From the Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706
Received for publication, October 4, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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The role of subunit a in proton
translocation by the Escherichia coli
F1Fo ATP synthase is poorly understood. In the
membrane-bound Fo sector of the enzyme, H+
binding and release occurs at Asp61 in the middle of the
second transmembrane helix (TMH) of subunit c. Protons are
thought to reach Asp61 via an aqueous access
pathway formed at least in part by one or more of the five TMHs of
subunit a. In this report, we have substituted Cys into a
19-residue span of the fourth TMH of subunit a and used
chemical modification to obtain information about the aqueous
accessibility of residues along this helix. Residues 206, 210, and 214 are N-ethylmaleimide-accessible from the cytoplasmic side
of the membrane and may lie on the H+ transport route.
Residues 215 and 218 on TMH4, as well as residue 245 on TMH5, are
Ag+-accessible but
N-ethylmaleimide-inaccessible and may form part of an
aqueous pocket extending from Asp61 of subunit
c to the periplasmic surface.
H+-transporting F1Fo ATP
synthases consist of two structurally and functionally distinct sectors
termed F1 and Fo (1). In the intact enzyme, ATP
synthesis or hydrolysis takes place in the F1 sector and is
coupled to active H+ transport through the Fo
sector. Structurally similar F1Fo ATP synthases
are present in mitochondria, chloroplasts, and most eubacteria (1). The
F1 sector lies at the surface of the membrane and in
Escherichia coli consists of five subunits in an
The structure of subunit a and its role in H+
translocation are poorly defined. Subunit a is known to fold
with five TMHs (24-26) with aTMH4 packing in parallel to
cTMH2, i.e. the helix to which Asp61
is anchored (27). The interaction of the conserved Arg210
residue in aTMH4 with cTMH2 is thought to be
critical during the deprotonation-protonation cycle of
cAsp61 (23, 28-32). The predicted
aTMH4/cTMH2 interactions are in accord with
second site revertant analysis (33), and cross-link analysis has
confirmed closest neighbor proximity of cTMH2 with
aTMH4 over a span of 19 amino acid residues (27). Both
modeling and cross-linking experiments indicate that helix 2 of subunit
c should be packed on the outside of the ring (17, 34).
Electron microscopic studies support the positioning of subunit
a and the two b subunits at the periphery of the
c ring (35-37).
The chemical labeling of cysteine side chains introduced by
site-directed mutagenesis has been used as a means of mapping aqueous
accessible regions on several membrane proteins (38-51). Several
reagents have been used to modify the genetically introduced Cys to
determine accessibility, including NEM (38, 41-44), MTS reagents (40,
48-50), and Ag+ (45-48). Modification of Cys by these
reagents depends upon ionization of the Cys sulfhydryl to its thiolate
form (52-55), and this is expected to occur preferentially in an
aqueous environment (38, 39, 41). In this report, a span of 19 residues
in aTMH4 were replaced with Cys and tested for accessibility
to water-soluble reagents NEM and Ag+. We found that
residues 206, 210, and 214 are NEM-accessible from the cytoplasmic side
of the membrane. In contrast, residues 215 and 218 on helix 4 and
residue 245 on helix 5 form an Ag+-accessible but
NEM-inaccessible pocket bridging aTMH4 and aTMH5. This pocket may form part of the aqueous access pathway extending from
Asp61 to the periplasmic surface.
Construction of Cys-substituted Mutants--
Cysteine
substitutions were introduced by a two-step PCR method using a
synthetic oligonucleotide that contained the codon change and two wild
type primers (56). Most substitutions had already been generated in
this lab (27) and were transferred to a plasmid containing the entire
unc (atp) operon, in which all endogenous Cys had
been substituted by Ala (57), and a hexahistidine tag on the C terminus
of subunit a (24), using two unique BamHI sites
that lie within the subunit a gene (nucleotide positions 1110 and 1727) (58). The presence of the mutation was confirmed by
sequencing the cloned fragment through the ligation junctions. For
these studies, the plasmids were transformed into JWP292 (2), a strain
with a chromosomal deletion of the entire unc operon. The
aH245C/D119H mutant described in Fig. 12 was constructed in a pDF163-like plasmid (59). Plasmid pDF163 contains the
HindIII (870) to SphI (3216) fragment of
unc DNA cloned between these sites in plasmid pBR322 and
encodes subunits a, b, c, and Comparative Growth Studies--
The Cys-carrying strains were
plated on glucose-containing minimal medium with 0.1 mg/ml ampicillin.
Single colonies were then tested for growth on succinate-containing
minimal plates with scoring for growth at 72 h, as well as growth
yield in liquid minimal medium containing 0.04% glucose.
