From the
The regulatory
The chloroplast coupling factor 1 (CF
There are six nucleotide binding sites on the enzyme
(5, 6, 7) probably located at interfaces
between the
The apparent asymmetry among the nucleotide binding
sites on CF
The
F
In addition to the selective removal of the
Nucleotides and tentoxin were purchased from
Sigma. Stock solutions of tentoxin were prepared by dissolving the
inhibitor in ethanol to a final concentration of 5 mM, and
stored at
Analytical ultracentrifugation
was performed on a Beckman model E664 analytical ultracentrifuge
equipped with schlieren optics. Electron microscopy was performed as
described elsewhere
(32) .
We found that
the
The
Conditions
were also identified for isolating large amounts of a complex, which
contained equal amounts of the
An
Nearly all F
The
results presented in this study show that the
Four of the six nucleotide binding sites on CF
The low ATPase activity observed with the
The catalytic activity that is manifested
by the chloroplast
The
In
conclusion, we have described and characterized procedures for
isolating and reconstituting the
subunit and an
complex were
isolated from the catalytic F
portion of the chloroplast
ATP synthase. The isolated
subunit was devoid of catalytic
activity, whereas the
complex exhibited a very low ATPase
activity (
200 nmol/min/mg of protein). The
complex
migrated as a hexameric
complex
during ultracentrifugation and gel filtration but reversibly
dissociated into
and
monomers after freezing and thawing in
the presence of ethylenediamine tetraacetic acid and in the absence of
nucleotides. Conditions are described in which the
and
preparations were combined to rapidly and efficiently reconstitute a
fully functional catalytic core enzyme complex. The reconstituted
enzyme exhibited normal tight binding and sensitivity to the inhibitory
subunit and to the allosteric inhibitor tentoxin. However,
neither the
complex nor the isolated
subunit alone
could bind the
subunit or tentoxin with high affinity. Similarly,
high affinity binding sites for ATP and ADP, which are characteristic
of the core
enzyme, were absent
from the
complex. The results indicate that when the
subunit binds to the
complex, it induces a three-dimensional
conformation in the enzyme, which is necessary for tight binding of the
inhibitors and for high-affinity, asymmetric nucleotide binding.
)
(
)
utilizes the energy of a transmembrane proton gradient to
catalyze ATP synthesis. CF
is composed of five different
subunits designated
to
in order of decreasing molecular
weight and with a subunit stoichiometry of 3
, 3
, 1
,
1
, and 1
(1) . CF
is a latent ATPase,
requiring activation either by removing the
subunit and/or
reducing the only disulfide bond of the enzyme located on the
subunit
(2) . The
subunit is readily removed from the
enzyme by treatment with ethyl alcohol, while CF
is bound
to an anion-exchange resin. The resulting
-deficient enzyme is
recovered from the resin as a fully active ATPase
(3) . The
subunit can also be removed by similar treatment
(2, 4) with no apparent effect on the catalytic activity of
isolated CF
. Thus the maximum number of subunits required
for ATPase activity is
.
and
subunits
(8, 9) . Freshly
isolated CF
has up to two molecules of ADP bound with
sufficient strength to resist removal during successive passage through
several gel filtration columns
(10) . Addition of MgATP results
in occupancy of four sites, two with ADP tightly bound and two with ATP
tightly bound. The remaining two sites do not retain nucleotides during
gel filtration and are thus assumed to be of lower affinity. Tightly
bound ADP can be released from the enzyme upon binding of MgATP at
another site, probably one of the lower affinity sites
(10, 11, 12) . This intersite cooperativity is
considered to be involved in an alternating sites mechanism in
which two
(12) or three
(13) catalytic sites switch
properties with each other with respect to their affinity for
nucleotides.
is considered to result from asymmetric
interactions between the three
and three
subunits and the
smaller single copy subunits,
,
, and
. Since the
asymmetry remains intact following removal of the two smallest
subunits,
and
, the
subunit is sufficient to provide
the necessary asymmetric interaction(s). However, some recent studies
of the reconstituted
and
subunits of the F
of
the thermophylic bacterium PS3
(14, 15, 16) indicated that an
complex retained asymmetric features resembling those of the
normal enzyme but in the complete absence of small subunits.
complexes of several bacteria have been successfully
disassembled in vitro and reassembled from their individual
subunits
(17, 18, 19, 20) . In vitro assembly of an F
enzyme from a eukaryotic source,
however, has not yet been reported in spite of intensive efforts in
many laboratories. This goal remains a very important one for
effectively studying subunit interactions for these enzymes, especially
since the eukaryotic enzymes exhibit several properties, including
subunit composition and regulatory mechanisms, that are different from
their bacterial counterparts (for reviews, see Refs. 1, 21, and 22).
