(Received for publication, June 5, 1995)
From the
Second site suppressor mutations at position 31 of F subunit
recouple ATP-driven H
translocation in the uncoupled Q42E mutant of subunit c of the Escherichia coli F
F
ATP
synthase (Zhang, Y., Oldenburg, M., and Fillingame, R. H.(1994) J.
Biol. Chem. 269, 10221-10224). This finding suggests a
functional interaction between subunit c and subunit
during the coupling of H
transport through F
to ATP synthesis of F
. However, the
physical proximity of the two subunits remained to be defined. In this
study, Cys residues were introduced into residues in the polar loop
region of subunit c surrounding Gln42 and at position 31 of
subunit
to see whether the subunits could be cross-linked.
Disulfide bridge formation between subunit c and subunit
was observed in membranes of three double mutants, i.e. cA40C/
E31C, cQ42C/
E31C, and cP43C/
E31C, but not in wild type membranes or in
membranes of the cA39C/
E31C double mutant. These results
indicate that the polar loop of subunit c and the region
around residue 31 of subunit
are physically close to
each other in the F
F
complex and support the
hypothesis that these two subunits interact directly in the coupling of
H
transport to ATP synthesis. Disulfide cross-linking
of the Q42C subunit c and E31C subunit
leads to
inhibition of ATPase coupled H
transport, as might be
expected in a model where the catalytic sites of the F
ATPase alternate during H
transport-coupled ATP
hydrolysis/synthesis. However, a quantitative relationship between the
extent of inhibition of transport and the extent of cross-linking could
not be established by the methods used here, and the possibility
remains that the
-c cross-linked F
F
complex retains residual H
transporting
activity.
The H-transporting, F
F
ATP synthase of Escherichia coli utilizes an
H
electrochemical gradient to drive ATP synthesis
during oxidative phosphorylation (Senior, 1988). Similar enzymes are
found in mitochondria, chloroplasts, and other bacteria. The enzymes
are composed of two sectors, termed F
and F
.
The F
sector contains the catalytic sites for ATP
synthesis, and when released from membrane, it shows ATPase activity.
The F
sector traverses the membrane and functions as the
H
transporter. When F
is bound to
F
, the complex acts as a reversible,
H
-transporting ATP synthase or ATPase. In E.
coli, F
is composed of five types of subunits in an
stoichiometry and
F
is composed of three types of subunits in an a
b
c
stoichiometry (Foster and Fillingame, 1982). Each
subunit is encoded by a single gene of the unc operon (Walker et al., 1984).
Subunit c is a protein of 79 amino
acid residues which folds in the membrane with a hairpin-like
structure. The two, hydrophobic transmembrane -helices are joined
by a more polar loop region that is exposed to the F
binding side of the membrane (Fillingame, 1990).
Asp
, lying in the center of transmembrane helix-2, is
believed to be essential for H
transport. The coupling
of H
transport in F
to ATP
synthesis/hydrolysis in F
is thought to occur via
conformational changes transmitted through the complex, since the
catalytic sites in F
may lie more than 50 Å away from
the surface of the membrane (Abraham et al., 1993, 1994;
Lucken et al., 1990). The Q42E mutation in the universally
conserved Arg
-Gln(or Asn)
-Pro
sequence of the polar loop of subunit c causes an
uncoupling of H
transport and ATP hydrolysis/synthesis
(Mosher et al., 1985). This ``uncoupled'' phenotype
of the cQ42E mutant can be suppressed by second site
substitutions in Glu
of F
subunit
(Zhang et al., 1994). A functional interaction between the polar loop
of subunit c and subunit
could play a key role in the
coupling process There is however no physical evidence that subunit c and
lie close to each other in the
F
F
complex. In this study, we report
on experiments where the polar loop region of subunit c and
residue 31 of subunit
were cross-linked by disulfide bridge
formation following introduction of Cys residues into these regions.
Wild type subunits c and
lack Cys, so the site of
cross-link formation must be between the sites of Cys substitution. The
experiments provide the first evidence of a contacting interface
between subunit c and subunit
and support the
hypothesis that the subunits may interact directly during the coupling
of H
transport to ATP synthesis.
