(Received for publication, May 31, 1995; and in revised form, July 10, 1995)
From the
The mechanisms of energy coupling and catalytic cooperativity
are not yet understood for H-ATPase (ATP synthase). An Escherichia coli
subunit frameshift mutant (downstream
of Thr-
277) could not grow by oxidative phosphorylation because
both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and
Futai, M.(1990) J. Biol. Chem. 265, 5043-5048). The
defect(s) of the
frameshift was obvious, because the mutant
subunit had a carboxyl terminus comprising 16 residues different from
those in the wild type. However, in this study, we surprisingly found
that an Arg-
52
Cys or Gly-
150
Asp replacement
could suppress the deleterious effects of the
frameshift. The
membranes of the two mutants (
frameshift/Cys-
52 with or
without a third mutation, Val-
77
Ala) exhibited increased
oxidative phosphorylation, together with 70-100% of the wild type
ATPase activity. Similarly, the
frameshift/Asp-
150 mutant
could grow by oxidative phosphorylation, although this mutant had low
membrane ATPase activity. These results suggest that the
subunit
mutation suppressed the defects of catalytic cooperativity and/or
energy coupling in the
mutant, consistent with the notion that
conformational transmission between the two subunits is pertinent for
this enzyme.
H-ATPase (ATP synthase,
F
F
) synthesizes ATP coupled with an
electrochemical gradient of protons (for reviews see (1, 2, 3) ). The catalytic site of the enzyme
is located in the
subunit of the F
sector
(
). The
membrane-intrinsic F
sector (ab
c
) functions as a
proton pathway. Studies on a negatively stained specimen labeled with
maleimidogold established that the central mass of F
contains the amino-terminal part of the
subunit(4) . The x-ray structures of the mitochondrial F
sector of bovine heart and rat liver have been studied by two
groups independently(5, 6, 7) . In the
crystal structure of bovine F
, the
and
subunits
are arranged alternatively around the amino and carboxyl-terminal
helices of the
subunit(7) . Consistent with its central
location in the F
sector, the active role of the
subunit in catalysis and its energy coupling with proton transport have
been supported by biochemical and molecular biological
studies(8, 9, 10, 11, 12) .
Mutational studies on the Escherichia coli enzyme suggested
the importance of the amino- and carboxyl-terminal regions of the
subunit (286 total residues) in catalysis and energy coupling. Amino
acid substitutions in the region between Gln-
269 and Thr-
277
decreased the membrane ATPase activity and growth by oxidative
phosphorylation(8, 10) . The amino-terminal mutants,
Met-
23
Lys and Met-
23
Arg, exhibited
substantial ATPase activities (65 and 100%, respectively, of the wild
type level), although they grew only very slowly by oxidative
phosphorylation(9) . The uncoupled phenotype of the Met-
23
Lys mutant was suppressed by eight different amino acid
replacements mapped between residues 269 and 280 in the
carboxyl-terminal domain(10, 13) . These results
suggest that the two terminal regions functionally interact to mediate
efficient energy coupling. The
subunit with the Ser-
8
Cys mutation cross-linked with a different
subunit region in the
presence of Mg
-ADP or Mg
-ATP,
suggesting that conformational changes related to the catalytic site
event occur around Ser-
8(11) . Furthermore, a fluorescence
probe introduced into the
subunit changed its spectrum on the
addition of ATP or AMPPNP(
)(12) . However, it is
unknown which region(s) of the
subunit interacts functionally
with the
subunit.
The interaction(s) between different
subunits within the F sector can be clearly shown by
mutation studies(14) ; suppression of the defective energy
coupling of the Ser-
174
Phe mutation by an Arg-
296
Cys replacement indicated the importance of the functional
-
interaction in energy coupling. In this study, a similar
approach was adopted for the
subunit-
subunit interaction.
We previously isolated an interesting frameshift mutant that had 16
unrelated residues due to a nucleotide deletion from the Gln-
278
codon(8) ; the
subunit frameshift has 7 additional
residues at its carboxyl terminus together with 9 altered residues
downstream of Thr-
277. The mutant cells exhibited low membrane
ATPase activity and could not grow by oxidative phosphorylation. Thus,
the long altered carboxyl-terminal domain may interact deleteriously
with the
subunit leading to lower catalytic cooperativity and
defective energy coupling. The defect of the
frameshift mutation
was suppressed by
R52C (Arg-
52
Cys) with or without a
V77A (Val-
77
Ala) or
G150D (Gly-
150
Asp) replacement in the
subunit, suggesting that the deleterious
-
interaction in the
frameshift was suppressed by the
second
subunit mutation. It is surprising that growth of the
three mutants can be supported by oxidative phosphorylation.
