From the
A role in coupling proton transport to catalysis of ATP
synthesis has been demonstrated for the Escherichia coli F
In the F
Through the use of mutagenesis, we established that the conserved
terminal regions of the
To assure that no extraneous
mutations accompanied the identified suppressor mutations, each
suppressor mutation was isolated on a restriction fragment and ligated
into the original pBMG15 (Gln-269
Molecular
biological manipulations
(23) and DNA sequencing
(24) were done by standard protocols.
For immunoblotting, membrane proteins were prepared as described by
Nakamoto et al.(29) and separated on a 12.5%
SDS-polyacrylamide gel
(30) . Proteins were then transferred to
nitrocellulose
(31) and reacted with polyclonal antibodies
raised against E. coli F
The suppression
behavior of each second-site mutation was tested by isolating it on a
restriction fragment and ligating into the original plasmid with either
In summary, the suppressor
mutations fell into two groups: three were found in the conserved
amino-terminal region between positions 18 and 35, and four in the
conserved carboxyl-terminal region between positions 236 and 246. The
mutations near the amino terminus changed residues that are
conservatively replaced in the known
Interestingly, the differences in activity between the
The behavior of the
The multiplicity of second-site mutations that suppress
The second important feature
derived from the crystal structure is the relative position of the
three
Our results also suggest that the structural
integrity of the domain is extremely important for efficient energy
coupling. We base this notion on two observations. First, several
different amino acid changes were able to suppress the same primary
mutations. These results indicate that suppression is not the repair of
a single, specific interaction between two residues, but the
restabilization of interactions between segments of the
We thank Dr. Alan Senior of the University of
Rochester for the gift of anti-
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
F
ATP synthase
subunit. Previously,
functional interactions between the terminal regions that were
important for coupling were shown by finding several mutations in the
carboxyl-terminal region of the
subunit (involving residues at
positions 242 and 269-280) that restored efficient coupling to
the mutation,
Met-23
Lys (Nakamoto, R. K., Maeda, M., and
Futai, M. (1993) J. Biol. Chem. 268, 867-872). In this
study, we used suppressor mutagenesis to establish that the terminal
regions can be separated into three interacting segments. Second-site
mutations that cause pseudo reversion of the primary mutations,
Gln-269
Glu or
Thr-273
Val, map to an
amino-terminal segment with changes at residues 18, 34, and 35, and to
a segment near the carboxyl terminus with changes at residues 236, 238,
242, and 246. Each second-site mutation suppressed the effects of both
Gln-269
Glu and
Thr-273
Val, and restored
efficient coupling to enzyme complexes containing either of the primary
mutations. Mapping of these residues in the recently reported x-ray
crystallographic structure of the F
complex (Abrahams, J.
P., Leslie, A. G., Lutter, R., and Walker, J. E.(1994) Nature 370, 621-628), reveals that the second-site mutations do not
directly interact with
Gln-269 and
Thr-273 and that the
effect of suppression occurs at a distance. We propose that the three
subunit segments defined by suppressor mutagenesis, residues
18-35,
236-246, and
269-280,
constitute a domain that is critical for both catalytic function and
energy coupling.
F
ATP synthase, energy coupling
between proton transport and catalysis of ATP synthesis occurs via
conformational changes transmitted through a complex made up of at
least eight different subunits (for reviews see Refs. 1-7). A key
subunit in the coupling mechanism is the
subunit, which appears
as a single copy in the complex. Changes in chemical cross-linking
patterns
(8, 9, 10, 11, 12, 13) ,
protease sensitivity
(14) , intensities of fluorescence
probes
(15) , and immunoelectron micrographic images
(16) have demonstrated that the
subunit undergoes
conformation changes in response to catalysis or proton motive force. A
structural model of bovine F
based on x-ray diffraction
verifies that the
subunit has specific interactions with the
and
subunits that contain the catalytic sites and suggests
that these interactions may be critical for function
(17) .
subunit are involved in coupling. By
changing conserved residue Met-23 of the Escherichia coli
subunit (286 amino acids in length) to lysine, energy
coupling was rendered extremely inefficient
(18) . Functional
interactions between terminal regions were realized by identification
of several second-site mutations near the carboxyl terminus that
restored efficient coupling to the
Met-23
Lys
mutant
(19) . Two such second-site mutations were the
replacements,
Gln-269
Arg and
Thr-273
Ser.