Figure 1: Plasmids used in polymerase chain reaction mutagenesis and in construction of mutagenized unc operon plasmids. Key restriction digest sites are shown. The unc fragments in the plasmids are shaded.
The ATPase and ATP-driven ACMA
quenching activities of membrane vesicles from these transformant
strains are shown in Table 3. Membrane vesicles were prepare
under nonreducing conditions in buffer lacking dithiothreitol. The
E31C substitution by itself led to a 2-fold elevation in ATPase
activity, whereas the polar loop substitutions by themselves had little
effect on ATPase activity. The combined effect of mutations on ATPase
activity was variable. The cP43C mutation by itself caused a
significant reduction in the ATP-driven ACMA quenching response. Three
of the four polar loop mutations, when combined with the
E31C
mutation, also led to significant reductions in the ACMA quenching
response. The significance of these changes in activity is considered
in greater detail below.
Figure 2:
Immunoblots of SDS-polyacrylamide gel of
membrane vesicles. Membrane vesicles (60 µg of protein) were
prepared and electrophoresed under nonreducing conditions before being
transferred to nitrocellulose paper. The blot was first probed with
monoclonal antibody against subunit . The blot was then stripped
of bound antibodies by submerging it in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM
-mercaptoethanol for 30 min at 50 °C, and the same blot
was reprobed with antiserum against subunit c. Mutations are
indicated by referring to the Cys residues at the positions of subunit c or/and
. Dashes indicate wild type subunit c or
. The positions of the subunit monomers and the
putative homo- or heterodimers are shown by arrows. The
positions of prestained molecular mass markers, with the molecular mass
given in kDa, is shown at the side of the
blots.
Figure 3:
Immunoblots of SDS-polyacrylamide gel of
partially purified F from oxidized membrane vesicles. The
F
were prepared and electrophoresed under nonreducing
conditions. Separate gels were run for each blot. The blots are
presented and marked in the same way as described in the legend for Fig. 2. The purified subunit
was a gift from Dr. S. Dunn
(University of Western Ontario, Canada).
Following dissociation of
F subunits with detergent, the putative c-
cross-linked product was precipitated by antibody to subunit
. The
immunoprecipitates were analyzed by immunoblotting (Fig. 4). In
the three double Cys mutants shown, the 25-kDa protein band was
detected both with antiserum against subunit c and antibody to
subunit
. A corresponding band was not detected in equivalent
preparations from wild type or
E31C single Cys mutant membranes (Fig. 4A). The 25-kDa protein band was not observed if
the sample was treated with
-mercaptoethanol before
electrophoresis. Instead, two protein bands were observed, and they
migrated to the positions corresponding to subunit c and
subunit
monomers (Fig. 4B). From these results we
conclude that a disulfide bridge is indeed formed between polar loop
residues of subunit c and position 31 of subunit
, after
introduction of Cys groups at these positions, and that these two
regions lie physically close to each other at the surface of the
membrane.
Figure 4:
Confirmation of components of 25-kDa
cross-linked product as subunits c and . Partially
purified F
prepared from oxidized membrane vesicles were
immunoprecipitated with subunit
monoclonal antibody preabsorbed
to protein A-Sepharose. Immunoprecipitates were solubilized in SDS
sample buffer in the absence (A) or presence (B) of
10%
-mercaptoethanol and analyzed by electrophoresis and
immunoblotting. Separate gels were run for each blot. The blots are
presented and marked the same way as in the Fig. 2. The
``control w/o Ab'' was prepared by
mock-precipitation of cA40C/
E31C F
with protein A beads lacking antibody. Purified subunit c was prepared as described (Hermolin and Fillingame,
1989).
Figure 5:
Effect of cross-linking on ATPase-coupled
H transport function of the enzyme. A,
ATP-driven quenching of ACMA fluorescence by cQ42C/
E31C
membranes prepared in the absence (trace 1) or presence (trace 2) of 5 mM dithiothreitol. Membranes were
diluted to 0.25 mg/ml in HMK buffer, pH 7.5, and incubated with 0.3
µg/ml ACMA. ATP was added to 0.94 mM and the uncoupler
SF6847 added to 0.3 µM. Membranes prepared in the absence
of dithiothreitol were incubated with 10 mM dithiothreitol in
HMK buffer for 30 min prior to the addition of ATP (trace 3).