Development of an in vitro assembly system is also essential
for our studies of CF
that involve assembling genetically
engineered subunits into an active enzyme complex for probe-labeling
studies aimed at examining protein dynamics
(23, 24) .
and
subunits
from CF
(3, 4) , conditions have been
described
(25) for isolating the CF
subunit
and reconstituting it with
-deficient membranes of
Rhodospirrillum rubrum to form an active hybrid F
complex. In this paper, we report the isolation of the
subunit and an
complex from CF
. The two protein
preparations were readily reconstituted with each other to form a fully
functional core enzyme complex. Using this reconstitution system, we
have been able to demonstrate that an
complex is likely to be the
minimum structure required for asymmetric nucleotide binding and,
therefore, for cooperative catalysis by CF
. Formation of
the core complex is also prerequisite for proper binding of the
inhibitory
subunit and for binding of the allosteric
CF
-specific inhibitor tentoxin.
Materials
CFand CF
lacking the
and
subunits,
CF
(
), were prepared from fresh market
spinach as described previously
(2, 26) and stored as
ammonium sulfate precipitates. Prior to use, the proteins were desalted
on Sephadex G-50 centrifuge columns
(27) . The isolated
subunit was stored in the isolation buffer at 4 °C
(2, 3) .
70 °C. Urea and guanidine hydrochloride were
purchased from Fluka. Hydroxylapatite was purchased from Bio-Rad.
Preparation of an
1-10 mg
of CF Complex
(
) were desalted on Sephadex G-50
centrifuge columns equilibrated with 30 mM potassium phosphate
(pH 7.0). ATP and MgCl
were added to final concentrations
of 5.5 mM and 5 mM, respectively. LiCl was added
slowly from a 10 M stock solution to a final concentration of
2 M. The final protein concentration was kept below 1 mg/ml.
The solution was incubated on ice for 1.5 h and then applied to a 1.5
10-cm hydroxylapatite column equilibrated with a solution
containing 30 mM potassium phosphate (pH 7.0), 1 mM
MgATP, and 0.3 M LiCl at 4 °C. The column was washed with
the same buffer solution. A mixture of
and
subunits, and
usually a trace amount of contaminating
subunit, was washed
straight through the column. This fraction was collected and dialyzed
at 4 °C against at least 5 volumes of a solution containing 10%
(v/v) glycerol, 20 mM Hepes-NaOH (pH 7.0), 1 mM
MgATP, and 2 mM dithiothreitol. The
complex was
further purified by application to a diethylaminoethyl-cellulose column
equilibrated with a solution containing 20 mM Hepes-NaOH (pH
6.5), 1 mM ATP, and 1 mM EDTA. A small amount of free
subunit was washed from the column with the equilibration buffer
containing 0.12 M NaCl. The
mixture eluted when the
column was washed with the same buffer containing 0.17 M NaCl.
Any residual undissociated
remained tightly bound to
the column until the NaCl concentration was raised to 0.4 M.
The
mixture was dialyzed at 4 °C against at least 5
volumes of a solution containing 20% glycerol, 20 mM
Hepes-NaOH (pH 7.0), and 1 mM MgATP and stored at
70
°C for further use. More than 50% of the
and
subunits
of the CF
(
) were routinely recovered in
the
complex. Isolation of the
Subunit from
CF
(
)-CF
(
)
was desalted on a centrifuge column of Sephadex G-50 equilibrated with
a solution containing 15 mM sodium phosphate (pH 7.0). ATP,
MgCl
, and urea were added to final concentrations of 5.5
mM, 5 mM, and 4 M, respectively. The protein
concentration was kept at or slightly below 1 mg/ml. The solution was
stirred gently on ice for 1 h and then applied to a 1.5
7-cm
hydroxylapatite column equilibrated with a solution containing 15
mM sodium phosphate (pH 7.0), 1 mM ATP, and 0.5
mM MgCl
at 4 °C. The protein (
) was
washed through the column with 50 ml of buffer containing 4 M
urea, 15 mM sodium phosphate (pH 7.0), 1 mM ATP, and
0.5 mM MgCl
. The column was washed with 20 ml of
15 mM NaH
PO
(pH 7.0) followed by 30 ml
of 200 mM NaH
PO
(pH 7.0). The
subunit was eluted as a sharp peak by washing the column with a
solution containing 4 M urea and 300 mM sodium
phosphate (pH 7.0). The
subunit tended to aggregate when the
protein concentration at this step was too high, requiring dilution
with the elution buffer. The
fraction was dialyzed at 4 °C
against a buffer containing 20% glycerol, 50 mM NaHCO
(pH 9.5), 5 mM dithiothreitol, and 0.3 M LiCl.
