(Received for publication, January 29, 1997, and in revised form, March 17, 1997)
From the Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229
The N-ethylmaleimide reactivity of c
subunits in Escherichia coli F1F0
ATP synthase (ECF1F0) isolated from five
mutants, each with a cysteine at a different position in the polar loop
region (positions 39, 40, 42, 43, and 44), has been investigated. The maleimide was found to react with Cys placed at positions 42, 43, and
44 but not at 39 or 40. All copies of the c subunit reacted similarly
when the Cys was at position 43 or 44. In contrast, the Cys in the
mutant cQ42C reacted as two classes, with 60% reacting relatively
rapidly and 40% reacting at a rate 40-fold slower. After removing
F1, all copies of the c subunit in this mutant reacted
equally fast. Therefore, the slow class in the cQ42C mutant represents
c subunits shielded by, and probably involved directly in, the
interaction of the F0 with and
subunits of the
F1 part. Based on the estimated stoichiometry of c subunits
in the ECF1F0 complex, 4 or 5 c subunits
are involved in this F1 interaction. N-Ethylmaleimide modification of all of the c subunits
reduced ATPase activity by only 30% in ECF1F0
from mutant cQ42C. Modification of the more rapidly reacting class had
little effect on ATP hydrolysis-driven proton translocation, and did
not alter the DCCD inhibition of ATPase activity. However, as those c
subunits involved in the F1 interaction became modified,
DCCD inhibition was progressively lost, as was coupling between ATP
hydrolysis and proton translocation.
F1F0 ATP synthases are found in the plasma
membrane of bacteria, the thylakoid membrane of chloroplasts, and the
inner membrane of mitochondria, where they use the energy of a proton
electrochemical gradient to drive ATP synthesis. These large
multi-subunit complexes are composed of two domains, as the name
implies: an F1 part, which is extrinsic to the membrane and
contains the catalytic sites, and an F0 part, which spans
the bilayer and contains a proton pore. The F1 part of the
Escherichia coli enzyme is composed of five subunits, ,
,
,
, and
, in the stoichiometry 3:3:1:1:1. The
F0 part contains three subunits, a, b, and c, in the molar ratios 1:2:9-12 (1-4).
The recent high resolution structure determination of bovine
mitochondrial F1 shows the and
subunits arranged as
a hexamer surrounding a central cavity in which the
subunit is
located. This
subunit extends from the
3
3 barrel into the narrow stalk region
that joins the F1 and F0 parts (4, 5). Recent
evidence indicated that the
subunit binds directly to the c
subunits of the F0 part (6, 7). Also present in the stalk
is the
subunit (8), which has direct interaction with c subunits (9). The
subunit has also been considered a component of the stalk
(10, 11). However, recent studies indicate that this subunit is bound
close to the top of the F1, at an
-
interface, where
it binds to the F0 part by interaction with the b subunit (12, 13).
The c subunit oligomer is arranged in F0 as a ring that is
70 Å in diameter based on atomic force microscopy (14). Each c
molecule is folded as a helical hairpin (15-17), with two helices traversing the membrane. The N and C termini are in the periplasmic space, and the polar loop region is in the cytosol (18, 19). A key
residue of the c subunit is Asp61.
DCCD1 reacts with this residue, blocking
proton translocation and inhibiting F1 catalytic function
(10, 20). How many of the c subunits are involved in the interaction
between the F0 and
and
subunits is not known. To
examine this question, we have reacted Cys residues introduced into
this polar loop region with [14C]NEM. Our results show
two types of c subunits within the ring: a group of 4-5 c subunits
that are shielded from reaction with the maleimide by interaction of
F1 subunits, and a second group of 6-7 c subunits that are
more readily accessible for modification. NEM reaction of these two
groups affected functioning differently, and this has significance for
the mechanism of energy coupling within
ECF1F0.
Mutants cA39C, cA40C, cQ42C, cP43C, and cD44C have been described previously (6, 9). The purification of ECF1F0 from these mutants was according to Aggeler et al. (21). Reconstitution of ECF1F0 into egg lecithin vesicles was as described previously (7) for the vesicles used in ACMA fluorescence quenching assays. F0 was prepared by KSCN extraction of purified F1F0 after reconstitution into egg lecithin vesicles (22). The F0 was resuspended in Buffer A (50 mM MOPS, pH 7.0, 10% glycerol, 2 mM MgCl2) by washing twice by centrifugation at 150,000 × g for 30 min at 4 °C.