The quenching response of cQ42C membranes prepared under
nonreducing conditions without dithiothreitol is also indicated (trace 4). B, immunoblots of SDS-polyacrylamide gel
of the cQ42C/
E31C membrane vesicles prepared in the
absence or presence of 5 mM dithiothreitol. Membrane vesicles
(50 µg of protein) were electrophoresed under nonreducing
conditions before being transferred to nitrocellulose paper for probing
with monoclonal antiboby against subunit
. Arrows indicate bands corresponding to subunit
and the c-
dimer. The positions of prestained molecular mass
markers are shown.
The ATP-driven ACMA quenching response of
the cQ42C/E31C membrane vesicles, prepared under reducing
and nonreducing conditions, was compared to assess the effect of
cross-linking on ATPase-coupled H
transport
function.
As shown in Fig. 5A, the
ATP-driven ACMA quenching response of cQ42C/
E31C
membranes prepared under nonreducing conditions was approximately 70%
of that given by membranes prepared under reducing conditions (curve 1 versus curve 2). A nearly complete quenching
response was restored when the oxidized preparation was assayed in HMK
assay buffer with 10 mM dithiothreitol (Fig. 5A, curve 3). The final experiment shown
in Fig. 5A (curve 4) is the quenching response
of cQ42C membrane vesicles prepared under nonreducing
conditions. These vesicles show a normal quenching response. This
control suggests that the oxidation induced inhibition of quenching
with cQ42C/
E31C vesicles can be attributed to formation
of the c-
cross-link rather than c-c cross-links.
Unfortunately, the ACMA quenching response does
not decrease linearly with decreases in ATPase activity (Miller et
al., 1990). Using the calibration curves previously described by
Miller et al.(1990), we estimate that a 30% reduction in
quenching response would occur under conditions where activity was
reduced by 50-90%. The reduction in quenching response is thus
approximately that expected from the extent of cross-link formation, if
the c- cross-linked F
F
is inactive in proton pumping. The results therefore do indicate
that c-
cross-link formation markedly reduces enzyme
function, but do not rule out the possibility that the c-
cross-linked enzyme retains residual proton pumping activity.
ATPase-coupled H transport by other membrane
vesicles prepared under nonreducing conditions was also measured. As
shown in Table 3, the quenching response by the cA39C
and cA40C single mutant membranes approached that of wild type
membranes, even though subunit c homodimer formation was
observed in both preparations (Fig. 2). The quenching responses
by the double mutant membranes, i.e. cA40C/
E31C and cP43C/
E31C, were decreased compared with that of their
subunit c single mutant membrane counterparts, whereas the
quenching response by the cA39C/
E31C mutant membranes was
not significantly different from that of cA39C single mutant membranes.
The cA39C/
E31C mutant was the only double mutant not
showing formation of a cross-link between subunit c and
subunit
. These results provide further support to the above
conclusion that cross-linking between subunit c and
inhibits the function of the enzyme.
In a previous study (Zhang et al., 1994), we
demonstrated that the uncoupled phenotype of the cQ42E
mutation could be suppressed by second site mutations in Glu of F
subunit
. This discovery suggested a
possible functional interaction between the polar loop of subunit c and subunit
in the coupling process. To test whether the
loop region of subunit c and residue 31 of subunit
are
actually physically close to each other in the F
F
complex, Cys residues were introduced into both regions and
cross-linking attempted by oxidation. Heterodimeric cross-linked
products were formed in the membrane vesicles of three of the double
Cys mutants, i.e.cA40C/
E31C, cQ42C/
E31C, and cP43C/
E31C, but not in the
membrane vesicles of the cA39C/
E31C mutant. These
experiments provide the first evidence of a contacting interface
between the polar loop of subunit c and subunit
and
support the hypothesis that these two subunits may interact directly.
Our conclusions are consistent with the previous cross-linking
experiments of Suss(1986), who concluded that subunit c and
of chloroplast F
F
could be
cross-linked with bifunctional imidoesters.
