(Received for publication, April 21, 1997)
From the Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229
A triple mutant of Escherichia coli
F1F0-ATP synthase, Q2C/
S411C/
S108C,
has been generated for studying movements of the
and
subunits
during functioning of the enzyme. It includes mutations that allow
disulfide bond formation between the Cys at
411 and both Cys-87 of
and Cys-108 of
, two covalent cross-links that block enzyme
function (Aggeler, R., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 13888-13891). A cross-link is also generated between the Cys at
2 and Cys-140 of the
subunit, which has no
effect on functioning (Ogilvie, I., Aggeler, R., and Capaldi, R. A. (1997) J. Biol. Chem. 272, 16652-16656).
CuCl2 treatment of the mutant
Q2C/
S411C/
S108C
generated five major cross-linked products. These are
-
-
,
-
,
-
-
,
-
, and
-
. The ratio of
-
-
to
the
-
product was close to 1:2, i.e. in one-third of
the ECF1F0 molecules the
subunit was
attached to the
subunit at which the
subunit is bound. Also,
20% of the
subunit was present as a
-
-
product. With
regard to the
subunit, 30% was in the
-
-
, 20% in the
-
-
, and 50% in the
-
products when the cross-linking
was done after incubation in ATP + MgCl2. The amounts of
these three products were 40, 22, and 38%, respectively, in
experiments where Cu2+ was added after preincubation in ATP + Mg2+ + azide. The
subunit is fixed to, and therefore
identifies, one specific
subunit (
). A
distribution of the
and
subunits, which is essentially random
with respect to the
subunits, can only be explained by rotation of
-
relative to the
3
3 domain in
ECF1F0.
F1F0-type ATPases are found in the plasma
membrane of bacteria, the inner membrane of mitochondria, and the
thylakoid membrane of chloroplasts. These enzymes can both use a proton
gradient to synthesize ATP and in the reverse direction hydrolyze ATP
to establish a proton gradient for subsequent substrate and ion
transport processes (1-3). The F1 part of the enzyme from
Escherichia coli, ECF1F0, is
composed of ,
,
,
, and
subunits in the stoichiometry 3:3:1:1:1. This part is linked by a narrow stalk to the F0
part that is composed of a, b, and c subunits in the stoichiometry 1:2:9-12 (3-6). The stalk contains a part of the
and
subunits (6, 7). Two other subunits,
and b, are required for linkage of the
F1 and F0 parts. These two subunits may provide
a second separate connection (8, 9).
Electron microscopy (10) and, more recently, a high resolution
structure of the mitochondrial F1 (11) have shown that the
and
subunits alternate in a hexagonal arrangement around a
central cavity. These two large subunits have a similar fold, each with
three domains, an N-terminal
barrel domain on top and away from the
F0, a middle nucleotide-binding domain, and a C-terminal
-helical domain (11). Three catalytic sites are present and located
predominantly on
subunits. The other three nucleotide binding sites
on the
subunits appear to have mostly a structural role. A part of
the
subunit is found within the
3
3
barrel and organized as two
helices. A third short
helical segment of this subunit was also resolved in the crystal structure of
mitochondrial F1 (11). This lies under the C-terminal
domain of one of the
subunits where it interacts with the so-called "DELSEED" region (the sequence of residues of this part).
The subunit in ECF1 is a two-domain protein (12). The
N-terminal 10-stranded
barrel region interacts with the
subunit through the length of the stalk (7, 13) and with the c subunit oligomer
at one end (14). The helix-loop-helix C-terminal domain of
lies
under the
3
3 subunit barrel and interacts
with the DELSEED region (15) of a different
subunit from the one
that binds to the short
helix of
(16).
There is now considerable evidence that F1F0-type ATPases are highly cooperative enzymes with all three catalytic sites involved (2, 17). This cooperativity of both ATP synthesis and ATP hydrolysis is currently best described by the alternating site model (18). In this model at any time during functioning, one catalytic site is involved in the bond cleavage reaction and is essentially closed; a second is opening to release the tightly bound product, and the third is closing to trap the substrate.