Membrane Preparation--
Plasmid transformants of strain JWP292
were grown in M63 minimal medium containing 0.6% glucose, 2 mg/liter
thiamine, 0.2 mM uracil, 0.2 mM
L-arginine, 0.02 mM dihydroxybenzoic acid, and 0.1 mg/ml ampicillin, supplemented with 10% LB medium, and harvested in the late exponential phase of growth (2). The cells were suspended
in TMG-acetate buffer (50 mM Tris acetate, 5 mM
magnesium acetate, 10% glycerol, pH 7.5) containing 1 mM
DTT, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml of
DNase I and disrupted by passage through a French press at 124 MPa and
membranes prepared as described (60). Following differential
centrifugation and washing, the final membrane preparation was
suspended in TMG-acetate buffer. The protein concentrations were
determined using a modified Lowry assay (61).
ATP-driven Quenching of ACMA Fluorescence--
The
membranes were suspended in 3.2 ml of the HMK buffers described below,
containing either chloride or nitrate as the counter ion. ACMA was
added to 0.3 µg/ml final concentration, and 30 µl of 0.1 M ATP, pH 7.0 was added to initiate quenching of
fluorescence. The reaction was terminated by addition of 8 µl of 288 µM nigericin (final concentration, 0.5 µg/ml). The
level of fluorescence obtained after the addition of nigericin was
normalized to 100% in calculating the percentage of quenching caused
by ATP-driven proton pumping. For NEM treatment, 80 µl of membranes
at 10 mg/ml in TMG-acetate were incubated at room temperature with 5 mM NEM for 15 min prior to dilution into HMK-chloride
buffer (10 mM Hepes-KOH, 5 mM
MgCl2, and 300 mM KCl, pH 7.5). For
AgNO3 treatment, 160 µl of membranes at 10 mg/ml were
suspended in HMK-nitrate buffer (10 mM Hepes-KOH, 1 mM Mg(NO3)2, and 10 mM
KNO3, pH 7.5) containing 40 µM
AgNO3 and incubated at room temperature for 15 min before
carrying out the quenching assay. Identical results were observed using
silver acetate instead of AgNO3. As a control, the
membranes were also pretreated at 10 mg/ml in TMG-acetate buffer with 5 mM NEM and then diluted into HMK-nitrate buffer before
carrying out the same quenching assay.
NADH-driven Quenching of Quinacrine Fluorescence in Stripped
Membrane Vesicles--
Membranes in TMG-acetate were centrifuged and
resuspended in TEG buffer (1 mM Tris acetate, 0.5 mM EDTA, 10% glycerol, pH 8.0) and incubated for 30 min at
30 °C. The membranes were centrifuged again, washed once with TEG
buffer, and then resuspended in TMG-acetate buffer at 10 mg/ml. The
stripped membrane vesicle suspension (480 µg) was added to 3.2 ml of
HMK-chloride buffer, and following addition of quinacrine to 1.875 µg/ml (final concentration), 16 µl of 10 mM NADH was
added to initiate quenching of fluorescence. The reaction was
terminated by addition of 8 µl of 288 µM nigericin (final concentration, 0.5 µg/ml). For NEM treatment, 480 µg of stripped membranes at 10 mg/ml were incubated at room temperature with
5 mM NEM for 15 min prior to dilution in HMK-chloride
buffer. For dicyclohexylcarbodiimide treatment, diluted stripped
membranes at 150 µg/ml in HMK-chloride buffer were treated with 18.75 µM dicyclohexylcarbodiimide at room temperature for 15 min prior to the quenching measurement.
[14C]N-Ethylmaleimide Labeling
Studies--
[14C]NEM (PerkinElmer Life Sciences; 34.2 mCi/mmol) in pentane was added to TMG-acetate, and the pentane was
evaporated by blowing a stream of argon over the solution. The
resulting aqueous solution of NEM was then added to 3 mg of membrane
vesicles in TMG-acetate such that the final concentration of membranes
was 10 mg/ml and the final concentration of [14C]NEM was
1 mM. The mixture was incubated at room temperature for
1 h, after which the reaction was stopped by the addition of
Cys Substitutions in TMH4 of Subunit a--
In this study, Cys
substitutions in the fourth TMH of subunit a were
transferred into a His-tagged version of subunit a in a
plasmid that coded the entire unc (atp) operon in
which all the endogenous Cys in F1 and Fo had
been substituted by Ala (57). These plasmids were transformed into
JWP292, a strain with a chromosomal deletion of the entire
unc operon. The growth of transformant strains was tested on
glucose and succinate-containing minimal medium. The Cys substitutions
in subunit a reported here have little effect on growth,
with the exception of R210C, G213C, and E219C, which grew poorly or not
at all on succinate and exhibited low growth yields with glucose as a
carbon source (Table I).