Other replacements of these two conserved residues invariably caused
reduced turnover and coupling efficiency
(20) ; the most severe
mutations were
Gln-269
Glu and
Thr-273
Val. In
this paper, we describe the identification of several intragenic
second-site mutations that suppress
Gln-269
Glu and
Thr-273
Val. Taken together, the suppressor mutations
reveal three
subunit regions that functionally interact to
mediate energy coupling.
Materials
Oligonucleotides were synthesized with
a Pharmacia LKB Gene Assembler Plus.
[-
P]dCTP (3000 Ci/mmol) and
[
P]P
were from Amersham Corp.
Restriction endonucleases and other DNA modifying enzymes were from
Takara Shuzo Co., Nippon Gene Co., Toyobo Co., or New England Biolabs.
Taq polymerase and deoxynucleotides were from Perkin-Elmer.
For all other chemicals and enzymes, the highest grades commercially
available were used.
Bacterial Strains, Plasmids, and Growth
Conditions
The subunit-deficient E. coli strain,
KF10rA (thi, thy, recA1, uncG10 (Gln-14
end)), was
described previously
(21) and grown as before
(20) .
Unless otherwise indicated, all strains were grown at 37 °C. To
assure that mutations in chromosomal or plasmid-borne copies of
uncG did not revert during an experiment, the phenotype of all
strains were checked after growths and plasmids were isolated and
sequenced. Expression and mutagenesis of uncG were performed
in derivatives of plasmid pBMG15
(18) .
Random Mutagenesis, Selection of Pseudo Revertants, and
Manipulation of pBMG15
The Gln-269
Glu and
Thr-273
Val mutations first described by Iwamoto et
al.(20) were moved to plasmid pBMG15 to facilitate
replacement of the entire uncG coding sequence with the
randomly mutagenized gene as was done by Nakamoto et
al.(19) . uncG (Gln-269
Glu or Thr-273
Val) was randomly mutagenized using a modified polymerase chain
reaction
(19, 22) . Transformation of KF10rA with
mutagenized plasmids, genetic selection and analysis of isolates able
to grow on succinate minimal medium (suc
)
were done as before
(19) .
Glu) and pBMG15 (Thr-273
Val). Second-site mutations near the amino terminus were
isolated on the NcoI to XbaI fragment, mutations
between codons 233 and 246 were isolated on the RsrII to
PstI fragment, and mutations of codon 269 were isolated on the
PstI to BglII fragment. Reconstructed plasmids were
sequenced to assure both mutations were present.
Biochemical Procedures
Membrane vesicles were
prepared from strains grown at 37 °C in minimal medium containing
0.2% glucose. Logarithmic phase cells were passed through a French
press at 16,000 p.s.i. and membranes isolated by differential
centrifugation as described previously
(25) .
Protein
(26) , ATPase activity
(25, 27) , and ATP
synthesis
(28, 32) were assayed as described previously.
and
subunits
(obtained from Dr. Alan Senior, University of Rochester) or the
subunit. Immunoreactive bands were detected using the TMB membrane
peroxidase system (Kirkegaard & Perry Laboratories, Inc.).
