of
the wild-type nhaA of pGM36. After verification that this DNA
fragment is responsible for the Na
resistant property
at alkaline pH, the cloned fragment was sequenced to identify the
mutation.
Table 2summarizes the results of the screen which
resulted in 45 mutants. Most of the mutants (71%) were revertants of
H226R back to His-226. These results re-emphasize the importance of
histidine at position 226 in the pH sensing capacity of NhaA. Eleven
mutants obtained from the hydroxylamine-treated cells contained
cysteine codons, 10 TGC and one TGT. Both at pH 7.5 and 8.5, the growth
phenotype of the H226C mutant in the presence of Na is
very similar to that of the wild type strain (Table 3).
The
Na/H
antiporter activity of the
mutant was determined at various pH values in everted membrane vesicles
isolated from
nhaA
nhaB transformed with the
mutated plasmids. Again this host proves very useful since it has no
background of Na
/H
antiporter
activity when transformed with the vector plasmid (pBR322, Fig. 1) but exhibits full activity with the plasmid bearing
wild-type nhaA (pGM36, Fig. 1and Fig. 2). The
data obtained from the mutant at pH 7.5 and 8.5 is shown in Fig. 1while the pH profile of the
Na
/H
antiporter activity throughout
the pH range from 7 to 9 is summarized in Fig. 2A. For
comparison, the Na
/H
antiporter
activity versus pH of pH226R (20) is also shown.
Figure 1:
Na/H
antiporter activity in everted membrane vesicles of the NhaA
mutants at codon 226. Membrane vesicles were prepared from a
nhaA
nhaB E. coli strain, EP432 (11) transformed with plasmid bearing wild-type nhaA,
pGM36 (21) , pBR322, or plasmids carrying the various mutations
in nhaA in which codon 226 was replaced with cysteine
(pH226C), serine (pH226S), aspartate (pH226D), or alanine (pH226A).
Cells were grown in LBK (pH 7.5) and
pH was monitored with
acridine orange in medium containing 140 mM KCl, 10 mM Tricine (titrated with Tris or HCl to the indicated pH), 5 mM MgCl
, acridine orange (0.5 µM), and
membrane vesicles (50 µg of protein). At the onset of the
experiment Tris-D-lactate (5 mM) was added (arrows pointing down) and the fluorescence quenching (Q) was recorded. NaCl (10 mM, arrows pointing
up) were then added and the new steady state of fluorescence
obtained (dequenching) after each addition was
monitored.
Figure 2:
pH dependence of the
Na/H
antiporter activity of the
His-226 mutants. Everted membrane vesicles were prepared and assayed as
described in the legend to Fig. 1at the indicated pH values. A, the data obtained for EP432/pH226 (control,
),
EP432/pH226C (
), and for EP432/pH226S (
), are plotted
each, as percent of the respective maximal activity which was
60-62% of the wild-type. B, the percent of dequenching
observed following addition of 10 mM NaCl is shown versus pH of the assay.
, EP432/pH226;
, EP432/pH226D;
, EP432/pH226A;
, EP432/pH226R, copied from (20) .
The maximal activity of the Na/H
antiporter in isolated membrane vesicles derived from the
H226C-mutant is between 60 and 62% of that of the wild type both at
neutral and alkaline pH ( Fig. 1and data not shown). However,
the pH dependence of this mutant antiporter is identical to that of the
wild type activity increasing between pH 7 and 8 (Fig. 2A). These results show that cysteine at position
226 can replace His-226 and confer growth parameters very similar to
that of the wild type as well as Na
/H
antiporter activity which is substantially activated by pH
between pH 7 and 8.
Most interestingly, the mutant screen after in vivo mutagenesis in the mutD genotype also
produced mainly first-site reversions to His-226. Furthermore, it
yielded a new mutation, serine at position 226 (Table 2). Both in
growth parameters (Table 3) and
Na/H
antiporter activity of everted
membrane vesicles ( Fig. 1and 2A) the H226S mutant is
very similar to the H226C mutant, and thus to the wild type. To assess
the expression of the various mutants we used anti-NhaA antibody and
found that both the H226C and H226S mutants are significantly expressed
in membranes, 40 and 90%, respectively (Table 4).
Hence,
either one or two properties shared by histidine, cysteine, and serine
is important for the activation of NhaA by alkaline pH. These
properties are polarity and capacity of forming hydrogen bonds.