The protein (
1 mg/ml) was stored indefinitely at
70 °C
without loss of reconstitutive activity.
Reconstitution of the
The purified and
Fractions
mixture was diluted to about
100 µg/ml with a solution containing 20% glycerol, 50 mM
Hepes-NaOH (pH 7.0), 1 mM MgCl
, 1 mM ATP,
and 2 mM dithiothreitol and kept on ice. The
subunit was
added slowly to the
and gently mixed. The mixture was left
to sit at room temperature (
22 °C) for at least 1 h before
assaying ATPase activity. In some experiments, any unreconstituted
subunits were separated from the reconstituted
by DEAE
chromatography as described in the preceding section.
Analysis of Bound Nucleotides
Release of bound
nucleotides upon denaturation and precipitation of the and
CF
(
) complexes was achieved essentially as
described elsewhere
(10) . 50 µl of 1.2 M
HClO
were added to each 100 µl of protein sample
containing between 100 and 200 µg of protein. Precipitated material
was removed by centrifugation in a Beckman Microfuge at full speed for
5 min. 25 µl of 1.3 M K
CO
were
added immediately to the supernatant to neutralize it. The
centrifugation was repeated, and the supernatant was subjected to HPLC
analysis on a Beckman System Gold gradient system equipped
with a Shimadzu CR601 integrator. Nucleotides were separated
isocratically on a Beckman 5-µm ODS column with an eluting solvent
containing 50 mM potassium phosphate and 5 mM
tetrabutylammonium hydroxide (pH 6.5) in 92.2% (v/v) distilled water,
7.8% (v/v) acetonitrile
(10) . The nucleotide absorbance was
monitored at 254 nm. Concentrations of nucleotides in extracts were
determined by comparing peak areas with those of standard nucleotides
of known concentration.
Other Procedures
ATPase activities were determined
by measuring phosphate release
(28) for 5-10 min at 37
°C. The assay mixture for calcium-dependent ATPase activity
contained 50 mM Tris-HCl (pH 8), 5 mM ATP, and 5
mM CaCl. That for magnesium-dependent ATPase
activity contained 40 mM Tricine-NaOH (pH 8), 4 mM
ATP, 2 mM MgCl
, and 50 mM
Na
SO
(29) . Protein concentrations were
determined by the method of Lowry et al. (30) or by
the method of Bradford
(31) .
Isolation and Properties of the
Treatment of
CF Subunit and
Subunit Complex
(
) with 2 M LiCl
followed by hydroxylapatite chromatography under the conditions
described under ``Experimental Procedures'' led to isolation
of two protein fractions, one containing the
subunit with an
almost equal concentration of contaminating
subunit, and another
containing a freely soluble and highly purified mixture of
and
subunits. Between 50 and 70% of the
and
subunits were
recovered from the starting material. A highly purified
subunit
was obtained using a similar procedure in which the LiCl was replaced
by 4 M urea. The
fraction obtained using this
alternative procedure was inactive in reconstitution. Therefore, the
LiCl
fraction and the urea
fraction were used for
reconstitution studies. Gel electrophoresis of these fractions is shown
in Fig. 1. Preliminary studies (not shown) indicated that when
the
fraction shown in Fig. 1was subjected to
analytical ultracentrifugation, it sedimented as a single species with
an apparent molecular weight of approximately 350,000 (the calculated
molecular weight of the
complex is
328,000). The
fraction was also shown to co-chromatograph
with CF
(
) on a 1
40-cm column of
Sephadex G-200, a considerable distance in front of standard bovine
serum albumin that was shown earlier
(33) to co-elute with free
subunit under the same conditions.