[14C]NEM Reaction with ECF1F0 and F0 Preparations[14C]NEM was added to
ECF1F0 and ECF0 at 1 and 0.3 mg/ml
of protein, respectively, in Buffer A. For time course experiments, an
aliquot was removed immediately, 2% SDS was added to act as a control
for full incorporation, and this was incubated for 2 h at
22 °C. At the times indicated, aliquots were removed and the NEM
reaction stopped by incubating at 22 °C for 30 min with 50 mM DTT. The data were analyzed using a curve fit that
assumed two independent rates for Cys alkylation with the KaleidaGraph data analysis and graphing program. The equation used is:
P1 + P2 = A·D B·D exp
(
k1·N·t)
C·D exp
(
k2·N·t), where
P1 and P2 = the
proportion of group1-NEM adduct and group2-NEM
adduct, respectively; A = B + C = 100; k1 and
k2 = second-order rate constants for
group1 and group2, respectively;
N = initial NEM concentration; D = protein concentration; and t = time.
The quantitation of the incorporation of [14C]NEM into the c subunits was as described previously (22, 23). The percentage of the Cys in all c subunits that reacted with [14C]NEM after different treatments was determined on the basis of the total incorporation of the maleimide into the appropriate detergent-solubilized control sample incubated with 800 µM [14C]NEM for 2 h at 22 °C to ensure full labeling. The reaction was stopped by the addition of 50 mM DTT.
DCCD Inhibition and NEM TitrationECF1F0 reconstituted in egg lecithin vesicles suspended in Buffer A (0.5 mg/ml protein) was reacted with 50 µM DCCD for 1 h at 22 °C. Aliquots were then mixed with equal volumes of various concentrations of NEM dissolved in DMSO, and samples were incubated for 1 h at 22 °C. The reaction was stopped by addition of 600 µM DTT with incubation at 22 °C for 30 min. Samples were assayed for ATPase activity in 5 mM ATP and 2 mM MgCl2 as described previously (24).
ACMA Fluorescence Quenching and NEM TitrationSamples treated with NEM were reacted with 600 µM DTT for 30 min to quench the maleimide reaction, and then 20 µg of protein from each sample was assayed for ACMA fluorescence quenching as described previously (22) at 1 mM ATP and 2 mM MgCl2.
Other MethodsSamples for SDS-PAGE were mixed with a one-half volume of dissociation buffer (10% SDS, 0.6 M Tris, pH 6.8, 30% glycerol) and 50 mM DTT and incubated for 1 h at room temperature. Polypeptides were separated using a 10-22% linear gradient (25), and protein bands were visualized by staining with Coomassie Brilliant Blue R according to the method of Downer et al. (26). Protein concentrations were determined by the BCA assay from Pierce. [14C]NEM was obtained from DuPont NEN (40 mCi/mmol).
Five different mutants (cA39C, cA40C, cQ42C, cP43C, and cD44C)
were used in this study. ECF1F0 isolated from
each of these mutants had activities in the range of 25-33 µmol ATP
hydrolyzed per min/mg, and each mutant was DCCD sensitive (85% or more
inhibition on addition of 50 µM DCCD). Enzyme from each
of the mutants was reconstituted into proteoliposomes at a ratio of
protein to lipid of 1:2 (w/w) and the reactivity of the Cys introduced
at different positions in the polar loop region of the c subunits
examined by [14C]NEM labeling. For each mutant, NEM
incorporation into the c subunits of native
ECF1F0 was compared with that of enzyme complex dissolved in 2% SDS. The labeling of dissociated and
detergent-denatured ECF1F0 should be a measure
of the number of copies of the c subunit in the complex when related to
the labeling of those subunits with known stoichiometry. The and
subunits are present in 3 copies each and have 4 and 1 intrinsic
Cys respectively. When the counts/min incorporated into the
subunit
was used as a control (by dividing by 12), the amount of
subunits
estimated in several experiments with each of the five mutants was the
same, i.e. 3.1 ± 0.3 mol. The number of c subunits
calculated by the same approach was 9.8 ± 0.2 (three experiments)
for cA39C, 12.2 ± 0.5 for cA40C (three experiments), 11.0 ± 1.3 (nine experiments) for cQ42C, 9.4 ± 0.6 (three experiments)
for cP43C, and 12.2 ± 0.9 (four experiments) for cD44C. The
variability in these values could be due to several factors. There may
be small differences in the binding efficiency of the F1 to
F0 in the various mutants. Any loss of F1
during the handling before gel electrophoresis, which includes
centrifugation steps, would increase the observed number of c subunits
relative to
or
. In contrast, irreversible cross-linking of c
subunits into homodimers or with other subunits, e.g.
and
, which are known to be formed in some mutants (see Ref. 7), would underestimate the number of c subunits when the value is determined by counting the monomer c band. Because of the observed variability, data for each mutant were analyzed separately, and the
labeling of c subunits in the native complex is reported as a
percentage of the labeling in SDS for that mutant.