Nevertheless we have previously shown that arginine, which also has
these properties but in addition bears an ionizable group with a pK at a more alkaline pH than that of histidine, shifts the pH
profile of NhaA toward acidic pH(20) . We therefore tested the
effect of aspartate, presumably bearing a negative charge, in addition
to the other common properties, and alanine with none of these
properties, by site-directed mutagenesis of His-226 to H226D or H226A,
respectively. The mutated plasmids were transformed into a
nhaA
nhaB strain so that the growth
phenotypes conferred by the mutated nhaA could be assessed
with no interference of chromosomally encoded
Na
/H
antiporters.
The growth
phenotype of the mutants is summarized in Table 3. H226D grows at
a growth rate very similar to that of the wild type at Na concentrations at least up to 400 mM and throughout the
pH range between pH 7.5 and 8.5. Growth on solid medium is observed up
to 700 mM NaCl at pH range between pH 7 and 8.5 (not shown).
At the lower pH H226A also behaves like the wild type. In marked
contrast, however, at pH 8.4 and above, H226A does not grow in the
presence of 400 mM Na
(Table 3) while
no difference was noted in the presence of 400 mM K
(not shown).
The two mutants were expressed significantly but to a different degree (Table 4). In comparison to the wild type, the highest expression was that of H226A amounting to 78% and the lowest expression (24%) was that of H226D.
The maximal
Na/H
antiporter activity in isolated
everted membrane vesicles conferred by pH226D at alkaline pH is lower
than that of the wild type (pGM36) by only 25%. However, the mutation
shifts the pH profile of the mutated protein by half of a pH unit
toward alkaline pH (Fig. 2B); whereas the maximal
difference in activity of the wild type occurs between pH 7 and 8,
H226D is activated between pH 7.5 and 8.5.
Strikingly, throughout the pH range between 7.5 and 9, H226A is not activated by pH and in contrast to both H226D and the wild type, exhibits very low and constant activity ( Fig. 1and Fig. 2B). Below pH 7.5 the very low activity of H226A decreases with lowering of the pH and vanishes at pH 7. Hence a polar group and/or the capacity to form hydrogen bonds, is essential for the activation of NhaA by alkaline pH. A charge shifts the pH profile.
The distinct pH dependence of NhaA
Na/H
antiporter of E. coli,
being practically inactive below pH 7.0 and increasing V
dramatically between pH 7.0 and 8.0, led us to
suggest the existence of a pH-sensitive domain in
NhaA(12, 20) . The pH sensitivity of the
Na
/H
antiporter was first
demonstrated in right-side out membrane vesicles measuring
Na efflux driven by imposed artificial
pH or
(15) and then in purified protein functionally
reconstituted in proteoliposome, measuring passive
Na
efflux (12) .
Since flux of cations via the
cation/H antiporters affects the
pH across the
membrane, cation induced changes in the fluorescence of acridine
orange, and similar probes measuring
pH, have been proven a fast
and reproducible way to monitor the activity of
H
/cation antiporters in isolated membrane
vesicles(26, 27) . Although calculation of the kinetic
parameters of an antiporter with this technique is complicated due to
the indirect nature of the measurement, when it is conducted at cation
concentrations above the K
of the antiporter, it
most probably reflects the V
of the system and
is thus most suitable for comparison of antiporters
activity(26) .
Indeed with this technique we found the same
drastic pH dependence of NhaA Na/H
antiporter as was found by direct flux measurement using
Na (20) . Furthermore, the experimental system
used was everted membrane vesicles isolated from the
nhaA
nhaB mutant transformed with multicopy
plasmid bearing nhaA. This system has proven most suitable to
identify (by site-directed mutagenesis) residues in NhaA involved in
the pH-sensitive domain of the protein. A mutation can be easily
introduced into the plasmidic NhaA and its effect tested both in
vitro, in everted isolated membrane vesicles, and in vivo, in both cases with no background of chromosomal encoded by either
NhaA or NhaB Na
/H
antiporters.
With this approach, we found that although none of the eight
histidines in NhaA are important for Na/H
antiporter activity, His-226 is essential for the strong pH
dependence of this protein. Thus NhaA bearing the mutation Arg-226
(H226R) has a pH profile shifted to the acidic pH range by half a pH
unit and in addition at pH 8.5 is practically inactive rendering cells
Na
sensitive at this pH.
In the present study we
have further tested the importance of His-226 in the pH sensitivity of
NhaA Na/H
antiporter. In one approach
we randomly mutagenized H226R-nhaA, and exploited its
Na
sensitive phenotype at alkaline pH to select for
mutations, restoring the capacity of the antiporter to function at
alkaline pH and promoting growth in the presence of 700 mM NaCl. Such mutations were assumed to occur either in codon 226,
the first-site (reversions and suppressions), or in other sites
(second-site suppressions) participating in the pH sensor domain of
NhaA.