Figure 1:
SDS gel electrophoresis of isolated
and
subunits. Proteins were separated by
electrophoresis on 12% polyacrylamide gels and stained with Coomassie
Brilliant Blue G. Lane 1,
CF
(
) before dissociation; lane 2, purified
complex; lane 3,
isolated
subunit; lane 4,
complex reconstituted from the
and
fractions shown in
lanes 2 and 3 and purified by anion-exchange
chromatography as described under ``Experimental
Procedures.''
The high molecular weight
of the fraction suggested that it may exist as a hexameric
complex similar to that isolated
from the thermophilic bacterial F
(34) and to that
isolated from CF
following treatment of thylakoid membranes
with LiCl
(35) . However, when the protein was examined by
electron microscopy, we did not observe the usual hexameric structure,
which is routinely observed in preparations of CF
and
CF
(
)
(32) . This was true despite a
number of trials in which the protein concentration, the ionic
strength, and the nucleotide concentration of the suspension medium
were varied. A likely explanation for the lack of structure is that the
complex is less stable than the
complex and
fell apart upon dehydration and/or binding to the carbon grid when
preparing the protein for electron microscopy. That the
mixture is less stable than CF
or
CF
(
) was evident when the protein was
analyzed by HPLC as shown in Fig. 2. In the experiment shown in
Fig. 2B, the
mixture was chromatographed in
the presence of Mg
and ATP, both of which are
required for reconstitution with the
subunit (see the following
section). Under these conditions most of the protein migrated with an
apparent molecular weight expected for a hexameric
complex. A small fraction of the
protein migrated as a species with a molecular weight of approximately
50,000, which was presumed to be monomeric
and
subunits.
When the
mixture was frozen and thawed in the absence of
nucleotides and in the presence of EDTA, only the lower molecular
weight monomeric form was present (Fig. 2 C). We did not
observe significant amounts of material migrating between these two
forms, which would have suggested the presence of stable
heterodimers.
Figure 2:
Gel filtration chromatography of the
complex. Standard proteins and the
complex were
chromatographed on a Phenomenex Biosep-SEC-S-2000, 600 mm
7.8-mm column equilibrated with 50 mM sodium phosphate (pH 7)
using a Spectra Physics SP8700 HPLC system. Proteins were separated
isocratically in the column equilibration buffer and detected by their
absorbance at 280 nm. A, a mixture of molecular mass markers
containing bovine thioglobulin (679 kDa, peak 1),
bovine
globulin (158 kDa, peak 2), chicken
ovalbumin (44 kDa, peak 3), equine myoglobin (17 kDa,
peak 4), and hydroxycobalamin (1.4 kDa, peak 5). B, freshly isolated
complex.
C, the
complex in B was desalted into 20
mM sodium phosphate buffer (pH 7) containing 1 mM
EDTA to remove unbound metal and nucleotide, frozen at
20
°C, and thawed prior to HPLC.
The complex exhibited a low rate of
magnesium- or calcium-dependent ATP hydrolysis (). The
activity persisted even after the
was recycled through the
isolation procedure to remove any residual traces of the
subunit
and after further purification by anion-exchange chromatography as
described under ``Experimental Procedures.'' In addition, the
activity was totally insensitive to the inhibitor tentoxin and to added
subunit (results not shown) under conditions where both strongly
inhibit the catalytic activity of CF
(
).
These observations precluded the possibility that the residual activity
of the
complex resulted from the presence of a small amount
of contaminating CF
(
).
subunit aggregated upon removal of the urea, which was used
to elute it from the hydroxylapatite column during isolation. In order
to keep
soluble, it was necessary to slowly exchange the urea for
a buffer containing a relatively low concentration of chaotropic ions
(0.3 M LiCl) at high pH (9.5). Under these conditions,
could be concentrated to as much as 5 mg/ml without apparent
aggregation or loss of reconstitutive activity. The cleanest
preparations of the
subunit were completely devoid of any
catalytic activity yet were fully functional in reconstitution.
Reconstitution of
By
mixing the and
Subunits
and
fractions, we were able to recover more
than 80% of the catalytic activity of the starting material
(). The reconstitution occurred equally well regardless of
whether the
subunits were in the hexameric or monomeric
form. Purification of the reconstituted
complex by
anion-exchange chromatography resulted in an enzyme with essentially
identical properties to CF
(
)
(). The reconstitution was complete within 2-3 h of
mixing (Fig. 3) and was not sensitive to variations in pH between
6 and 8 (). However, if the pH of the reconstitution
mixture was kept at pH 9 or above, such as the conditions used to store
the
subunit, almost no activity was recovered ().
