(Received for publication, November 8, 1995; and in revised form, January 5, 1996)
From the
Site-directed mutagenesis was used to investigate the roles of a
short series of hydrophobic amino acids in the b-subunit of the Escherichia coli FF
-ATPase. A mutation
affecting one of these, G131D, had been previously characterized and
was found to interrupt assembly of the
F
F
-ATPase (Jans, D. A., Hatch, L., Fimmel, A.
L., Gibson, D., and Cox, G. B.(1985) J. Bacteriol. 162,
420-426). To extend this work, aspartic acid was substituted for
each one of the residues from positions 124 to 132. The properties of
mutants in this series are consistent with the region from Val
to Gly
forming an
-helix. Two of the
mutations, V124D and A128D, resulted in a similar phenotype to the
G131D mutation. This suggested that Val
,
Ala
, and Gly
form a helical face which may
have a role in inter- or intrasubunit interactions. This was tested by
overexpressing and purifying the cytoplasmic domains of the wild type
and A128D mutant b-subunits. Sedimentation equilibrium centrifugation
indicated that the wild type domain formed a dimer whereas the mutant
was present as a monomer.
The FF
-ATPase enzyme complex catalyzes
the terminal step in oxidative phosphorylation and photophosphorylation
and is located in mitochondrial, chloroplast, and bacterial membranes.
In Escherichia coli the enzyme comprises eight non-identical
subunits, a, b, c,
,
,
,
, and
, encoded by
the genes uncB, F, E, A, D, G, H, and C,
respectively(1) . The a, b, and c subunits are integral
membrane proteins and form the F
portion of the complex
which can function as a proton pore. The
,
,
,
,
and
subunits are peripheral membrane proteins forming the
F
-ATPase portion of the complex and which retains ATP
hydrolytic activity when removed from the membrane.
The a, b, and c
subunits are all required for the formation of a proton pore (2) and are present in a stoichiometry of
1:2:6-12(3) . The b-subunit consists of a single
transmembrane helix at the N terminus with the rest of the protein
extending into the F-ATPase. The cytoplasmic domain is
strongly hydrophilic and is predicted to form two long
-helices
separated by a turn(4, 5) . Existing evidence is
consistent with this structure. The cytoplasmic domain has been
expressed and purified and an elongated structure with a high
-helical content was indicated by sedimentation equilibrium
centrifugation and circular dichroism spectroscopy(6) . The
purified cytoplasmic domain of the b-subunit was found to form a dimer
which was capable of interacting with the F
(6) .
Cryoelectron microscopy of the complex formed between the F
and the purified dimer indicated that one of the
subunits
showed an increased density, suggesting that the b-subunit may interact
with a specific
subunit(7) . Interactions between the
hydrophilic domain of the b-subunit and F
subunits may also
be required for assembly of the F
F
-ATPase. A
number of b-subunit mutations which affect assembly of the ATPase
complex have been
isolated(8, 9, 10, 11) . One of
these, G131D, resulted in the b-subunit being able to assemble into the
membrane but assembly of the whole complex was blocked at the
1
2
stage, suggesting that this mutation interfered with the
b-subunit's interaction with one of the minor
subunits(8) .
Although the cytoplasmic domain of the
b-subunit is extremely hydrophilic, Gly lies in a short
stretch of hydrophobic residues (Val
to
Ala
). These lie near the end of a larger region predicted
to be
-helical. In the present study the effect of replacing each
of these hydrophobic residues with aspartic acid was examined.
Turbidities of cultures were measured with a Klett-Summerson colorimeter. Growth yields were measured as turbidities after growth had ceased in medium containing limiting (5 mM) glucose.
One transformant from each was purified and retained for further work. A similar plasmid carrying the G131D mutation was generated by digesting pAN257 (8) with HindIII and ClaI and subcloning the 2.2-kilobase fragment into pAN174 as described above. Coupled and uncoupled controls were produced by transforming strain AN1440 with pAN495, which carries the wild type uncB, uncE, and uncF genes(17) , and pAN174, respectively.
