From the Department of Biological Chemistry, Weizmann
Institute of Science, Rehovot 76100, Israel and the
§ Department of Molecular Biosciences, the University of
Kansas, Lawrence, Kansas 66045
Received for publication, August 18, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Refolding together the expressed The F0F1-ATP synthases catalyze the
synthesis of ATP from ADP and Pi at the expense of a
transmembrane electrochemical proton gradient generated by the
respiratory or photosynthetic electron transport chains. Its
membrane-bound F0 sector functions as the proton pathway
and has, in bacterial and photosynthetic cells, a subunit composition
of a1b2c9-12 and
a1b1b'1c9-12, respectively. The catalytic F1 sector, which functions as a
soluble ATPase, has in all cells a subunit composition of
A high resolution x-ray structure of the bovine heart mitochondrial
MF1 (12) demonstrated the alternating arrangement of the
Full elucidation of the detailed mechanism of action of the
F0F1-ATP synthase will depend on the
identification of the specific domains that participate in its
proton-coupled ATP synthesis and hydrolysis as well as in the
regulation of these reversible activities and their possible
correlation with the In this investigation, we have used our earlier developed procedure for
refolding and assembly of the Materials--
The recombinant Assembly and Isolation of
Assembly of Assembly and Isolation of the Hybrid WT and Mutant
Assays of ATPase Activities--
The activities of
RrF1 and both hybrid
Other Procedures--
SDS-PAGE was carried out on the Novex
Pre-Cast 10-20% Tris glycine gradient gels. The protein bands were
visualized by staining with Coomassie Brilliant Blue R-250. Protein
concentrations were determined by the Bradford method (32) or according
to Lowry et al. (33), using bovine serum albumin as a standard.
Stepwise Assembly of
RrF1-
The closed
A search for compounds or conditions that can stabilize the
noncatalytic RrF1 Assembly of Hybrid WT and Mutant
F1-
The hybrid
Modulation of the Hybrid WT and Mutant
F1- The Catalytic Properties of the Isolated RrF1 Dimers,
Hexamers, and Both Hybrid WT and Mutant
The properties of the CaATPase activities of the RrF1 and
both hybrid WT and mutant complexes were very different from their MgATPase activities (compare Figs. 5 and
6). They were dependent on the presence
of CaCl2, reaching saturation at a Ca/ATP ratio of 1.0, but
showed no inhibition even at a ratio of 5.0. They were thus rather
similar to the MgATPase activities of the dimers and hexamers (see Fig.
5, inset).
A detailed comparison of the Ca2+- and
Mg2+-dependent ATPase activities of
RrF1 and all assembled complexes (Table
I) demonstrated a number of additional
most interesting differences as follows:
1) Between the dimers and hexamers. The dimers have practically no
CaATPase activity, although their MgATPase is similar in its rates and
properties to that of the hexamers (Fig. 5 and Table I). These results
illuminate clear differences in the structure of catalytic
nucleotide-binding sites occupied by Ca2+ versus
Mg2+, since CaATP can bind to these sites and enable the
appearance of CaATPase activity only in the closed hexamer. On the
other hand, the identical and very low dimer and hexamer MgATPase
activities, which in the dimers cannot have any catalytic
cooperativity, suggest its absence also in the hexamers. Indeed, both
dimer and hexamer MgATPases do not respond to the usual MgATPase
effectors of CF1 and RrF1. They are not
stimulated by sulfite nor inhibited by azide (Table I).
2) Between both dimers and hexamers and the RrF1
(±AlFx) as well as the hybrid WT complex. The
3) Between the RrF1 (+AlFx) Ca2+-
and Mg2+-dependent ATPase activities. Surprisingly,
although AlFx does not affect the RrF1-MgATPase
(Fig. 5 and Table I), it inhibits by 70% the 10-fold higher CaATPase
(Table I). In light of these results the similar Mg- and CaATPase
activities in the
4) Between the hybrid WT and mutant complexes. Both RrF1
and the hybrid WT showed a 3-10-fold higher CaATPase than MgATPase activities. On the other hand, the hybrid mutant showed a much higher
MgATPase activity than either RrF1 or the hybrid WT complex but a lower CaATPase (Figs. 5 and 6). Its CaATPase, as all other CaATPase activities, was not regulated and did not respond to any
tested MgATPase effector. But the very high MgATPase activity of this
mutant was also much less responsive to these effectors (Table I).