Sulfhydryl-specific Reagents Inhibit Function of the aN214C
Mutant--
We wished to examine the aqueous accessibility of residue
214. Previously, Jiang and Fillingame (27) demonstrated that
aN214C forms a cross-link with cM65C and
cA62C, indicating that this residue may be in close
proximity to the proton-binding Asp61 residue of subunit
c. The capacity of several sulfhydryl-specific reagents to
inhibit ATP-driven quenching of ACMA fluorescence by aN214C
inverted membrane vesicles was tested. NEM and nonpolar MTS reagents,
i.e. ethylamino-MTS and carboxyethyl-MTS, inhibited quenching in aN214C membranes (Fig.
1) but not in control membranes lacking
the aN214C (data not shown). The larger, charged MTS
reagents, i.e. trimethylammonium-MTS and sulfonatoethyl-MTS,
were not inhibitory. NEM was found to inhibit quenching in
aN214C membranes maximally at 5 mM but also
quite significantly at 1 mM (Fig.
2). The [14C]NEM labeling
studies described below were carried out with 1 mM NEM. The
inhibition of ATP-driven quenching observed here may be due to a direct
block in proton transport through Fo because NEM also
blocked passive, Fo-mediated proton translocation by stripped membrane vesicles (Fig. 3). This
is indicated by the increase in NADH-driven quenching of quinacrine
fluorescence after NEM treatment, which can be attributed to a decrease
in the proton leakiness of the stripped membrane vesicles.
NEM Inhibition of Quenching with Other aTMH4 Cys
Substitutions--
The Characterization of NEM Reaction with Residues aS206C and
aN214C--
In the models of subunit a discussed elsewhere
(24-27, 32), residue 206 is located on the cytoplasmic face of the
membrane, whereas residue 214 is located near the center of the lipid
bilayer. Because NEM reacts preferentially with the ionized form of Cys (52, 53), the pH dependence of NEM inhibition of the two residues was
examined. When S206C membranes were treated with NEM at pH 7.5, inhibition of quenching was observed. However, NEM treatment at pH 7.0 resulted in no inhibition of quenching and indicated a higher level of
protonation of the sulfhydryl side chain at this pH (Fig.
5A). In contrast, quenching
with N214C membranes was inhibited by NEM treatment at either pH,
indicating that this Cys residue is subject to ionization at pH 7.0 (Fig. 5B). The Cys214 residue must therefore
have an unusually low pKa and be in an unexpectedly
hydrophilic environment for a residue centered in the middle of the
membrane. The Cys214 side chain may be close to the
essential aR210 residue, which could provide charge
neutralization, leading to the low pKa.
[14C]NEM Labeling of aTMH4 Cys Substitutions--
To
check whether NEM reacted with other residues without causing
inhibition of quenching, inside-out membrane vesicles from the
aTMH4 mutants were treated with 1 mM
[14C]NEM for 1 h to determine the aqueous
accessibility of each residue. The hexahistidine-tagged subunit
a was then purified by Ni2+ affinity
chromatography, and the amount of label incorporated into subunit
a was quantified by scintillation counting and
PhosphorImager analysis (Fig. 6). Cys
substitutions at positions 206, 210, and 214 were strongly labeled with
NEM, indicating that this hydrophilic reagent has access to these
residues, possibly via the same aqueous channel by which
protons move through the enzyme.
Silver as a Probe of Aqueous Accessibility to aTMH4--
Silver
has been used to map aqueous pores of several membrane proteins
(45-48), and it was shown to inhibit the
Na+,K+-ATPase (63). Silver ion has an ionic
radius of 1.26 Å, which is close to that of Na+ (0.97 Å)
and H3O+ (1.54 Å) (64). It forms a covalent
bond with sulfhydryl groups of Cys (45, 55, 65). In our initial
experiments with aN214C membranes, Ag+ treatment
caused an inhibition of ATP-driven quenching of ACMA fluorescence, the
extent of inhibition varying with the amount of Ag+ used
(Fig. 7), whereas membranes lacking the
Cys substitution were unaffected by Ag+ treatment. The
extent of inhibition also proved to depend upon the amount of membrane
added to the cuvette (Fig. 8). The amount of AgNO3 needed for a given extent of inhibition increased
proportionally with the amount of membrane present and suggested that
the silver may partition to the membrane phase rather than remaining
soluble in the aqueous phase. Following AgNO3 treatment,
the sample was centrifuged, and atomic absorption analysis was
performed on the membrane pellet and the buffer. We found that 80% of
the AgNO3 added to the solution was associated with the
membrane pellet, further supporting the idea that silver complexes with
the membrane.