The vector used to construct GST fusion proteins was p2GEX-4T, which carries two copies of the gene encoding glutathione S-transferase(20) . A BamHI site was introduced into the wild type and A128D mutant b-subunit genes by site-directed mutagenesis, replacing the codons for Pro-27 and Pro-28. This creates a thrombin cleavage site at the junction between GST and the b-subunit portion, allowing the release of the b-subunit domain by digestion with thrombin. A 1.0-kilobase BamHI/EcoRI fragment carrying the portion of the uncF gene encoding the cytoplasmic domain of the b-subunit and the uncH gene was then subcloned into the BamHI/EcoRI sites of the vector. After confirmation of the presence of the insert by restriction analysis, plasmids were used to transform strain AN3347 which also contains the plasmid pGroESL (Table 1).
where R is the gas constant, T is the absolute temperature,
is the angular rotation in radians per second,
is the
partial specific volume of the protein, 0.73 in this case, and r is the
density of the buffer. The density of SMP buffer, which is
significantly higher than that of water due to the presence of the
sucrose, was determined using a digital precision densitometer to be
1.048 g/ml. The procedure followed in analyzing the data was to obtain
the best fit molecular weight for the sample using the program XLAEQ
which assumes the sample is homogeneous and thermodynamically ideal and
evaluates the molecular weight corresponding to the best fit straight
line through the A versus r data from . Point
average molecular weights as a function of concentration were also
evaluated from using the program XLAMW which fits groups
of points along the A versus R scan and uses the slope of the
ln A versus r
data at the central point of the
group to give a point average molecular weight at the corresponding
concentration. Examination of the plot of molecular weight versus concentration indicated whether there was size heterogeneity in
the sample. Dependence of weight average molecular weight on
concentration was interpreted in terms of the known subunit molecular
weight of the protein as described in the text.
Figure 1: Hydropathy plots of b-subunits from different species. Hydropathy plots were calculated using the algorithm of Kyte and Doolittle (24) and a window size of 9 amino acids. The hydrophobic region discussed in the text is underlined. Sequences used were from: E. coli(1) , Vibrio alginolyticus(25) , Bacillus megaterium(26) , and the thermophile, Bacillus PS3(27) .
Figure 2: Alignment of the conserved hydrophobic regions and properties of mutants of E. coli in which these residues were replaced by aspartic acid. A, the percentage homology of between the E. coli b-subunit and each of the others was calculated using BestFit in the GCG Sequence Analysis Software Package. Sources of the sequences were as for Fig. 1. Conserved amino acids are in bold. B, properties of mutants of E. coli in which each residue in this region was replaced by aspartic acid. The ability of each strain to grow on solid succinate medium is indicated as + or -. The values for the growth yield on 5 mM glucose and ATPase activity of each mutant are expressed as percentages, with the values for the coupled and uncoupled controls being set at 100% and 0, respectively. The ATPase activity of the coupled control was 0.8 µmol/min/mg protein. Atebrin fluorescence quenching activities are given as percentages of the maximum quench. The wild type values were 85% for NADH-dependent quenching activity and 92% for ATP-dependent quenching activity.
To further investigate the role of this region, a series of mutants
was constructed in which aspartic acid replaced each residue from
Val to Ala
. Each mutant plasmid was
transformed into strain AN1440 (uncF469) which carries the
chromosomal mutation Trp
stop(18) .
Membranes were prepared from each strain and the growth properties of
the mutant strains and the ATPase activity and proton pumping
activities of the membranes were determined. The results are shown in Fig. 2B and examination of all the properties of the
mutant strains suggests a helical periodicity for this region. The most
severely affected strains were those carrying the V124D and A128D
mutations. These two strains were unable to grow on succinate and had
growth yields typical of the uncoupled control. The membranes had no
ATPase activity or ATP-dependent fluorescence quenching activity. The
NADH-dependent quenching activity was reduced, indicating that the
membranes were permeable to protons. If this region was helical,
Val
and Ala
would be one helical turn apart
and would lie on the same face of the helix as Gly
. The
G131D mutation resulted in similar properties to V124D and A128D
although the effects were slightly less severe.
On the opposite side
of the helix are Ile and Ala
and mutation
of either of these residues resulted in strains which were similar to
the wild type. The introduction of an aspartic acid residue at these
positions did have some effect as both the I126D and A130D strains had
reduced ATPase activity and reduced levels of ATP-dependent
fluorescence quenching. However, both strains showed significant growth
on succinate and had growth yields which approached the wild type
level. The NADH-dependent fluorescence quenching activity of these two
strains was similar to the wild type.