In this study large amounts of highly active hybrid WT and mutant
photosynthetic F1- This hybrid WT
The hybrid WT
An additional, most important difference between Ca2+- and
Mg2+- or Mn2+-dependent ATPase
activities was recorded in the membrane-bound RrF0F1 where the CaATPase was not coupled to
proton translocation and Ca2+ did not enable any ATP
synthesis (23). So in the hybrid WT Another interesting target for such studies is the hybrid mutant
In the membrane-bound RrF1 this
and
subunits of the Rhodospirillum rubrum
F1(RF1)-ATPase led to assembly of only
1
1 dimers, showing a stable low MgATPase
activity. When incubated in the presence of AlCl3, NaF and
either MgAD(T)P or CaAD(T)P, all dimers associated into closed
3
3 hexamers, which also gained a low
CaATPase activity. Both hexamer ATPase activities exhibited identical
rates and properties to the open dimer MgATPase. These results indicate
that: a) the hexamer, as the dimer, has no catalytic cooperativity; b)
aluminium fluoride does not inhibit their MgATPase activity; and c) it
does enable the assembly of
RrF1-
3
3 hexamers by
stabilizing their noncatalytic
/
interfaces. Refolding of the
RrF1-
and
subunits together with the spinach
chloroplast F1 (CF1)-
enabled a simple
one-step assembly of two different hybrid
RrF1-
3
3/CF1
complexes, containing either wild type RrF1-
or the
catalytic site mutant RrF1
-T159S. They exhibited over 100-fold higher CaATPase and MgATPase activities than the stabilized hexamers and showed very different catalytic properties. The hybrid wild type MgATPase activity was, as that of RrF1 and
CF1 and unlike its higher CaATPase activity, regulated by
excess free Mg2+ ions, stimulated by sulfite, and inhibited
by azide. The hybrid mutant had on the other hand a low CaATPase but an
exceptionally high MgATPase activity, which was much less sensitive to
the specific MgATPase effectors. All these very different ATPase
activities were regulated by thiol modulation of the hybrid unique
CF1-
disulfide bond. These hybrid complexes can provide
information on the as yet unknown factors that couple ATP binding and
hydrolysis to both thiol modulation and rotational motion of their
CF1-
subunit.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
1
1
1
and six nucleotide-binding sites located on the
and
subunits. Its three catalytic
sites show high negative cooperativity in substrate binding and strong positive cooperativity in catalysis (1-5). The minimal F1 subcomplex, which resembles the
whole F1-ATPase in its catalytic properties, is the
F1-
3
3
-ATPase. Such
subcomplexes were reconstituted from either native (6, 7) or
recombinant (8, 9) individual subunits of the respiratory
TF1-1 and
EcF1-ATPases. Similar highly active pure photosynthetic
subcomplexes have been assembled up to now only by incubating an
isolated native CF1(
) complex with a native (10) or
recombinant (11)
C subunit.
and
subunits in a closed hexamer, with the resolved N- and
C-terminal
-helices of the
subunit embedded in its central cavity and all six nucleotide-binding sites residing at alternating
/
interfaces. The three catalytic sites, located mainly on the
subunits, appeared in three different conformational states that,
in association with the unique resolved part of the
subunit, imposed an asymmetric structure on the
3
3
hexamer (12). This MF1-
3
3
structure is compatible with the binding change mechanism (3, 13),
which proposed that ATP synthesis involves transitions between
different but interacting catalytic sites, via
energy-dependent affinity changes in substrate binding and
product release. Such transitions were first suggested to occur via
movement or rotation of a cluster of the catalytic
3
3 subunits around a core of the single
copy
or
subunits (14). The reversible proton-translocating ATP
hydrolysis was later proposed to generate rotation of the F1-
subunit which, when transmitted to F0,
could result in pumping of protons back across the membrane (15),
possibly via coupled rotation of the F1-
subunit with
the F0-c subunits (3, 16). MgATPase-induced rotation of
within immobilized
3
3 hexamers was
observed in genetically engineered respiratory TF1- and
EcF1-
3
3
complexes (17,
18). But its further coupling with F0-c rotation, which has
been tested in some recent reports, did not yield clear results
(19-21). So there is at present no direct correlation between
rotation and proton-coupled ATP synthesis and hydrolysis.
or
-c rotation. Tight regulation of ATP
hydrolysis is especially important in photosynthetic cells, where it
prevents the depletion of essential cellular ATP pools in the dark (1,
4, 5). Plant chloroplasts and bacterial chromatophores have a number of
such regulatory pathways, which operate in their membrane-bound
F0F1- as well as the solubilized F1-ATPases. Both chloroplasts (22) and chromatophores (23) show a high sensitivity to inhibition by excess free Mg2+
ions, which results in a drastic decrease of their MgATPase activities at Mg/ATP ratios above 0.5. A unique chloroplast regulatory system, termed thiol modulation, involves reduction-oxidation of a disulfide bond (24) formed between Cys199 and Cys205 in a
region of its
C subunit that does not appear in any
other F1-
subunits (25). But there are as yet no
assembled CF1-
3
3
or
similar photosynthetic complexes that can be engineered for studies
aimed at elucidating the molecular mechanism of their regulatory
systems and the possible ATPase-induced rotation of their
subunit.
R and
R
subunits of the photosynthetic bacterium Rhodospirillum
rubrum into
R1
R1 dimers (26)
to follow their further association into
R3
R3 hexamers,
and with the recombinant chloroplast
C (11), into hybrid
R3
R3
C-ATPases.
Two types of such highly active hybrids were assembled, containing WT
R or the catalytic site mutant
R-T159S
(27), and both retained the specific
C redox regulation.