Suppression of
Previously,
residues Met-23
Lys by Various Amino
Acids at Positions 269 and 273 of the
Subunit
Gln-269 and
Thr-273 were implicated in energy
coupling because mutations
Gln-269
Arg and
Thr-273
Ser were able to suppress the effects of the primary mutation,
Met-23
Lys, and restored efficient energy
coupling
(19) . Interestingly, a number of other mutations at
these same positions also suppressed
Met-23
Lys and
resulted in oxidative phosphorylation-dependent growth when succinate
was used as the sole carbon source (suc
). In
addition to the original suppressor mutations, replacement of
Gln-269 with Leu and Glu, and
Thr-273 with Gly and Val
conferred intragenic suppression of
Met-23
Lys
(). Of these,
Gln-269
Glu and
Thr-273
Val were the most deleterious and, as single mutations, did not
allow growth on solid succinate medium at 37 °C. Clearly, the
suppression between either of these mutations and
Met-23
Lys was mutual. Similar to previously described mutations at these
positions (19),
Gln-269
Glu and
Thr-273
Val
mutant strains were temperature-sensitive and were able to grow on
succinate at 30 °C.
In turn, we searched for
second-site mutations that would suppress the effects of Subunit Mutations That Suppress
Gln-269
Glu and
Thr-273
Val
Gln-269
Glu and
Thr-273
Val. Random mutations were generated
in uncG (
Gln-269
Glu or
Thr-273
Val)
and screened for the ability to grow by oxidative phosphorylation.
Twelve stable suc
colonies arose, and
plasmids were isolated and sequenced. As listed in , six
different mutations that resulted in amino acid changes were found as
single second-site mutations. Two other second-site mutations,
Glu-233
Gly and
Ala-240
Val, were accompanied
by changes of
Glu-269. Finally, two second-site mutations,
Asp-36
Gly and
Met-246
Leu, were found on the
same plasmid with
Thr-273
Val. To assure that the
clustering of suppressor mutations near the ends of the gene was not
due to a bias of the mutagenesis system, several randomly selected
plasmids were sequenced. Mutations were found distributed throughout
the mutagenized segment which included the entire uncG coding
sequence (data not shown). These observations indicated that mutations
were randomly introduced throughout the gene.
Gln-269
Glu or
Thr-273
Val. Based on increased
growth yields in liquid succinate medium, we confirmed that
Ser-34
Leu,
Gln-35
Arg,
Ala-236
Thr,
Glu-238
Gly,
Arg-242
His, and
Met-246
Leu partially suppressed the effects of the original primary
mutation. In addition, we found that each of these mutations suppressed
both
Gln-269
Glu and
Thr-273
Val.
Although the growth yield was lower than the others,
Lys-18
Met was able to suppress
Gln-269
Glu, but only imparted a
slight increase in growth to
Thr-273
Val. The remaining
mutations listed in were unable to suppress; however,
strains carrying
Glu-269
Lys or Gly as single mutations
grew on succinate (data not shown).
subunit sequences and are
adjacent to residues that are completely conserved, while those near
the carboxyl terminus changed residues that are conserved or nearly so.
In general, the second-site mutations near the amino terminus were not
as effective as ones near the carboxyl terminus in suppressing effects
of the primary mutations.
Suppressor Mutations Restore Efficient
Coupling
The ATPase activities of Gln-269
Glu or
Thr-273
Val mutant enzymes were greatly reduced compared to
wild-type enzyme (I), as were ATP-dependent proton pumping
(Fig. 1A) and NADH-dependent ATP synthesis rates
(I). The
Val-273 enzyme generated a smaller
electrochemical gradient of protons and had a lower rate of ATP
synthesis than the
Glu-269 enzyme despite having similar ATPase
activities. Both properties suggested that the
Val-273 enzyme was
less efficient at coupling proton transport to catalysis. As an
indicator of coupling efficiency, ATP synthesis and ATP hydrolysis
rates were compared. Assuming that hydrolysis rates represent the
catalytic competence of the enzyme, we can use synthesis:hydrolysis
ratios to indicate the ability of the F
F
complex to couple energy between proton transport and catalysis.
The synthesis:hydrolysis ratios listed in I suggest that
the coupling efficiency of the
Glu-269 mutant enzyme was similar
to wild type, whereas the
Val-273 mutant enzyme was significantly
lower.