Ag+ Inhibition of Quenching in aTMH4 Cys
Substitutions--
Cys-substituted aTMH4 membranes were
treated with 40 µM AgNO3 in chloride-free
assay buffer, and ATP-driven quenching of ACMA fluorescence was
measured. As with NEM treatment, Ag+ treatment inhibited
quenching by aS206C and aN214C membranes. Additionally, and in striking contrast, Ag+ treatment also
resulted in dramatic inhibition with several mutants that were NEM
insensitive. For example, the aM215C and aG218C mutants were very sensitive to inhibition by Ag+ but were
NEM-insensitive (Fig. 9). Other Cys
substitutions showing a lesser sensitivity to Ag+ but still
>50% inhibition include 207, 213, 216, 217, 219, and 220 (Fig.
10). The Ag+ inhibition
observed in several of these mutants, e.g. 213 and 219, may
be due in part to the generally feeble quenching response (Table
II).
Characterization of Ag+ Reaction with Residues aS206C
and aN214C--
The ability of DTT to reverse the inhibition of
quenching caused by treatment of membranes with AgNO3 was
also explored (Fig. 11). We found that
the addition of DTT rapidly reversed AgNO3 inhibition with
aS206C membranes. In contrast, inhibition was very slowly reversed with aN214C membranes, indicating that the
hydrophilic DTT may have more direct access to the cytoplasmically
located S206C than the membrane-buried Cys214 residue.
Involvement of aTMH5 in Proton Conductance--
In the models of
subunit a suggested previously (20, 22, 24-26, 32),
Gly218 in TMH4 is thought to be adjacent to
His245 in TMH5. This model is supported by several second
site suppressor pairs between TMHs, including the aG218D
mutation, which was corrected by a second mutation, aH245G
(66). We wondered whether the Ag+-accessible region of
aTMH4 was also bounded by aTMH5. The
aH245C substitution, located in aTMH5, has been
characterized as a nonfunctional mutant. However, partial function can
be restored by the introduction of a second mutation, aD119H
(24). Membranes carrying the aH245C/D119H double mutant were
treated with silver, and ATP-driven quenching of ACMA fluorescence was
measured (Fig. 12). Ag+
inhibits proton pumping in this mutant, suggesting that several helices
of subunit a may be involved in the formation of an
Ag+-accessible cavity extending into the interior of
subunit a. The boundaries of this cavity will be
investigated further in the future.
In this paper, we report on the aqueous accessibility of cysteine
residues substituted into the fourth transmembrane helix in subunit
a of the rotary ATP synthase. aTMH4 is thought to
interact with Asp61 of subunit c during proton
translocation and may play a part in forming an aqueous access channel
to cAsp61 from one or both sides of the
membrane. The proximity of aTMH4 to cTMH2 was
initially postulated on the basis of second site suppressor analysis
(33) and is now supported by intermolecular Cys-Cys cross-links between
cTMH2 and aTMH4 (27). Aqueous access to
Asp61 of subunit c had been postulated
previously based upon the pH-sensitive function of the cA24D
mutant (67) and is further supported by the discovery that simultaneous
mutation of three residues surrounding Asp61 makes the
enzyme sensitive to inhibition by Li+ (68). Additionally,
the analogous enzyme from Propiogenium modestum, with
a very homologous subunit c, alternatively transports Na+, Li+, or H+ (69, 70). Thus, it
seems likely that these various ions gain access to the
membrane-embedded carboxyl of subunit c via a water-filled channel. We have substituted cysteine over the length of
aTMH4 and tested the susceptibility of each substitution to
modification with water-soluble, thiol-modifying reagents. The approach
has been used previously to define surfaces of membrane proteins with aqueous accessibility (38-51).
Reactivity of Cys Substitutions with NEM, Ag+, and MTS
Reagents--
The ionized sulfhydryl group of cysteine is the form
that preferentially reacts with the thiol-specific reagents used here. For example, the reactivity of MTS reagents with the thiolate is
preferred by a factor of 109 over reaction with a
nonionized thiol group (54). Similarly, NEM (52, 53) and
Ag+ (45, 55) react preferentially with ionized thiolates
rather than with neutral thiols. The differential reactivity of
substituted cysteines can thus provide information about the ionization
state of different residues, which in most cases should be related to aqueous accessibility, i.e. the interpretation given in
similar studies of other membrane proteins (38, 39, 41). The means by
which NEM penetrates the membrane to react with Cys residues is not
certain. It may be sufficiently lipid-soluble to gain access to
transmembrane Cys via the hydrophobic phase of the membrane. However,
modification should only be observed with those residues subject to
ionization. It is also conceivable that uncharged MTS reagents could
access reactive Cys residues via the lipid phase, although
reactivity would again depend upon the ionization state of the
sulfhydryl group. It is of interest that Ag+ reacts with
transmembrane residues that are NEM-insensitive. This may indicate that
NEM-sensitive residues need to be bounded by larger aqueous cavities,
sufficient in size to accommodate the bulkier NEM molecule.