Interestingly, all 45 mutations obtained were in the first-site, 32 of which reversed back to His-226. Both after chemical mutagenesis of the H226R bearing plasmid with hydroxylamine or after in vivo mutagenesis by its proliferation in a mutD host bacteria, the majority of the first-site mutations reversed back to His-226, although in different proportions. Hydroxylamine acts by deaminating cytosine residues and converting them to thymine(23) . This mutagenesis is therefore expected to be biased; cytosine containing codons such as that of H226R (GCG) would be changed back to that of histidine GTG. However, since the mutD dependent in vivo mutagenesis, which is random(25) , gave mainly His-226 revertants we suggest that the high frequency of reversions to histidine at position 226 reflects the importance of this residue in the pH sensitivity of NhaA.
Three non-histidine
first-site suppressions were obtained by the random mutagenesis
approach. The hydroxylamine treatment produced two mutants with
cysteine in position 226 (H226C) each encoded by a different codon: TGC
and TGT, respectively. The mutD mutagenesis yielded the H226S
mutation encoding serine at position 226. These first-site suppressants
exhibit an identical phenotype, very similar to that of the wild type
both in terms of growth in the presence of Na at
alkaline pH, as well as Na
/H
antiport
activity which is activated by pH between pH 7 and 8. As compared to
the wild-type strain, the only difference observed is that the maximal
activity of the Na
/H
antiporter of
both mutants (obtained at pH 8.5) is about 60-62% of that of
wild-type. This difference cannot be accounted for by a difference in
expression of the mutated proteins since expression of the Cys-226
antiporter in the membrane was 40% and that of serine 226 90%. It
should be noted that the phenotype of the reversion mutations was
tested on multicopy plasmid, i.e. large excess of the
antiporter. Therefore it cannot be excluded that a single copy of the
mutation could have allowed us to discern subtle differences among the
mutants and between them and the wild-type strain. Nevertheless we may
conclude that cysteine and serine at position 226 can efficiently
replace histidine.
In solution both cysteine and histidine have a pK in the physiological range (Table 5.1 in (31) ), whereas serine does not ionize under these conditions. Both histidine and cysteine have been shown to be ionized in proteins with pK not very different from that observed in solution(31, 32) . Nevertheless it is impossible to predict the pK of an aminoacid in a protein neither from its ionization constant in solution, nor from its pK in another protein. It has been suggested that serine is also capable of ionization in a protein of the bacterial photosynthetic reaction center, but only when its electrostatic environment is drastically changed by electron transport(33) , an unlikely situation in the case of NhaA. Therefore, we suggest that ionization of residue 226 is not essential for pH regulation of the antiporter, rather it is the polarity and/or capacity to form hydrogen bonds, properties which histidine, serine, and cysteine share, which is essential.
Our results with the site-directed mutagenesis approach substantiate this suggestion: two mutations were introduced at position 226, one encodes a polar residue but now with a negative charge, aspartate (H226D), the other codes for a non-polar residue alanine (H226A). H226D is almost as active as His-226 but shifts the pH profile of the antiporter toward the basic side by half a pH unit. Remarkably, the H226A mutation, which is expressed almost as the wild-type gene, produces a carrier with a very low activity (about 10% of that of the wild-type), which is not activated by pH.
Taken together it appears that at position 226 of NhaA charge, polarity and/or hydrogen bonding affect the pH-sensitive domain of NhaA; a negative charge shifts the sensitivity toward the alkaline range, a postive charge toward the acidic range. As yet we do not know how these residues exert their effects. Amino acid residues are known to cause micro changes within proteins reflected in the electrostatic microenvironment, stability of acidic or basic residues, density of local protons, and even binding of water molecules(31, 32, 33) . It should be emphasized, however, that as long as the structure of the protein is unsolved we cannot exclude short range steric effects or long range conformational effects not related directly to the ``pH sensor.''
It is of interest that the difference between the
mutants in the activity of the antiporter at acidic pH is not expressed
in growth phenotype. Even H226D with the lowest activity at this pH
range grows like the wild type. On the other hand, at alkaline pH
growth is performed only by the mutants which are substantially
activated by pH, thus H226A stops growing beyond pH 8.4. These results
corroborate our previous results with H226R (20) emphasizing
the physiological importance of NhaA at alkaline pH in the presence of
Na.