Figure 1:
Effect of subunit
mutations on formation of ATP- or NADH-dependent electrochemical
gradients of protons in membrane vesicles. 100 µg of membrane
vesicle protein from strain KF10rA harboring the indicated mutant
subunits were suspended in 1.0 ml of the buffer described in Table III.
Fluorescence intensity at 530 nm (excitation at 460 nm) was monitored
at 37 °C. A, ATP-driven quenching. At the indicated times
(arrows), 5 µl of 0.2 M ATP (1 mM final
concentration) or 1 µl of 1 mM
carbonylcyanide-m-chlorophenylhydrazone (CCCP) (1
µM final) were added. B, NADH-driven quenching.
At the indicated times (arrows), 20 µl of 0.1 M
NADH (2 mM final), 10 µl of 0.3 M KCN (3
mM final), or 1 µl of 1 mM
carbonylcyanide-m-chlorophenylhydrazone (CCCP) were
added.
When the Glu-269 and
Val-273 mutations were
combined with each of the second-site mutations, the ATPase hydrolysis
rates were essentially unchanged; however, proton pumping and ATP
synthesis rates were increased (Fig. 1A and
I). Significantly, the ATP synthesis:hydrolysis ratios for
the double mutant enzymes (
Glu-269 or
Val-273 plus a
suppressor mutation) were 2-4-fold higher than for the single
mutants. These data indicate that coupling between proton transport and
catalysis became more efficient and even exceeded that of wild-type
enzyme.
Glu-269 and
Val-273 mutant enzymes and wild type were not as
striking as the differences in oxidative phosphorylation-dependent
growth (). A possible reason was that activities were
measured in conditions optimal for the
Glu-269 and
Val-273
enzymes (pH 7.5 and 200-300 mM KCl),(
)
and that in vivo conditions, especially during oxidative
phosphorylation-dependent growth, may have caused the
Glu-269 and
Val-273 mutations to perturb enzyme function to a greater extent.
Glu-269 and
Val-273 mutant complexes
could be explained by loosely associated or unstable F
,
which readily dissociates from the membrane leaving F
to
passively conduct protons. Mutant membranes were tested for the ability
to generate a proton motive force from NADH via electron transport
(Fig. 1B). Similar electrochemical gradients of protons
were generated regardless of the mutation present; therefore, the
mutant F
complexes appear to remain bound to the membranes
and no free F
exposed under our experimental conditions. In
fact, immunoblot analysis of membranes using polyclonal antibodies
against
,
, and
subunits demonstrated that the F
subunits were membrane-associated in all mutant complexes except
when
was not synthesized (Fig. 2). Interestingly, a small
but significant amount of
and
subunits were
membrane-associated even in the case of strain KF10rA harboring plasmid
pBR322, which lacks uncG and expresses no
subunit.
Figure 2:
Immunoblot detection of ,
, and
subunits in membrane preparations from strain KF10rA harboring
pBMG(
Glu-269 or
Val-273) with selected suppressor mutations
(
Ser-34
Leu,
Glu-238
Gly, and
Arg-242
His). 25 µg of membrane protein from strains grown at 37
°C were separated on a 12.5% SDS-polyacrylamide gel and
,
, and
subunit polypeptides detected by immunoblotting (see
``Experimental Procedures''). Lane1, wild
type (pBWG15); lane2, no
subunit (no uncG on plasmid pBR322); lane3,
Glu-269;
lane4,
Glu-269/
Ser-34
Leu; lane5,
Glu-269/
Glu-238
Gly; lane6,
Glu-269/
Arg-242
His; lane7,
Val-273; lane8,
Val-273/
Ser-34
Leu; lane9,
Val-273/
Glu-238
Gly; lane10,
Val-273/
Arg-242
His.