To compare the relative reactivity of Cys at different
positions in the c subunit loop, native ECF1F0
from each of the mutants was reacted with 100 µM
[14C]NEM for 1 h at room temperature. Fig.
1 summarizes the results obtained. The labeling of the
Cys at positions 39 and 40 was negligible. In contrast, Cys at position
43 or 44 was labeled completely under the reaction conditions chosen.
With the mutant cQ42C, only around 70% of the c subunits were labeled,
implying that in this mutant, there are two populations of c subunits,
one class that reacts readily and a second class that is protected from
labeling. In Fig. 1, the shaded bars show the labeling of
F0 preparations from the various Cys mutants. After the
removal of the F1 part, the Cys at 39 and 40 remained
unreactive, indicating that these residues are not buried by contact
with F1 subunits but, instead, either by protein-protein
contacts within the F0 or by lipid-protein interactions.
After removal of F1 from the mutant cQ42C, all of the c
subunits became reactive to NEM.
The Interaction of the F1 Part Differentiates a Subclass of c Subunits
The reactivity of the c subunit in
ECF1F0 from the mutant cQ42C was examined in
more detail by both concentration dependences and time courses of
[14C]NEM labeling. Fig. 2A
shows the reaction of the c subunit in this mutant with 0-200
µM NEM. Modification of all of the c subunits occurred at
significantly lower NEM concentrations in the isolated F0
than in the intact F1F0 complex. A time course
of reaction of c subunits in ECF1F0 with 100 µM [14C]NEM is presented in Fig.
2B. Two phases of the reaction were observed. A fraction of
the c subunits reacted less readily than the remainder. The best fit to
these kinetic data was obtained with 60% of the c subunits reacting at
the same rate of 62 s1 M
1, and
40% reacting with a second rate of 1.5 s
1
M
1. When c subunits were reacted with NEM
under identical conditions, but in isolated F0, all reacted
rapidly, with a rate of 94 s
1
M
1. For comparison, 100% of c subunits of
ECF1F0 from the mutant cD44C reacted with
[14C]NEM at one rate of 100 s
1
M
1.
Effect on Functioning of NEM Modification of the Cys in the Mutant cQ42C
The activity effects of NEM modification of the Cys at
position 42 of the c subunit was examined as a function of the
percentage of c subunits reacted. The extent of the modification was
established by altering the concentrations of NEM used (as in Fig.
2A). Reaction of all copies of Cys42 (obtained
by prolonged incubation at 200 µM NEM) caused 30%
reduction in the rate of ATP hydrolysis. DCCD inhibition of ATPase
activity is also shown in Fig. 3. NEM incorporation of
60% of the c subunits had essentially no effect. However, when the
extent of labeling was increased above this amount, DCCD inhibition of
ATP hydrolysis was progressively lost.
The effect of NEM modification on ATP hydrolysis-coupled proton
translocation by ECF1F0 from cQ42C is presented
in Fig. 4. Modification of the more reactive c subunits
(curve 2) had very little effect on ATP-driven proton
translocation as measured by the ACMA fluorescence quenching assay.
However, when the modification of c subunits exceeded 60%, coupled
proton translocation was essentially lost (curves 3-5).
We have been using mutants of ECF1F0 in which Cys residues have been introduced into various subunits to probe structure-function relationships, using cross-linkers (e.g. Ref. 27), binding fluorescent probes (e.g. Ref. 28), or, as in this study, by monitoring the reactivity of the Cys and the effect of the modification on functioning (see also Ref. 29). Here, we have examined the arrangement and role of the polar loop region of the c subunits by taking advantage of various mutants that have a Cys at residue position 39, 40, 42, 43, or 44 in this loop (7-9). Position 41 is a highly conserved Arg, and its replacement destroys function, so this specific site was not studied here.
A comparison of the reactivity of the introduced Cys to [14C]NEM in the native and denatured forms of ECF1F0 provides insight into the arrangement of the c subunit oligomer and into the interaction of the F1 part with the F0. NMR data, as well as biochemical and genetic studies, have established that each c subunit in ECF1F0 is arranged as a helical hairpin with N- and C-terminal regions in the periplasmic space. The polar loop, which is predicted to include residues 39-44, is in the cytosol pointing toward the F1 part (18). Our results show that residues 39 and 40 are buried, not by interaction of the F1 part, but either by lipid-protein interaction or protein-protein contacts within the F0 part. In the latter case, these must be interactions between c subunits given the low copy number of a and b subunits.