An important tenet of the binding change mechanism is that catalytic
sites are sequentially linked to the proton channel for energy coupling
by a rotation of the small subunits. Early evidence for such rotation
came from cryoelectron microscopy studies on ECF1 that
showed the subunit distributed at all three
subunits rather
than fixed at one site (19, 20). Consistent with the idea of rotation
of
within the
3
3 domain,
cross-linking of this subunit to
or
subunits was found to fully
inhibit the functioning of ECF1 (16, 21).
Additional evidence for rotation of the subunit relative to the
3
3 domain has been provided more recently
by Duncan et al. (22). These authors isolated a complex
containing a (unlabeled)
and
subunit, stably linked by a
disulfide bond between Cys-87 of
and a Cys introduced at position
380 of
. They mixed this
-
complex with a
35S-labeled
subunit along with
,
, and
subunits to regenerate a functional F1. This reconstituted
enzyme was then shown to undergo subunit switching when the disulfide
bond was broken and MgCl2 + ATP was added, i.e.
the
moved from unlabeled to labeled
subunits. Sabbert et
al. (23) have also provided evidence of rotation of the
subunit in chloroplast F1. Finally, rotation of the
subunit in the
3
3
subcomplex of
TF1 has been visualized directly in recent elegant single
molecule studies (24).
However, all of the above studies have focused on the movements of the
subunit in F1, and in terms of functional relevance it
remains important to show that rotation of the
subunit in conjunction with other subunits (e.g. the
subunit)
occurs in the intact F1F0. Also, for an
understanding of the coupling mechanisms, it is necessary to establish
which subunits are moving with the
subunit and which are fixed with
respect to the
3
3 subdomain. One attempt
to examine rotation in ECF1F0 has been reported
recently by Cross and colleagues (25) using the same cross-linking and reconstitution approach they used before for F1. They
showed an ATP-driven and dicyclohexylcarbodiimide-sensitive scrambling
of
relative to
subunits, although the level of this scrambling was lower than would be expected if all enzyme molecules were active.
We have previously observed that disulfide bonds can be formed from an
subunit via a Cys at position 2 to the
subunit (in the mutant
Q2C (26)) and via a Cys at residue 411 to the
and
subunits
(in the mutant
S411C/
S108C (21)). Here, we report cross-linking
studies with ECF1F0 from the mutant
Q2C/
S411C/
S108C where cross-linking from the
subunit to
all three small subunits can be obtained at the same time. The
combinations of cross-linked products obtained provide information
about which subunits have to be moving in the ATP synthase and which do
not.
The triple
mutant pRA170 (Q2C/
S411C/
S108C) was obtained by ligating the
5.8-kilobase XhoI/NsiI fragment of pRA140 (21), which contains the mutations
S411C and
S108C, to the 6.8-kilobase XhoI/NsiI fragment of pIO1 (26), which contains
the mutation
Q2C. Mutant pRA141 (
S411C) was created by ligation
of the 2.9-kilobase SstI/NsiI fragment of pRA100
(27) with the 9.7-kilobase SstI/NsiI fragment of
pRA140. We have recently found that both ECF1 and ECF1F0 can be obtained in purer form by using
E. coli strains that do not make cytochrome bo
(28). For this reason pRA170 was incorporated in RA1, a
unc
/cyo
E. coli
strain that was created by P1 transduction of
(uncB-uncC)ilv::Tn10 from DK8 (29)
to GO104 (F
, thi, rpsL,
cyo::Kan). GO104 was a kind gift from Dr.
Robert Gennis, University of Illinois. Successful transduction was
shown by Southern blotting (30). ECF1 and
ECF1F0 were isolated as described by Gogol
et al. (31) and Aggeler et al. (32),
respectively.