Figure 1:
Construction of a recombinant plasmid
(pBMUG15fs) carrying the unc (H-ATPase)
operon with the
subunit frameshift mutation and introduction of
random mutations into the
subunit. a, an SpeI
site was introduced by PCR between the
and
subunit genes
carried by pBWU1; primers I/III and II/IV were used. The product was
subjected to a second PCR (primer I/II). The resulting amplified
fragment was digested with RsrII and SacI and then
inserted into the corresponding site of pBWU1. The NdeI
fragment was deleted from the resulting plasmid, and the final
construct was named pBWU15. b, an SpeI site was also
introduced downstream of the
subunit gene of pBMG293fs using
primers I and FS. The Rsr II-SpeI fragment was
obtained and introduced into the corresponding site of the derivative
of pBWU15 constructed in a. The derivative was named
pBMUG15fs. It should be noted that five restriction sites were
introduced previously into the carboxyl-terminal region without changes
in the amino acid residues coded(8) . c, the SpeI-SacI fragment of pBMUG15fs was transferred
to pBluescript II SK+ and then subjected to PCR mutagenesis with
sequence primers. The amplified products were digested with SpeI and SacI and then ligated with the large SpeI-SacI fragment of pBMUG15fs. Three plasmids
conferred the ability of oxidative phosphorylation-dependent growth on
strain DK8 lacking the unc operon.
For further
studies, the frameshift subunit gene was transferred from
pBMG293fs into pBWU15 (Fig. 1, a and b). The
resulting plasmid, pBMUG15fs, carried all cistrons of the unc operon for H
ATPase with the
frameshift
mutation. Similar to KF10rA/pBMG293fs, DK8 harboring pBMUG15fs could
not grow by oxidative phosphorylation and exhibited about 23% of the
membrane ATPase activity of the same strain with wild type plasmid
pBWU15 (Table 1). As shown previously(22) , DK8 harboring
pBWU15 had 10-fold more enzyme than the regular E. coli strain. Thus, mutant cells even with enzyme having 10% of the
specific activity of the wild type can grow by oxidative
phosphorylation, if they can couple H
transport and
ATP synthesis properly. The Ser-
174
Leu mutant is an
example of such cells; this mutant exhibited 10% membrane ATPase
activity and a 50% growth yield on oxidative
phosphorylation(14) . Therefore, it is clear that DK8/pBMUG15fs
(with 23% membrane ATPase activity) is defective in energy coupling
because no growth by oxidative phosphorylation could be observed.
Consistent with no growth, the rate of ATP synthesis by DK8/pBMUG15fs
membrane vesicles was less than 5% of the wild type level (Table 1).
Figure 2:
Polyacrylamide gel electrophoresis of
F-ATPase fractions from DK8 harboring various mutant
plasmids. Cells were grown in a synthetic medium with glycerol as the
sole carbon source. Their membranes (40 µg of protein) were
subjected to polyacrylamide gel electrophoresis (12%) in the presence
of sodium dodecyl sulfate and then stained with Coomassie Brilliant
Blue (a). The solubilized F
sectors (EDTA extract,
15 µg of protein) were also subjected to electrophoresis, and
portions of the gel are shown to indicate mobility differences between
the wild type and frameshift
subunits (b). The positions
of F
subunits are indicated by arrows, together
with the frameshift
(
fs). Lane 1, purified
F
(15 µg); lane 2,
frameshift; lane
3,
frameshift/R52C; lane 4,
frameshift/R52C/V77A; lane 5,
frameshift/G150D; lane
6, wild type; lane 7, DK8/pBR322 (no unc operon); lane 8, G150D.
Membrane vesicles from the
frameshift/
R52C mutant and that with
V77A exhibited
high membrane ATPase activities (70 and 100% of the wild type level,
respectively) and could synthesize ATP at about 15 (
frameshift/
R52C) and 30% (
frameshift/
R52C/
V77A) of
the wild type rate (Table 1). These results suggest that the
subunit mutations suppressed the defective catalytic
cooperativity of the
frameshift and increased the membrane ATPase
activity. Consistent with higher growth by oxidative phosphorylation,
the
frameshift/
R52C/
V77A mutant exhibited higher ATPase
activity and ATP synthesis than the
frameshift/
R52C.
Membrane vesicles from the
frameshift with
subunit
mutations exhibited lower ATP synthesis than the wild type, suggesting
that they are still defective in energy coupling.
The membrane
ATPase activity of frameshift/
G150D was found to be low when
assayed by the standard procedure. The
frameshift/
G150D
enzyme was unstable under these conditions. As shown
previously(14) , the Arg-
296
Cys/Ser-
174
Phe enzyme was quickly inactivated but stabilized with high
concentration of KCl(14) . Similar to this mutant enzyme, the
frameshift/
G150D enzyme was stabilized by KCl, and ATPase
activity was found to be highest (2.6 units/mg protein, about 10% of
the wild type level) when assayed in the presence of 70 mM KCl (Fig. 3a). The optimal pH values of the mutant enzyme
with and without KCl were 7.0-8.0 and 6.5, respectively, whereas
that of the wild type was 8.5 regardless of the presence of KCl (Fig. 3, a and b); the specific activity of
the mutant ATPase was found to be about 40% of the wild type level when
assayed at pH 6.5. Thus the high growth yield of the mutant by
oxidative phosphorylation may be due to the restored energy coupling
because the mutant exhibited lower ATPase activity than the wild type
even assayed under different conditions. Repeated trials to obtain
membrane vesicles with ATP synthesis from the double mutant were not
successful; we tried buffers with different pH values and ionic
strengths for the preparation of membranes and assaying ATP synthesis,
but the mutant membranes were always leaky to protons. The altered
properties of the mutant were due to the combination of the
frameshift and
G150D; as separate mutants, the
frameshift
and
G150D exhibited 23 and 17% of the membrane ATPase activity of
the wild type, respectively, when assayed at pH 8.0, and membranes of
the
G150D single mutant were not leaky to protons, showing 13% of
the ATP synthesis level of the wild type (Table 1).