NEM Reactive TMH4 Substitutions--
Residues 206, 210, and 214 on
aTMH4 are preferentially modified with NEM, as indicated by
direct labeling with [14C]NEM and also by inhibition of
ATP-driven quenching in the case of the 206 and 214 mutants. The
radioactive labeling studies were carried out to address the
possibility that residues might be modified with NEM without effect on
function. Additionally, we were able to examine the labeling of a Cys
at position 210 in a mutant that is nonfunctional in proton transport.
These three residues fall on one face of an
The Cys206 residue appears to titrate as the pH of the
medium is lowered from 7.5 to 7.0, as indicated by the dramatic
decrease in NEM reactivity, suggesting that this residue is accessible to the bulk solvent. The solvent accessibility of this residue is also
supported by the rapid reversal of Ag+ inhibition by
dithiothreitol. On the other hand, the reactivity of Cys214
is unaffected by lowering the pH of the medium to 7.0, suggesting that
this residue remains ionized at a lower pH than Cys206,
even though topological analysis would place residue 214 in the middle
of the membrane. Despite its low apparent pKa, Cys214 does not appear to be generally accessible to the
aqueous medium based upon the slow reversal of Ag+
inhibition by DTT. We suggest that the low pKa of
Cys214 at the center of the membrane may be due to salt
bridge formation with the proximal Arg210 residue.
Silver Used to Probe aTMH4 Cys Substitutions--
Silver has been
used previously as an irreversible covalent modifier of Cys introduced
into several membrane proteins (45-48). The irreversibility of the
Ag+ inhibition under the conditions used here is indicated
by an experiment where Ag+ pretreatment of membrane
vesicles protected Cys215 from becoming labeled with
[14C]NEM after solubilization of the membrane with SDS
(experiment not shown). Further, if membranes are treated with silver
and centrifuged to remove the assay buffer, inhibition is still
observed on resuspension in Ag+-free buffer with
Cl
As seen in Fig. 12, aH245C/D119H shows inhibition of
ATP-driven quenching when treated with silver. This is the first direct evidence that the aqueous access pathway to
cAsp61 may involve more than one helix of
subunit a. Looking at the silver-sensitive residues
highlighted on the model of subunit a in Fig. 13, it is
certainly possible that these residues form a pocket that is accessed
from an aqueous channel extending to the cytoplasmic side of the
membrane. The aqueous pathway to the cytoplasm appears to include
NEM-sensitive residues 206, 210, and 214 on one
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
1
1
1 stoichiometry. The Fo sector spans the membrane and in
E. coli consists of three subunits in an
a1b2c10
stoichiometry (2). The structures of several types of F1
have been solved by x-ray crystallography (3-8). In the case of the
bovine mitochondrial enzyme crystallized in the presence of
Mg2+ and adenosine phosphates, the three
and three
subunits alternate around the central
subunit, with subunit
interacting asymmetrically with the three catalytic sites formed at the
interface (3-5). In the widely accepted binding change
mechanism for ATP synthesis, the alternate tight binding of ADP and
Pi and subsequent release of product ATP are mediated by
subunit rotation between the alternating catalytic sites (9-11).
Rotation of the
subunit during ATP hydrolysis was demonstrated by
attaching an actin filament to an immobilized
3
3 complex (12, 13). In the complete
membranous enzyme, the rotation of subunit
is proposed to be driven
by H+ transport-coupled rotation of a connected ring of
c subunits in the Fo sector of the enzyme, which
extend through the lipid bilayer and maintain a fixed linkage with the
subunit. Rotation of the c ring was also demonstrated
using the filament rotation assay (14, 15). The structure of monomeric
subunit c has been solved by NMR in a membrane mimetic
solvent mixture (16), and the structure of the oligomeric
c10 ring was predicted from this structure and cross-linking constraints (17, 18). The proposed subunit
packing is now supported by a 3.9 Å x-ray diffraction map of an
F1c10 subcomplex from yeast
mitochondria (19). The c subunit spans the membrane as a
hairpin of two
-helices and in the case of E. coli
contains the essential aspartyl 61 residue at the center of the second
TMH.1 Asp61 is
thought to undergo protonation and deprotonation as each subunit of the
oligomeric ring moves past a stationary subunit a. Subunit a is believed to provide access channels to the
proton-binding Asp61 residue, but the actual proton
translocation pathway remains to be defined (20-23).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. For this experiment, the plasmid was transformed into strain JWP109 (27), a
strain with a chromosomal deletion of unc genes coding subunits a, b, c, and
.