Gln-269
Glu and
Thr-273
Val, in addition to
those that suppress
Met-23
Lys
(19) , reveals three
regions of the
subunit that are involved in coupling
(Fig. 3). One region is between residues 269-280 near the
carboxyl terminus and was defined by a series of second-site mutations
that suppressed
Met-23
Lys
(19) . The other two
regions encompassing residues 18-35 and 236-246 were
described in this study. We further note that changes at position
Arg-242 suppressed
Met-23
Lys as well as
Gln-269
Glu and
Thr-273
Val. We conclude that the three
regions functionally interact because each shares primary
mutation/suppressor mutation combinations with the other two regions.
Moreover, because the primary mutations,
Met-23
Lys,
Gln-269
Glu, and
Thr-273
Val, affect coupling
and the second-site mutations restore coupling to varying degrees, we
propose that the three regions are directly involved in coupling
transport to catalysis. Interestingly, the three regions coincide with
the conserved portions of the
subunit (see Ref. 5).
Figure 3:
Three interacting regions of the
subunit involved in energy coupling. The
-helical termini of the
E. coli
subunit (residues 1-45 and 223-286)
are indicated by the stripedverticalbars.
The position of the helices relative to one another was approximated
from the structural model of Abrahams et al. (17) based on
x-ray crystallographic analysis. In the model, the two helices are in a
coiled-coil conformation. The three interacting regions involved in
coupling and defined by suppressor mutagenesis are indicated by the
brackets (see ``Discussion''). The position of the three
primary mutations,
Met-23
Lys,
Gln-269
Glu, and
Thr-273
Val, are indicated by the smallnumbers.
We are
fortunate that the three subunit regions are found in the partial
x-ray crystallographic structure of bovine F
that was
recently presented by Abrahams et al.(17) . Two
important features of the structural model apply to our mutagenesis
results. First, the structure shows that
Gln-269 (E. coli numbering; equivalent of bovine residue
Gln-255) forms a
hydrogen bond with one of the
subunits near its nucleotide
binding site (17). The effect of replacing this residue confirms its
significance. Although formation of the hydrogen bond in not essential
(Arg, Gly, Leu, and Lys replacements retain function), turnover of the
enzyme is greatly reduced as evidenced by low membrane ATPase and ATP
synthesis rates (I and Refs. 19-20). Because the
coupling efficiency of the
Gln-269
Glu mutant enzyme was
the same as wild type, this mutation appears to disrupt catalytic
turnover in both hydrolysis and synthesis directions. One turn of the
helix away, replacement of the conserved
Thr-273 with valine
caused the same decrease in ATP hydrolysis rate and, in addition, a
proportionally larger decrease in ATP synthesis rate. These results
suggest that changes of
Thr-273 can influence catalysis and
coupling, possibly by perturbing the conformational changes involved in
linking proton transport to catalysis.
subunit regions identified by suppressor mutagenesis. Even
though the coordinates of the structure are not yet
available
(17) , the model clearly shows that the amino acid side
chains of residues 269-280 do not directly contact those of
residues 236-246 or 18-35 (see Fig. 3). Instead, the
18-35 segment appears to be adjacent to residues 236-246 in
the coiled coil and interaction with the 269-280 segment is
through the intervening
-helix. Considering the structure and the
functional interactions, we propose that the three regions of the
subunit defined by the suppressor mutations constitute a domain
responsible for transmitting energy between proton transport and
catalytic sites.
subunit
and possibly the
subunit as well. Second, the temperature
sensitivity caused by the three primary mutations suggests that the
structural stability of the complex was perturbed. Further structural
and mutagenesis studies are required to decipher the mechanism by which
the mutations affect coupling and catalysis. This information should
provide an understanding of the mechanism by which proton transport is
coupled to catalysis.
Table:
Oxidative phosphorylation-dependent growth of
Gln-269 and
Thr-273 mutations with or without
Met-23
Lys
Table:
Second-site mutations found in plasmid-borne
uncG (Gln-269
Glu or
Thr-273
Val)
Table:
Activities of mutant FF
in membrane vesicles from strains grown at 37 °C
/
antiserum and Alistair
Erskine for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.