ECF1F0 was reconstituted in egg lecithin on a Sephadex G-50 column (medium, 1.5 × 60 cm) in 50 mM Tris, pH 7.5, 2 mM MgCl2, 2 mM DTT1 and 10% glycerol, and cross-linking of the enzyme was carried out with CuCl2 in 50 mM MOPS, pH 7.0, 2 mM MgCl2, 2 mM ATP, 10% glycerol as described by Aggeler et al. (16). Cross-linked products were separated by electrophoresis on SDS-containing polyacrylamide gels according to Laemmli (33). Two-dimensional SDS-polyacrylamide gel electrophoresis was carried out by resolving cross-linked products in a first dimension without prior treatment with reducing agents on an 8% polyacrylamide gel. A portion of a lane was cut out and exposed to 50 mM DTT in dissociation buffer for 2 h at room temperature. The gel piece was rotated 90° and positioned with agarose on a stacking gel of a 10-18% polyacrylamide gel for the second dimension. Protein concentration was determined with the BCA protein assay from Pierce. Gels were stained with Coomassie Brilliant Blue R (34). Cross-linked products were identified with Western blotting, using monoclonal antibodies against F1 subunits (35).
The experiments described here utilize the mutant
Q2C/
S411C/
S108C. The ATP hydrolysis rates of this mutant were
in the range of 25-30 µmol of ATP hydrolyzed per min per mg, which
is the same as for wild-type enzyme. Also, the
ECF1F0 displayed efficient proton pumping
activity as determined by ATP-dependent
9-amino-6-chloro-2-methoxyacridine fluorescence quenching in inner
membranes (result not shown). Therefore, the introduction of three
different mutations did not disrupt the functioning of the enzyme.
Previous work with ECF1 and ECF1F0
from the mutant
S411C/
S108C has shown that CuCl2 treatment induces cross-linking between the Cys at 411 of one
subunit and the intrinsic Cys-87 of the
subunit and between the
Cys-411 of a second
subunit and Cys-108 of
(21). Cross-linking of
to
, as obtained in an
S411C mutant, or of
to
obtained at low CuCl2 concentrations in the
S411C/
S108C mutant fully inhibited ATPase activity. Studies using
the ECF1 and ECF1F0 from the mutant
Q2C have shown that CuCl2 causes full yield
cross-linking between an
and the
subunit involving the Cys at
position 2 of
and the two intrinsic Cys residues of
,
i.e. Cys-64 and Cys-140. The cross-linking of
to
is
without significant effect on either ATPase activity or ATP-coupled
proton translocation as measured by the
9-amino-6-chloro-2-methoxyacridine fluorescence quenching method
(26).
The effect of CuCl2 treatment on
ECF1F0 isolated from the triple mutant
Q2C/
S411C/
S108C is shown in Fig.
1. These experiments were conducted using
50 or 200 µM CuCl2 with incubation for 1 h at room temperature. All of the subunits of the complex are resolved
in Fig. 1A. The
and
subunits are each essentially missing, and the
subunit is much reduced in the gel, having been
cross-linked with the
subunit. The band of the
subunit is also
greatly reduced (by around 85%). In Fig. 1B, the
cross-linked products are optimally resolved. Each was identified by
immunoblotting with monoclonal antibodies against each of the subunits.
Five main cross-linked products were generated; in order of decreasing apparent molecular weight, these are
-
-
,
-
,
-
-
,
-
, and
-
, respectively. Other cross-linked products were
obtained but only in small amounts. These include an
-
product
that has also been resolved after cross-linking of the
Q2C mutant
and probably involves a disulfide bridge from Cys-2 of one
and
Cys-90 of a second
subunit.
In several different experiments the cross-linked products involving
the subunit, i.e.
-
-
,
-
-
, and
-
,
appeared to be in almost equal amounts based on immunostaining of the
Western blots with the anti-
monoclonal antibody (e.g.