-mercaptoethanol to 10 mM. SDS was added to 1%,
and unlabeled NEM was added to 20 mM. His-tagged subunit
a was purified from the SDS-solubilized membranes as
follows. After 10 min at room temperature, 660 µl of binding buffer
(50 mM Tris-HCl, 0.3 M NaCl, 0.1% SDS, pH 8)
and 40 µl of nickel-nitrilotriacetic acid-agarose (Qiagen) were
added, and the solution was incubated at room temperature with mixing
for 1 h. The beads were washed twice with 1 ml of wash buffer (50 mM Tris-HCl, 0.3 M NaCl, 1% SDS, pH 8), then
100 µl of elution buffer (62.5 mM Tris-HCl, 20 mM EDTA, 10% glycerol, 2% SDS, 0.01% bromphenol blue,
2.5%
-mercaptoethanol, pH 6.75) was added, and the sample was
boiled for 3 min prior to loading 30 µl on a 12% Tris-Tricine gel
(62). The resulting gel was stained with Coomassie Blue and dried. The
dried gel was exposed to a storage phosphor screen and scanned with a
PhosphorImager (Molecular Dynamics) to quantitate radioactivity
incorporated into subunit a. To determine the amount of
protein in each band, the stained, dried gel was scanned using a
flatbed scanner linked to a Macintosh computer using Epson Twain 5 software. The scan was quantitated using the public domain NIH Image
program.2 Alternatively, the
band containing subunit a was excised from the gel following
Coomassie Blue staining, and the gel slice was incubated with 12%
H2O2 at 90 °C for 5 h to dissolve the
gel. The amount of radioactivity incorporated into subunit a
was determined by scintillation counting. The scintillation counting
experiments confirmed the general accuracy of the PhosphorImager analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Growth properties of aTMH4 substitutions
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Fig. 1.
ATP-driven quenching of ACMA fluorescence by
aN214C membranes is inhibited by a variety of
sulfhydryl-specific reagents. An 80-µl aliquot of membranes at
10 mg/ml in TMG-acetate were treated with sulfhydryl-specific reagents
for 15 min at room temperature before being diluted into 3.2 ml of
HMK-chloride buffer containing 0.3 µg/ml ACMA. ATP was added to 0.94 mM (first arrow), and the uncoupler nigericin
was added to 0.5 µg/ml (second arrow) at the times
indicated. The traces indicate no treatment (trace
1), 20 mM trimethylammonium ethyl-MTS (trace
2), 20 mM sulfonatoethyl-MTS (trace 3), 20 mM carboxyethyl-MTS (trace 4), 2 mM
ethylamino-MTS (trace 5), and 5 mM NEM
(trace 6).
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Fig. 2.
Concentration dependence of NEM inhibition of
ATP-dependent quenching by aN214C
membranes. Membranes at 10 mg/ml in TMG-acetate were treated for
15 min at room temperature with the indicated amounts of NEM, added
from a 0.5 M stock in ethanol, prior to the quenching
measurement as described in the legend to Fig. 1. The traces
indicate no treatment (trace 1), 0.5 mM NEM
(trace 2), 1 mM NEM (trace 3), and 5 mM NEM (trace 4).
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Fig. 3.
NEM inhibits passive proton flux through
stripped Cys214 membrane vesicles. Stripped membranes
at 10 mg/ml in TMG-acetate were treated for 15 min with 5 mM NEM or 18.75 µM dicyclohexylcarbodiimide
at room temperature before being diluted into 3.2 ml of HMK-chloride
buffer containing 1.875 µg/ml quinacrine. NADH was added to 50 µM, and nigericin was added to 0.5 µg/ml at times
indicated. The traces indicate no treatment (trace
1), dicyclohexylcarbodiimide treatment (trace 2),
and NEM treatment (trace 3).
unc strain was transformed with
the set of plasmids containing single Cys substitutions in
aTMH4. Inside-out membrane vesicles were prepared from these
strains. The membranes were treated with 5 mM NEM, and
ATP-dependent quenching of ACMA fluorescence was tested for
each substitution. Representative quenching traces for several mutants
are presented in Fig. 4. NEM inhibited
quenching most strikingly with two Cys substitutions, aS206C
(Fig. 4) and aN214C (Fig. 1), whereas proton translocation
activity remained largely unchanged in the other 17 mutants tested
(Table II).
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Fig. 4.
Differing sensitivity of Cys substitutions in
aTMH4 to NEM inhibition. Membranes from various
aTMH4 Cys mutants were treated at 10 mg/ml in TMG-acetate
with 5 mM NEM for 15 min prior to the quenching measurement
as described in the legend to Fig. 1. A, aS206C
membranes; B, aR210C membranes; C,
aM215C membranes; D, aE219C membranes.
In all panels, trace 1 represents no treatment of the
membranes, whereas trace 2 represents membranes that have
been treated with 5 mM NEM.
Quenching activity ratios of aTMH4 mutants with or without NEM
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Fig. 5.
Residues Cys206 and
Cys214 differ in pH sensitivity to NEM inhibition.