Figure 3:
Time-dependence for reconstitution of
and
subunits. The isolated
complex and
subunit were reconstituted and assayed under the conditions
described under ``Experimental Procedures'' except that the
time allowed for reconstitution was varied. Magnesium-dependent ATPase
activities were determined at the times
indicated.
The reconstituted core enzyme complex was fully sensitive to
inhibition by tentoxin and was strongly inhibited by the subunit
( Fig. 4and ). It was observed that exposure of the
subunit to urea-containing solutions for several hours or
overnight resulted in a significant loss of sensitivity to inhibition
by the
subunit, without a significant loss of
-dependent
reconstitution of ATPase activity. This suggested that part of the
binding site for
is present on the
subunit and that this
site may have been chemically modified ( e.g. carbamylation of
one or more amino groups) during prolonged incubation of
in the
presence of urea. This explained the higher residual activity observed
for the reconstituted
complex than for
CF
(
) following addition of normally
saturating concentrations of the
subunit (). To avoid
such modification, the
subunit isolation procedure was performed
as quickly as possible, and more recently (results not shown) it was
found that 2 M guanidine hydrochloride can effectively
substitute for urea in the isolation procedure, eliminating the
time-dependent loss of
binding.
Figure 4:
Sensitivity of the reconstituted
complex to tentoxin. The
and
subunits
were reconstituted as described under ``Experimental
Procedures.'' 10-µg samples of reconstituted
complex (
) or CF
(
) (
) were
preincubated for 5 min at 22 °C with the indicated amounts of
tentoxin in 0.5 ml of a solution containing 50 mM Tricine-NaOH
and 1 mM ATP. At the end of the preincubation, 0.5 ml of
double strength calcium-ATPase assay mixture, prewarmed to 37 °C,
was added to initiate the assay. The control rate was 17.5
µmol/min/mg of protein.
MgATP was present throughout
the preparation and storage of the complex, so in order to
test the effects of magnesium and ATP on reconstitution, the
complex was first desalted by column centrifugation. Reconstitution was
found to be almost completely dependent on the presence of both
magnesium and ATP (). The small residual activity observed
in the absence of added nucleotide probably resulted from a small
amount of ATP or ADP remaining bound to the
complex during
the desalting procedure (see I). ADP could substitute
quite well for ATP, whereas GTP was a poor substitute. AMP and CTP were
ineffective.
The
The Complex Lacks Tight ADP Binding
Sites
protein complex was routinely isolated and
stored in the presence of both magnesium and ATP. To examine the
complex for the presence of tightly bound nucleotides, free and loosely
bound nucleotides were first removed by passing the protein
sequentially through two centrifuge columns. The columns were warmed to
room temperature prior to addition of the protein to avoid dissociation
of the
into monomers. Nucleotides that remained bound to the
protein following chromatography on two consecutive Sephadex G-50
centrifuge columns were identified using a HPLC procedure similar to
that described by Shapiro et al. (10) . The results are
shown in I. The
complex retained approximately
1 mol of MgATP/mol of
under these
conditions. When EDTA was included in the buffer used to equilibrate
the centrifuge columns, the residual MgATP was completely removed. This
is in contrast to CF
(
), which contains
nearly four tightly bound
nucleotides/
, two of which remain
tightly bound even after continuous exposure to EDTA during isolation
(I). The nucleotide binding properties of
CF
(
) were essentially identical to those
described previously for CF
(10, 26) .
High Affinity Binding of Tentoxin and
The isolated Subunit
Requires the
Subunit
subunit alone was
shown
(26) not to bind tentoxin with sufficient strength to
compete with CF
(
) for binding of the
toxin. This result suggested that CF
subunits other than,
or in addition to, the
subunit are required for tentoxin binding.
We undertook similar competition experiments to test for a binding
interaction between tentoxin and the
subunit or the
subunit complex. As shown in Fig. 5, neither the
complex nor the
subunit, when preincubated with tentoxin, could
reduce the effective concentration of the toxin, even at molar
concentrations more than 10-fold higher than the concentration of
latent CF
, which was needed to completely block tentoxin
inhibition. As an additional control, some of the
CF
(
) was pretreated with DCCD (see the
legend to Fig. 6) before it was reconstituted with the
subunit. The resulting
complex was devoid of catalytic
activity, but it was still capable of binding tentoxin and thus
effectively blocking tentoxin inhibition of the active
CF
(
) (results not shown).