The number of c subunits per ECF1F0 remains unclear; values of 10-12 have been reported (18, 19). The labeling data here for enzyme denatured in SDS are no more definitive for technical reasons. The value obtained was different for each mutant, but in the range of 9.4-12.2.
The key result of the present study is that the c subunits in
ECF1F0 are not all equivalent, but fall into
two classes based on the NEM reactivity of the Cys at position 42. On
labeling of ECF1F0 from the mutant cQ42C, 60%
of the c subunits reacted relatively rapidly with maleimide, all at a
similar rate of around 62 s1
M
1. The other 40% reacted much more slowly
(rates of 1.5 s
1 M
1). These
more slowly reacting c subunits are involved in F1 binding because after removal of the F1, all of the c subunits were
equally accessible.
No subset of shielded c subunits was observed on labeling ECF1F0 from mutants cP43C or cD44C with [14C]NEM, suggesting that the interaction of the F1 part with c subunits is relatively limited and involves mainly Arg41 and Gln42.
The effect of NEM incorporation at position 42 on DCCD inhibition of
ATP hydrolysis supports the idea that only a few of the c subunits
interact with the F1 part. Modification of the faster reacting 60% of c subunits has little or no effect on the DCCD sensitivity of ATP hydrolysis in the mutant. However, with reaction of
the last 40%, there was a progressive loss of the inhibition caused by
the covalently bound carbodiimide. The implication is that NEM reaction
with the more shielded Cys disrupts the F1F0 interaction and causes loss of DCCD sensitivity. Genetic studies have
shown that Gln42 can be replaced by Val, Ala, or Cys but
not by more bulky side chains without disrupting the
F1F0 interface (30). Presumably the size
difference between Cys and the Cys-NEM adduct is sufficient to alter
binding of the and
subunits with the c subunit oligomer. Reaction of the Cys at position 42 was also found to affect ATP-driven proton translocation in the mutant cQ42C. As the number of c subunits modified by NEM was increased, the coupling of ATP hydrolysis to proton
translocation as measured by ACMA fluorescence quenching was decreased.
This assay is not quantitative, but it is clear that modification of
more than 60% of the c subunits is required before proton pumping is
greatly reduced. The uncoupling of ATP hydrolysis from proton
translocation is explained if NEM modification of the Cys at position
42 disrupts the F1 and F0 interface.
The question of how many c subunits are involved in binding of F1 to F0 is not answered absolutely by the present studies because of uncertainties about the stoichiometry of the c subunit in the complex. However, if there are 10 copies, 4 are involved, whereas if there are 12, then 5 are involved. The organization of c subunits in the F0 part remains to be defined precisely. However, the recent single-particle, atomic force microscopy studies show the c subunit oligomer as a ring within which is a pit (14). This would appear to preclude the idea that the c subunits are a tightly packed bundle with an inner core set that binds the F1 part.
If, as proposed, the c subunits are arranged in a single ring, then the
fact that two classes of c subunits can be detected in activity assays
has important functional implications. The emerging model of the
coupling of catalytic site events with proton translocation includes
the rotation of the stalk subunits (31-33) and
(32, 34). This
rotation of
and
could be relative to a fixed c subunit ring, or
there could be concerted movements of a domain that includes both
and
subunits and the c oligomer. We have previously found that the
cross linking of
(via Cys at 205) to the c subunit ring does not
significantly affect ATP hydrolysis rates. We interpreted these results
in favor of a model in which the
and
subunits oligomers, along
with the c subunit oligomer, move together (7). The results presented
here are consistent with this idea. If the
and
subunits were
moving relative to the c subunit ring, all of the c subunits should
become equivalent during enzyme turnover and modification of any group of the c subunits by NEM should have the same overall effect. As a
consequence, NEM incorporation into the faster-reacting class of c
subunits would be expected to disrupt the F1F0
interface as the
-
domain moves around to interact with these.
This is not the case, as both the DCCD inhibition of ATPase activity
and the coupling of ATP hydrolysis to proton translocation are not lost
until more than half of the c subunits have been modified.
In summary, evidence is presented that 4 or 5 c subunits are
involved in the binding of the and
subunits to the
F0. Furthermore, it appears that this interaction is fixed
such that the
and
subunits do not switch between c subunits
during functioning. Our working model is that rotational catalysis
involves movements of a complex of the
,
, and c subunits and is
relative to other parts of the F1 and F0 that
are linked by a stator constituted by the
and b subunits (see Ref.
13).
Dr. Robert Fillingame (University of Wisconsin) kindly provided the c subunit mutants used in this study. We thank our colleagues, Drs. Robert Aggeler, Stephan Wilkens, and Gerhard Grüber for helpful discussion and Kathy Chicas-Cruz for technical assistance.