S206C (A) and N214C (B) membranes were treated at
10 mg/ml in TMG-acetate with 5 mM NEM for 15 min at room
temperature at either pH 7.0 or 7.5 prior to the quenching measurement
as described in the legend to Fig. 1. The traces indicate no
treatment at pH 7.5 (trace 1), 5 mM NEM
treatment at pH 7.0 (trace 2), and 5 mM NEM
treatment at pH 7.5 (trace 3).
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Fig. 6.
Residues Cys206,
Cys210, and Cys214 preferentially label with
[14C]NEM. Membranes from various aTMH4
Cys mutants were treated at 10 mg/ml in TMG-acetate (pH 7.5) with 1 mM 14C NEM for 1 h at room temperature.
The membranes were then solubilized with SDS, and subunit a
was purified with nickel-nitrilotriacetic acid-agarose. Samples were
subjected to SDS-PAGE, and the radioactivity in the dried gel was
quantitated with a PhosphorImager. Top panel (A),
Coomassie stain of dried gel, used to normalize the labeling
intensities. Middle panel (B), phosphorimage of
dried gel. Bottom panel (C), bar graph
of labeling intensity normalized to Coomassie staining intensity.
View larger version (13K):
[in a new window]
Fig. 7.
Concentration-dependent
inhibition of ATP-driven ACMA quenching in aN214C
membranes by Ag+. An 80-µl aliquot of membranes at
10 mg/ml in TMG-acetate were diluted into 3.2 ml of HMK-nitrate buffer
(pH 7.5), and AgNO3 was added to the concentration
indicated. Samples were incubated at room temperature for 15 min before
doing the quenching measurement as described in the legend to Fig. 1.
The traces indicate no treatment (trace 1), 5 µM AgNO3 (trace 2), 10 µM AgNO3 (trace 3), 20 µAgNO3 (trace 4), and 40 µM
AgNO3 (trace 5).
View larger version (8K):
[in a new window]
Fig. 8.
Concentration of AgNO3 required
for maximal inhibition of ATP-driven quenching increases in proportion
to the membrane concentration. Varying volumes of N214C membranes
(80, 160, or 320 µl) at 10 mg/ml in TMG-acetate were diluted into 3.2 ml HMK-nitrate buffer (pH 7.5), and AgNO3 was added to the
concentration indicated. Samples were incubated for 15 min at room
temperature prior to the quenching measurement. The relative inhibition
brought about by AgNO3 was plotted as a function of
AgNO3 concentration for each set. , 0.8 mg of membranes;
, 1.6 mg of membranes;
, 3.2 mg of membranes.
View larger version (12K):
[in a new window]
Fig. 9.
ATP-driven ACMA quenching by
Cys215 and Cys218 membranes is inhibited by
Ag+ but insensitive to inhibition by NEM. M215C
(A) and G218C (B) membranes at 10 mg/ml in
TMG-acetate were diluted into 3.2 ml of HMK-nitrate buffer, and
AgNO3 was added to 40 µM, or NEM was added at
5 mM to membranes in TMG-acetate buffer prior to dilution
into HMK-nitrate buffer. Following incubation for 15 min at room
temperature, the quenching measurement was made as described in the
legend to Fig. 1. The traces indicate no treatment
(trace 1), 5 mM NEM treatment (trace
2), and 40 µM AgNO3 treatment
(trace 3).
View larger version (27K):
[in a new window]
Fig. 10.
Cys215 and Cys218
are Ag+-sensitive and NEM-insensitive, whereas
Cys206 and Cys214 are both Ag+- and
NEM-sensitive. A 160-µl aliquot of membranes at 10 mg/ml in
TMG-acetate was diluted into 3.2 ml of HMK-nitrate buffer, and
AgNO3 was added to 40 µM for 15 min, or NEM
was added to 5 mM for 15 min prior to dilution into
HMK-nitrate, as described in the legend to Fig. 9. The results are
presented as the ratios of quenching in the presence of Ag+
or NEM to the quenching in the absence of a reagent. The gray
bars represent the quenching ratio ± Ag+
treatment, whereas the black bars represent the quenching
ratio ± NEM treatment. Each bar represents the average
ratio from n 2 determinations ± S.D.
View larger version (16K):
[in a new window]
Fig. 11.
Cys206 is more accessible to DTT
than Cys214. An 80-µl aliquot of membranes at 10 mg/ml in TMG-acetate were diluted into 3.2 ml of HMK-nitrate buffer,
AgNO3 was added to 20 µM, and the sample
incubated for 15 min at room temperature. DTT was then added to 1 mM from a 1 M stock, and the reaction was
incubated for the indicated length of time prior to the quenching
measurement. A depicts quenching traces from S206C
membranes; B depicts traces from N214C membranes. The
traces indicate control not treated with AgNO3
(trace 1), DTT treatment 1h (trace 2), DTT
treatment 15 min (trace 3), DTT treatment 1 min (trace
4), and no DTT treatment (trace 5).