Figure 5:
Competition between
CF(
), the
complex, and the
isolated
subunit for tentoxin binding. The effectiveness of the
complex (
), the free
subunit (
), or latent
CF
(
) to compete with
CF
(
) for tentoxin binding was determined
by preincubating fixed concentrations of tentoxin with varying amounts
of the competing protein. Each incubation mixture contained, in a total
volume of 0.5 ml, 50 mM Tricine-NaOH, 1 mM ATP, 0.6
µM tentoxin (except for the control sample), 10 µg of
CF
(
) plus the indicated amounts of
competing protein. After 5 min of incubation at 22 °C, 0.5 ml of
double-strength CaATPase assay mixture, preincubated at 37 °C, was
added to begin the ATPase reaction. Controls samples lacking the
CF
(
) were performed at each concentration
of the added competing proteins (
,
, and latent
CF
) in order to adjust for the low catalytic activities
(all <1 µmol/min/mg of protein) of these protein
preparations.
Figure 6:
Competition between
CF(
), the
complex, and the free
subunit for
binding. The effectiveness of the
complex (
), the free
subunit (
), or DCCD-inhibited
CF
(
) (
) to compete with
CF
(
) for
subunit binding, was
determined by preincubating a fixed concentration of the
subunit
with varying amounts of the competing protein. Each incubation mixture
contained, in a total volume of 0.5 ml, 50 mM Tricine-NaOH, 1
mM ATP, 10 µg of CF
(
),
varying amounts of the competing protein, and 1.9 µg of purified
subunit (added last to all but the control sample), which was
sufficient to inhibit the activity of the
CF
(
) by 51%. After 5 min of incubation at
22 °C, 0.5 ml of double strength CaATPase assay mixture,
preincubated at 37 °C, was added to begin the ATPase reaction. The
DCCD-inhibited CF
(
) sample was prepared by
treating CF
(
) with 200 µM
DCCD for 1 h in buffer containing 50 mM Hepes-NaOH (pH. 7.0)
(47). The CaATPase activity of the DCCD-treated protein was <0.1
µmol/min/mg of protein.
Competition
experiments were also used to examine the structural requirement for
binding. This was done by preincubating the
subunit with
the
subunit, or with the
complex, prior to addition of
CF
(
). If the
subunit binds to either
of these fractions, then their presence should reduce the effective
concentration of the
subunit, thus reducing the extent of
inhibition of the ATPase activity of CF
(
).
The results (Fig. 6) indicate that at high concentrations the
subunit did somewhat reduce the effectiveness of the
subunit, suggesting a weak binding interaction between these two
subunits. The
complex, however, even at a very high molar
excess, did not reduce the effective concentration of the
subunit, suggesting the absence of a strong binding interaction between
the
subunit and the
complex. In contrast, inactivated
(DCCD-treated) CF
(
) bound the
subunit strongly, effectively competing for free
subunit
(Fig. 6).
subunit has been isolated in reconstitutively
active form from the F
enzymes of several different
bacterial species including Escherichia coli (17) , the
thermophilic bacterium PS3
(18) , and R. rubrum (19) . In each case, the
subunit could be
reconstituted with the other F
or F
-F
subunits to restore, to varying extents, the catalytic activity
to the
-less enzymes. Repeating these experiments with eukaryotic
F
enzymes has proven much more difficult, due largely to
the aggregative properties of the
and
subunits, especially
under the conditions that were effective in reconstitution of the
bacterial subunits. In this report, we have described conditions for
isolating the CF
subunit in a highly purified form,
which can be stored indefinitely at a high concentration without
aggregation and which retains its ability to reconstitute with the
and
subunits to form an active core enzyme complex. Large
amounts of reconstituted, fully active
complex could be
obtained using a remarkably simple and quick protocol.
and
subunits and which
migrated as an
hexamer during
analytical ultracentrifugation and gel filtration. The complex is very
similar to that described by Avital and Gromet-Elhanan
(30) ,
which was one of several CF
subcomplexes isolated following
treatment of thylakoid membranes with 2 M LiCl. Their
complex chromatographed as two species, one with a high
molecular weight, consistent with an
structure, and a lower molecular weight species, which migrated
during gel filtration as an
heterodimer. In our case, the higher molecular weight
form predominates unless the protein
is frozen and thawed in the absence of metal ions and nucleotides, in
which case it reversibly disassembles into
and
monomers.