The cA39C/E31C mutant was the only double mutant not forming
a cross-link between subunit c and subunit
. The Cys of
the A39C subunit c does appear to be reactive in forming
subunit c dimers (see Fig. 2and Fig. 3). The
residue also appears to be accessible to
Cu(II)-(1,10-phenanthroline)
oxidation since c-c dimerization was increased by this reagent. On the other hand, the
A39C sulfhydryl reacted less readily with N-[
H]ethylmaleimide than the other polar
loop Cys residues (data not shown). The Cys
sulhydryl may
be buried in the F
complex in a less reactive, more
hydrophobic environment, relative to the other polar loop
substitutions.
A direct interaction between the polar loop of
subunit c and subunit has important implications in the
mechanism of coupling H
transport to ATP
synthesis. The conformation of the polar loop of subunit c is
hypothesized to change with the ionization state of Asp
in
the middle of the membrane (Fillingame, 1990; Fillingame et
al., 1992). The conformation of subunit
and its position
within F
also changes with the occupancy of
catalytic sites (Capaldi et al., 1992). Binding of
Mg-ADP-P
at catalytic sites results in a simultaneous
association of
and
with
(Gogol et al.,
1990), an association that is manifested by increased N-ethyl-N`-dimethylaminopropylcarbodiimide catalyzed
cross-link formation between Ser
of subunit
and
Glu
in the conserved DELSEED sequence of subunit
(Dallman et al., 1992; Mendel-Hartvig and Capaldi, 1991).
According to the recently published, atomic resolution crystal
structure of the
domain of beef
heart mitochondrial F
ATPase (Abraham et
al., 1994), the DELSEED sequence lies below the catalytic site at
the bottom of subunit
. When ATP is bound in the catalytic site of
subunit
, the DELSEED region of the same subunit directly contacts
several residues of subunit
to form a ``catch.'' We
envision that a conformational change in the polar loop of subunit c, caused by protonation or deprotonation of Asp
,
is transmitted to subunit
via a direct interaction with the
position 31 region. Further conformational changes may then be
transmitted, perhaps through subunit
, to subunits
and/or
. Movement of helical bundles initiated by changes in the DELSEED
region could then be envisioned as altering the conformation of the
catalytic site to promote release of ATP product. The role of the
Ser
region of subunit
in the coupling process is
still unclear. Kuki et al.(1988) have shown that mutant
F
F
with truncated versions of subunit
,
terminating after residue 78, are still active in oxidative
phosphorylation and ATP-driven proton transport.
The ATPase-coupled
H transport function of three double Cys mutants
membranes, i.e.cA40C/
E31C, cQ42C/
E31C and cP43C/
E31C, was decreased
under conditions where cross-link formation between subunits c and
was observed. The inhibition of H
transport appears to relate to c-
heterodimer formation rather than c-c dimer formation. In the cA39C, cA40C, and cQ42C single mutants, c-c dimers were formed but normal activity observed. Normal
activity was also observed in the cA39C/
E31C mutant where c-c dimers, but not c-
dimers, were formed.
Formation of c-
dimers might be expected to inhibit
activity by fixing the conformation of the two regions and preventing
further coupling at alternating catalytic sites (Boyer, 1993).
We
were not able to proportionally relate the extent of inhibition of
ATPase-coupled H transport function to the extent of c-
cross-link formation by the methods used here. The
major problem stems from the nonlinear decrease in ACMA quenching
response with ATPase function (Miller et al., 1990). The
cross-linked F
F
studied most thoroughly in cQ42C/
E31C membranes was generated by spontaneous
oxidation during membrane preparation, and the extent of
incorporation into the
-c dimer was approximately 70%. It
might be possible to relate
-c cross-link formation to
inhibition more easily under conditions where the cross-linking of
subunits
to c approached 100%. Although further
cross-linking could be achieved by mild treatment with
Cu(II)-(1,10-phenanthroline)
and activity further reduced,
the inhibition following this treatment was not effectively reversed by
dithiothreitol treatment. In conclusion, cross-link formation between
subunit
and a single subunit c might be expected to
totally inhibit ATPase-coupled proton transport by preventing
alternation between catalytic sites in F
. The inhibition
observed here is suggestive of such a phenomenon, but we cannot rule
out residual activity in an c-
cross-linked complex. The
relatively high ATPase activity of the cross-linked
F
F
complexes may reflect mutationally induced
uncoupling of F
from F
.