Of the remaining mutants, all
except the A132D mutant were unable to grow on succinate. Replacement
of Ala, Leu
, or Val
with
aspartic acid was not as deleterious as at positions 124 or 128 because
some ATPase function was retained in each of these mutants. The L127D
and V129D mutants had reduced NADH-dependent fluorescence quenching
activities indicative of proton permeable membranes. The only exception
to the apparent helical periodicity of this region was the A132D mutant
which had growth properties identical to the coupled control. Residue
132 is the last hydrophobic residue in this stretch. The A128D mutation
was selected for further experimentation to investigate the possible
role of this region in interaction between the two b-subunits.
Figure 3:
SDS-polyacrylamide gel electrophoresis
analysis of the purification of the GST/b-subunit (cytoplasmic portion)
fusion. Cells were grown and induced with
isopropyl--thio-galactopyranoside as described under
``Experimental Procedures.'' Samples were taken at various
stages of the purification procedure and run on a 4-20%
polyacrylamide gel which was then stained with Coomassie Blue. Lanes 1 and 8, molecular weight markers; lane
2, cell debris; lane 3, cytoplasmic fraction; lane
4, unbound material following exposure of the cytoplasmic fraction (lane 2) to glutathione-agarose beads; lane 5,
material bound to the beads; lane 6, material bound to the
beads following digestion with thrombin; lane 7, material in
supernatant following centrifugation to remove beads. The A128D mutant
b-subunit behaved similarly to the wild type during
purification.
We have shown that a single mutation, A128D, is sufficient to
prevent dimerization of the cytoplasmic domain of the b-subunit. Our
results with the wild type b-subunit are consistent with an earlier
study (6) which examined the properties of the cytoplasmic
domain of the b-subunit. This study found that the soluble portion of
the b-subunit could form a dimer which was capable of binding to
F-ATPase, although with a low affinity. This suggested that
it formed a structure similar to the native b-subunits. In both this
study and the present one, gel sieve HPLC of the cytoplasmic portion of
the wild type b-subunit gave a high apparent molecular mass, consistent
with an elongated structure. The native structure was proposed to be a
coiled-coil(6) , based on a high
-helical content, as
indicated by circular dichroism spectroscopy, and on the presence of
the characteristic heptad repeat in parts of the b-subunit. Since
coiled-coils are stabilized by multiple interhelix contacts, our
finding that dimer formation can be prevented by a single mutation
would suggest that other structures should be considered.
Ala lies in the center of a short stretch of
hydrophobic amino acids which is conserved among b-subunits from many
non-photosynthetic bacteria (Fig. 1). Photosynthetic organisms
possess two different homologues of the b-subunit (28, 29) and this region is not present in either one
of them. In these organisms it is thought that one of each of the two
b-subunit homologues may replace the pair of identical b-subunits found
in non-photosynthetic bacteria. The presence of the conserved
hydrophobic region only in species where the
F
F
-ATPase contains two identical b-subunits is
consistent with its role in dimerization. In E. coli, this
region is predicted to form part of an extended
-helix (5) and our data suggests that the residues from Ala
to Gly
do form a helix. The most severe effects
occurred when aspartic acid was placed on the helical face defined by
positions 124, 128, and 131. On the opposite face, defined by
Ile
and Ala
, the mutants showed normal or
near normal behavior. Aspartic acid at positions 125, 127, and 129
resulted in intermediate phenotypes, as would be expected if this
region does form a helix. The only mutant where results were
inconsistent with a helix was the A132D mutant which was similar to the
wild type. However, Ala
is the last hydrophobic residue
in this stretch and may mark the end of the helix or aspartic acid at
this position may interact with neighboring charged residues.
In the
normal b-subunit Val, Ala
, and Gly
may form a helical face which interacts with another hydrophobic
surface. In the mutants, this interaction would be disrupted by the
introduction of an aspartic acid residue. The finding that the
cytoplasmic domain of the A128D mutant b-subunit is unable to form a
dimer suggests that this interaction occurs between the two b-subunits.
An earlier study of the G131D mutation (8) reported results
consistent with those described here. However, data from
two-dimensional gels indicated that assembly of the
F
-ATPase was blocked at the 1
2
stage(8) ,
implying that one of the minor subunits was unable to interact with the
mutant b-subunit. The observation that the membranes prepared from the
A128D mutant were permeable to protons implies that the hydrophobic
domains of the mutant b-subunits were correctly located in the
F
. The inability of the hydrophilic domains to dimerize may
therefore provide a rational explanation for the failure of the mutant
to complete F
assembly.