Both the hexameric and monomeric forms could be reconstituted with the
subunit to form an active core enzyme complex.
complex has been assembled from the F
subunits of the
thermophilic bacterium PS3. The structure of the complex was confirmed
by electron microscopy as an
hexamer
(34) , which dissociated into
heterodimers in the presence of micromolar concentrations of
nucleotides
(14, 34) . Removal of the nucleotides
resulted in reassembly of the hexameric species
(36) . A stable
complex has also been isolated from the photosynthetic
bacterium R. rubrum following treatment of chromatophore
membranes with 2 M LiCl
(37) . The chromatographic
properties of the complex suggested that it was an
heterodimer. All of the
preparations described have been reported to exhibit low levels of
ATPase activity ranging between about 30 nmol/min/mg of protein for the
heterodimers to about 300 nmol/min/mg of protein for the hexamers. With
the exception of PS3 F
(34) , these catalytic rates
represent only a small percentage of the maximum catalytic rates
obtained with the parent F
enzymes. For the CF
complex, the activity reached a maximum of about 0.5%
of the fully activated CF
complex.
enzymes have been shown to contain six nucleotide binding sites.
The recently published crystal structure of the beef heart
mitochondrial F
at 2.8 Å resolution revealed that the
six sites are located at the six interfaces formed between the
alternating
and
subunits of the hexameric F
complex
(9) . The
subunits contribute the bulk of
the structure of three putative catalytic sites, the
subunit
contributing a small section of the adenine binding portion of the
catalytic pocket. This situation is reversed for the three putative
noncatalytic sites, which reside mainly on the
subunits with a
small contribution from each of the three
subunits. Since the
and
subunits are well conserved among different organisms
(38) it should be safe to assume that the arrangement of the six
nucleotide binding sites is essentially identical in all F
enzymes. The fact that both subunits contribute to nucleotide
binding at each site would explain why the
and
subunits
isolated from various F
enzymes are unable to catalyze ATP
hydrolysis on their own even though they are able to bind nucleotides
(39, 40, 41, 42, 43) . It also
explains the observation that complexes containing both
and
subunits are catalytically active, albeit at very low levels.
subunit is
required, in addition to the
and
subunits, for the high
rates of ATP hydrolysis that are normally associated with activated
CF
. Similar results have been obtained for several
bacterial systems in which it has been possible to reconstitute the
active ATPase complex from isolated subunits
(17, 18, 19, 20) . High catalytic rates
are usually associated with cooperative interactions among different
nucleotide binding sites that involve either two
(12) or three
(for review, see Ref. 13) catalytic sites alternately switching their
conformational states during the course of a single catalytic cycle.
The occurrence of heterogeneous nucleotide binding sites requires
structural asymmetry, which is generally assumed to result from binding
of the smaller, single-copy subunits to the
hexamer. Since the core
complex of CF
is
fully active
(26) , the necessary asymmetry must be provided by
binding of the
subunit to the
hexamer. Indeed, the crystal structure of the beef heart F
revealed a direct interaction between the
subunit and one
of the three
subunits, suggesting that the
subunit forces
that
subunit into an alternative conformation, altering its
nucleotide binding properties, and thereby introducing structural and
functional heterogeneity among the three potential catalytic sites
(9) .
or CF
(
) can, at any one time, be
occupied by nucleotides (2 ADP and 2 ATP) that remain firmly bound
following extensive desalting of the enzyme
(12) . In contrast,
the isolated
(33, 40) and
(
)
subunits bind nucleotides with a lower affinity
(dissociation constants in the range of 0.5-2 µM),
such that the nucleotides dissociate completely from the proteins
during gel filtration. Our results have shown that the
hexamer lacks the very tight ADP
binding sites that are characteristic of the core
complex (I).
Therefore, the
complex lacks the
asymmetry necessary for tight ADP binding and consequently must lack
the intersite catalytic cooperativity in which tightly bound ADP is
considered to be an essential intermediate
(13, 11) .