View larger version (11K):
[in a new window]
Fig. 12.
ATP-driven ACMA quenching in
aH245C/D119H membranes is inhibited by
Ag+. A 160-µl aliquot of membranes at 10 mg/ml in
TMG-acetate was diluted into 3.2 ml of HMK-nitrate buffer, and
AgNO3 was added to 40 or 80 µM. Samples were
incubated for 15 min at room temperature prior to the quenching
measurement. The traces indicate control not treated with
AgNO3 (trace 1), 40 µM
AgNO3 (trace 2), and 80 µM
AgNO3 (trace 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix (Fig.
13), and it seems likely that NEM
modification results in a block to proton transport through this region
of the protein. Inhibition of proton translocation by attachment of the
ethylmaleimide group is consistent with evidence from mutagenesis
studies that bulky substitutions are not as well tolerated as smaller
ones at these positions (Table III). The
fact that the three residues found to label with NEM line one face of
aTMH4 suggests that NEM could be gaining access to these
residues via an aqueous pathway formed at this face of an
-helix.
View larger version (37K):
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Fig. 13.
Location of NEM- and
Ag+-sensitive residues in a hypothetical model of subunit
a. Subunit a modeled by Rastogi and
Girvin (Ref. 22; Protein Data Bank code 1c17). Positions of
NEM-reactive Cys residues are shown in red; the
Arg210 side chain is also indicated.
Ag+-sensitive, NEM-insensitive residues are shown in
yellow. The model was constructed by standard NMR structure
calculation methods using -helical backbone constraints for TMHs
2-5 and five helix-helix contacts suggested from second site
suppressor analysis. In topological models (24, 32) the loops at the
top of the structure are thought to extend into the cytoplasm, and loop
4,5 is thought to extend into the periplasm, placing residue 214 in
aTMH4 at the center of the membrane. The figure is drawn
from the program MOLMOL (77).
Properties of aTMH4 mutations constructed previously in NEM or
Ag+-sensitive residues
present to precipitate any free silver released to
solution. As would be expected (65), the reaction and inhibition can in some cases be reversed by competing sulfhydryl reagents such as DTT,
e.g. in the case of the aS206C substitution. It
is not clear from our studies exactly how silver gains access to these
sulfhydryls. In this experimental system, we have shown by atomic
absorption analysis that 80% of silver added to membrane vesicles
becomes complexed with the membrane. Conceivably, the silver may be
transported to the interior of the membrane vesicles, where it gains
access to subunit a. Alternatively, the silver may bind to
other membrane proteins or sites of unsaturation on fatty acid tails
(71, 72). To test whether other proteins in the membrane were
transporting silver or were necessary for silver absorption by the
membrane, F1Fo was purified and reconstituted
into liposomes and tested for Ag+ inhibition of ATP-driven
proton pumping by Ag+. The Cys214 and
Cys215 mutant enzymes still exhibited inhibition by
Ag+, suggesting that the silver may gain access to these
cysteines without the aid of other membrane proteins and that the
inhibitory Ag+ does not need to be transported to the
interior of these vesicles by an inner membrane Ag+
transport system.
-helical face of
aTMH4. To the periplasmic side of
aAsn214, residues 215-220 are characterized
here as being NEM-inaccessible but Ag+-sensitive, with
residues 215 and 218 showing the greatest silver sensitivity. Residues
215 and 218 would fall on the
-helical face of aTMH4
opposite to residues 206, 210, and 214. If an aqueous pocket bridges
these residues and residue 245 in aTMH5, then
aTMH4 may have to swivel to gate access between the
periplasmic pocket and Asp61 of subunit c. As we
have discussed elsewhere (76), such swiveling may be coupled
mechanically to other helical movements that drive stepwise rotation of
the c ring.
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ACKNOWLEDGEMENT |
---|
We thank Kelly Gostomski for excellent technical assistance in some of the experiments reported.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant GM23105 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a traineeship awarded through National Institutes of
Health Biotechnology Training Program Grant 5T32 GM08349 to the
University of Wisconsin.
§ To whom correspondence should be addressed: Dept. of Biomolecular Chemistry, 587 Medical Sciences Bldg., University of Wisconsin, Madison, WI 53706. Tel.: 608-262-1439; Fax: 608-262-5253; E-mail: rhfillin@facstaff.wisc.edu.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M210199200
2 The program was developed at the National Institutes of Health and is available on the World Wide Web at rsb.info.gov/nih-image.
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ABBREVIATIONS |
---|
The abbreviations used are: TMH, transmembrane helix; DTT, dithiothreitol; MTS, methanethiosulfonate; NEM, N-ethylmaleimide; ACMA, 9-amino-6-chloro-2-methoxy acridine; Tricine, N-[2-hydroxy- 1,1-bis(hydroxymethyl)ethyl]glycine.
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