Fig. 1C). To better quantitate the yields of these
cross-linked products, CuCl2-treated enzyme was subjected
to two-dimensional SDS-polyacrylamide gel electrophoresis with
separation in the first dimension in the absence of DTT and in the
second dimension in the presence of DTT. A typical result is shown in
Fig. 2A. It is clear from
these data that the amounts of
,
, and
not in the major
products identified in the figure are very small, i.e. less
than 5% for each subunit. A scan of the three peaks of
subunit
released from
-
-
,
-
-
, and
-
products by DTT
dissociation is shown in Fig. 2B. The relative percentage of
these three peaks was 30, 20, and 50% (averages of two experiments),
respectively. Similar quantitation of percentages of the
subunit in
the
-
-
and
-
bands gave 31 and 69%, respectively, and
for the
subunit in the
-
-
and
-
gave 19 and 81%,
respectively. The experiment in Fig. 2 involved cross-linking of
ECF1F0 after preincubation in MgCl2 + ATP. When cross-linking was performed after preincubation in
MgCl2 + NaN3 + ATP the distribution of
-
-
,
-
-
, and
-
was 40.3 ± 1.0, 21.7 ± 0.9, and 38.0 ± 0.2% (averages of three experiments),
respectively.
The question of whether there is rotation of the subunit
between the three
-
pairs during the functioning of
F1 has been convincingly answered as reviewed in the
Introduction. ATP hydrolysis clearly drives a movement of the
subunit such that it visits all three
-
subunit pairs during
cooperative catalysis (23, 24). A priori, this rotational
movement could be an artifact of a freedom of the
subunit that is
allowed only when the F1 is dissociated from the
F0. However, the results of Zhou et al. (25) and
the data presented in this study are evidence that this is not the
case. The key observations here are that in
ECF1F0 from the mutant
Q2C/
S411C/
S108C
there is cross-linking of
and
subunits each separately to the
same
subunit that binds the
subunit (Fig.
3). However, the
and
subunits are
never bound to the same
subunit. The significance of these results is clear when the activity effects of cross-linking of the
,
, or
subunits to
subunits are considered. Covalent cross-linking of
the
to an
subunit has been found to have little or no effect on
either cooperative ATP hydrolysis or on the proton pumping function of
ECF1F0 (26). This is in contrast to the
cross-linking of
or
to
subunits, which completely blocks
functioning (21). The conclusion from these activity data is that
movements of
and
but not
are an essential part of the
functioning of the enzyme. It follows that the
subunit must be
fixed with its interaction thereby identifying one of the three
subunits (
) that can be visited by both
and
since both
-
-
and
-
-
cross-linked products were
obtained.
The distribution of cross-linked products observed is understandable
when it is considered that the experiments reported here involve a
population of ECF1F0 molecules that are not
synchronized. Thus, at any time during enzyme turnover after ATP
hydrolysis has stopped, around one-third of any rotating subunits
should be at each of the three -
pairs. This is approximately the
observed distribution of both
and
subunits in our experiments
whether enzyme activity was ended by substrate depletion or by addition of azide. In previous studies it was shown that cross-linking of
to
the
subunit did not block activity (7, 27), indicating that these
two subunits move together as a mobile domain. In electron microscopy
studies (19), we have seen movements of the
subunits relative to
, but this is because the antibody fragments to
and
used to
tag specific subunits release
from
so that it is fixed at one
-
subunit pair.
In summary, the scrambling of and
subunits with respect to the
three
subunits, one of which is clearly distinguished by
interaction of the
subunit, is evidence for rotational movements of
the main stalk forming subunits in ECF1F0. The
only other explanation of the data, that ECF1F0
assembles with a fixed random distribution of the small subunits, does
not seem tenable. The
and
subunits appear to move as one
domain, although there may be small movements of the two relative to
one another as part of the coupling between catalytic sites and proton
channel functioning (6).
For rotational movements of the and
subunits to occur within
ECF1F0, the
3
3
domain must be fixed relative to the F0 part by a stator.
Recent evidence suggests that this stator is contributed by the
with the b subunits (9, 26, 36). For coupling, ATP hydrolysis-driven
movements of the
-
domain must be linked to proton translocation.
It has been established that both the
and
(Refs. 7 and 14,
respectively) interact directly with the c subunit oligomer of the
F0 subunit. The covalent cross-linking of
or
to the
c subunit ring does not block ATP hydrolysis (7, 14), which implies
that the rotatory element in ECF1F0 is a
-
-c oligomer domain moving relative to the
3-
3-
-a-b2 complex.
The excellent technical assistance of Kathy Chicas-Cruz is gratefully acknowledged.