The fact that some MgATP (<1 mol/mol of
) remained bound to the complex after
desalting in the absence of EDTA may reflect a slightly enhanced
affinity of
pairs for MgATP over that of the individual
and
subunits.
complex probably results from the presence of three
structurally and functionally equivalent catalytic
sites/
hexamer that are capable of
slow catalytic turnover. Each site presumably operates independently of
the other two, since the
heterodimers from CF
(35) exhibit catalytic
rates of the same order of magnitude, indicating that formation of the
complex does not result in a
significant cooperative rate enhancement. Thus, the observed activity
represents single-site catalysis. This is in contrast to
``unisite'' catalysis (for review, see Refs. 13 and 21),
which involves one site on the F
complex that exhibits
single site catalytic turnover but binds nucleotides with a very high
affinity ( K
<10
M). We have shown that formation of such tight sites on
CF
requires the presence of the
subunit. These
results are at odds with studies of the PS3 F
hexamer, which suggest that
the activities of the catalytic sites on this complex are
interdependent. Inhibition of a single site by chemical modification of
a tyrosine residue on one of the three
subunits, blocks the
function of the entire complex
(16) . Interestingly, the
isolated PS3 F
further differs from other F
enzymes in that it is devoid of tightly bound endogenous
nucleotides
(44) .
subunit complexes is atypical of CF
in that it is insensitive to the potent inhibitors tentoxin and
azide
(35) and also insensitive to the
subunit that is a
naturally occurring ATPase inhibitor of CF
(3) .
Avital and Gromet-Elhanan
(35) reported that, although the
activity of the
complex was not inhibited by tentoxin,
tentoxin was still able to bind to the complex as it co-eluted with the
complex during chromatography on a Sephadex centrifuge
column. Our results have shown that the chloroplast
complex
is unable to effectively compete with the core
complex for tentoxin binding
and, therefore, that the
complex has a significantly lower
(at least 2 orders of magnitude) affinity for tentoxin than the core
complex, which is known to bind tentoxin with an
apparent dissociation constant of between 5 and 100
10
M(45, 26) . We had shown
earlier
(25) that reconstitution of spinach chloroplast
subunit with
-deficient chromatophores of R. rubrum conferred tentoxin sensitivity upon the normally
tentoxin-insensitive R. rubrum F
-F
complex. This result indicated that the
subunit may contain
all or part of the tentoxin binding domain. Studies by Avni et al. (46) confirmed this result and identified a region of the
N terminus of the
subunit that is important for tentoxin binding.
More recently, however, we showed that the isolated
subunit is
unable to compete with CF
for tentoxin binding, suggesting
that the
subunit provides only part of the tentoxin binding site
(26) . The fact that the
complex binds tentoxin with
a much lower affinity than the
complex indicates that
the same structure that is required for cooperative interactions among
nucleotide binding sites must also be required for normal tight binding
of the inhibitor.
subunit binds to CF
with an
apparent dissociation constant of <10
M(2) . Like tentoxin binding, normal high affinity binding of the
subunit also required the minimum
structure, and
could not
bind tightly to the
subunit or the
complex alone.
Tentoxin
(26) and the
subunit
(3) are both
allosteric effectors that inhibit the cooperative interactions between
nucleotide binding sites on CF
. The fact that neither
inhibitor binds with high affinity to the
complex nor
effects its low catalytic activity is in accord with the lack of
structural asymmetry in the
complex. Thus the normal high
affinity binding of both inhibitors requires an asymmetric
conformational state of CF
that results from an interaction
between the
subunit and the
and
subunits.
subunit with an
subunit mixture of the chloroplast ATP synthase. The reconstitution
procedure is unique in that it is simple and rapid and therefore will
be a valuable tool for studying interactions among the different
CF
subunits. It is noteworthy that, while this manuscript
was under review, Chen and Jagendorf
(48) published a method
for reconstituting cloned, over-expressed CF
subunits with
the help of molecular chaperonins. Together, these techniques have
opened the way for studies, which are currently underway in our
laboratory, that involve incorporating genetically engineered
,
and
subunits into an active core eukaryotic F
enzyme complex.
Table:
Reconstitution of an active core enzyme complex
from isolated and
subunits of CF
Table:
Conditions
for reconstitution of the complex with the
subunit
Table:
Analysis of tightly bound nucleotides
,
chloroplast coupling factor 1; CF
(
),
CF
lacking the
and
subunits; Tricine,
N-[2-hydroxy-1-bis(hydroxymethyl)ethyl]glycine;
HPLC, high performance liquid chromatography; DCCD,
N, N`-dicyclohexylcarbodiimide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.