Figure 3:
Effects of pH and KCl on the membrane
ATPase activity of the frameshift/
G150D mutant. pH activity
profiles of the
frameshift/
G150D (a) and wild type (b) membrane enzymes are shown. Membranes were diluted with
TKDG buffer and then ATPase activity was measured with (closed
symbols) and without (open symbols) 70 mM KCl.
The standard assay conditions were used except that different buffers
were used: pH 6 and 6.5, 20 mM MES-NaOH; pH 7 and 7.5,
MOPS-NaOH; pH 8 and 8.5, Tris-HCl.
Protons transported through F may cause a series
of conformational changes in different subunits and finally drive ATP
synthesis in the
subunit. Similarly in the reverse reaction, ATP
hydrolysis causes the
subunit conformational change(s), which is
transmitted through other subunits and finally to the H
transport pathway in the F
sector. The
frameshift mutant had two defects: a defect in catalytic cooperativity,
giving low ATPase activity, and one in energy coupling, resulting in
low efficiency in ATP synthesis. The frameshift
subunit had a
longer altered sequence downstream of Thr-
277. With two
methods(23, 24) , it was predicted that the mutant
carboxyl-terminal forms a
-strand, whereas with the same methods a
wild type
helix similar to the x-ray structure was
predicted(7) . The
-strand may possibly extend about 60
Å from Thr-
277 and interact with the upper
-barrel
and/or a part of the catalytic domain of the
subunit (Fig. 4). Thus, the mutant enzyme became defective in energy
coupling and catalytic cooperativity, possibly because the long
carboxyl-terminal
-strand inhibited the proper conformational
transmission between the
and
subunit. It may also be
possible that the transmission is carried out through the rotation of
the
subunit (7) and such mechanical movement was
inhibited in the
frameshift by the interaction of its carboxyl
-strand with the
subunit.
Figure 4:
A model of F with the
frameshift mutation and suppression by the
subunit mutations. A
model was drawn based on the x-ray structure of bovine F
(7) . The
frameshift has an unrelated sequence of
16 residues (additional 7 residues at the carboxyl terminus together
with 9 altered residues downstream of Thr-
277). With two
methods(23, 24) , it was predicted that the
carboxyl-terminal region of the
frameshift forms a
-strand
of about 60 Å (shaded arrow). The
R52C (with or
without
V77A) and
G150D mutations restored membrane ATPase
activity and ATP synthesis. Mutant residues (C
, A
,
and D
) are located following the bovine
F
structure. The amino (N) and carboxyl (C) termini of the wild type and the frameshift (Cfs)
subunit are shown. The glycine-rich phosphate loop (25, 26) containing the catalytic residues is also
shown (P loop).
The defective energy coupling
and catalytic cooperativity of the frameshift mutant were
suppressed by amino acid replacements in the
subunit, indicating
that the altered
-
interaction in the
frameshift was
restored by the
subunit mutations. Two different replacements,
R52C with or without
V77A and
G150D, suppressed the
frameshift, possibly through different mechanisms. The
Arg-
52 residue is conserved in all the
subunit so far
sequenced (57 different species; SWISS PROT rel. 30), and the
corresponding bovine residue is located in the
-barrel
domain(7) . Val-
77 corresponds to bovine Ile-84, which is
located in the
-sheet connecting the
-barrel and nucleotide
binding domains. Therefore, the deleterious interaction between the
putative
-strand of the carboxyl-terminal domain of the
frameshift and the
-barrel was restored by the second mutation
(
R52C) in the same domain. This is further supported by an
additional suppressing effect of the
V77A mutation.
Gly-150 is located in the phosphate loop containing the
catalytic residues(25, 26) . The corresponding bovine
loop may show a large conformational change during catalysis, because
structures of this region in the nucleotide-bound and empty
subunit are strikingly different(7) . Thus, the
G150D
mutation may affect the orientation of another loop located above the
glycine-rich sequence or that of the carboxyl-terminal
helical
domain. These structural considerations suggest that the defective
energy coupling of the
frameshift was suppressed by the
Asp-
150 mutation, possibly because the
subunit mutation
changed the mode of conformational transmission between the
catalytic site and the
subunit. Further studies with the present
approach will be helpful for clarifying the conformational transmission
among subunits during ATP synthesis.