These hybrid complexes provide the first photosynthetic F1
assemblies that can be genetically engineered for probing rotational catalysis. Both hybrids show, besides a very high MgATPase also a
CaATPase activity, that in the hybrid WT
R3
R3
C
is much higher than the MgATPase activity but does not respond to any
tested MgATPase effector. This hybrid WT complex thus supplies an
additional interesting system for assaying rotation, which has been
induced up to now (17-21) only by MgATP hydrolysis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R (26, 28), WT
R (29), and the mutant
R-T159S (27)
subunits were expressed in insoluble inclusion bodies and solubilized
by urea, as described by Du and Gromet-Elhanan (26). Recombinant
C was expressed and refolded as described by Sokolov
et al. (11). RrF1 was prepared from R. rubrum chromatophores according to Weiss et al. (30).
All other reagents were of the highest purity available.
R1
R1
Dimers--
The dimers were assembled by refolding the
urea-solubilized
R and
R together
according to the procedure developed for their optimal refolding into
functional monomers (26). The refolded mixture was concentrated to
about 1 mg/ml by Centriprep-10 (Amicon), precipitated with 60%
saturated (NH4)2SO4, and
resuspended in TGN buffer containing 50 mM Tricine-NaOH, pH
8.0, 20% glycerol, and 50 mM NaCl. The remaining insoluble
aggregates were removed by centrifugation, and the refolded mixture was
loaded on the size-exclusion HPLC Superdex-200 column (Amersham
Pharmacia Biotech) and eluted with 100 mM NaPi,
pH 7.0, containing 10% glycerol at a flow rate of 0.5 ml/min. The
pooled
1
1 dimer peak was concentrated,
transferred to TGN buffer by elution-centrifugation through Sephadex
G-50 columns, and stored at
80 °C.
R3
R3 Hexamers--
The isolated
R1
R1 dimers could
be fully converted into the
R3
R3 hexamers
only when incubated, at >1 mg of protein/ml for 1 h at 22 °C,
in TGN buffer containing also 10 mM NaF, and 0.5 mM AlCl3, in the presence of either 1 mM of CaCl2 or MgCl2 and 1 mM ATP or ADP (see Fig. 1D). Their activities
could therefore be assayed directly by diluting samples assembled in the presence of each cation and ADP into the same cation-ATPase assay mixtures.
R3
R3
C
Complexes--
The hybrid WT
R3
R3
C
was assembled by two procedures as follows: 1) incubation of the
isolated
R1
R1
dimers with refolded
C for 1 h at 22 °C in TGN
buffer in the presence of 1 mM MgCl2 or
CaCl2 and 1 mM ADP which in this mixture, as in
the hexamer assembly mixture, is as effective as ATP. Each incubated
sample could therefore be assayed directly for its ATPase activity as well as size-exclusion HPLC. 2) Refolding the urea-solubilized recombinant
R and
R each at 50 µg/ml,
together with urea-solubilized
C at 20 µg/ml,
according to the procedure developed above for assembly of
R1
R1 dimers. The
assembled hybrid complex was isolated by size-exclusion HPLC as
described for the dimers, except that the hybrids were eluted with
buffer containing 50 mM Tricine-NaOH, pH 8.0, 50 mM NaCl, and 10% glycerol. The peak containing the hybrid
complex was pooled, concentrated, exchanged into TGN buffer as
described for the dimers, and stored at
80 °C. The hybrid mutant
R3(
R-T159S)3
C
complex was assembled and isolated as described for the hybrid WT.
R3
R3
C
WT and mutant complexes were measured with 4-20 µg of protein for 5 min at 35 °C in 0.5 ml of an assay mixture containing 50 mM Tricine-NaOH, pH 8.0, 50 mM NaCl, 4 mM ATP, and 2 mM of either MgCl2 or
MnCl2 or 4 mM CaCl2. The activities
of the isolated
R1
R1 dimers were measured with
30 µg of protein for 30 min under the conditions described above. To
compare the ATPase activities of the
R3
R3 hexamers
with those of RrF1, the hexamers were first assembled by
incubating the dimers as described above, and RrF1 was
incubated under identical conditions. The ATPase activities of this
RrF1 and the freshly assembled
R3
R3 hexamers
were measured by diluting each incubation mixture into the relevant
assay mixtures, which also contained 10 mM NaF and 0.5 mM AlCl3. All ATPase activity assays were
started by adding the protein complexes and stopped by adding 50 µl
of 2 M trichloroacetic acid, and the released
Pi was measured as described by Taussky and Shorr (31). The
effect of reduction or oxidation on the ATPase activities of
RrF1 and the hybrid complexes was tested by their
preincubation for 1 h at 35 °C in TGN buffer containing either
10 mM DTT or 100 µM CuCl2,
followed by dilution into the relevant assay mixtures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R1
R1
Dimers and
R3
R3
Hexamers--
Active dimers, but no hexamers, have been assembled by
incubation of the isolated
R and
R
monomers for 5 min at 35 °C in the presence of MgATP (26). These
dimers showed a maximal MgATPase rate of 0.14 units/mg of protein,
which remained linear for at least 1 h at 35 °C. In search for
conditions that might enable the assembly of
R3
R3 hexamers,
the urea-solubilized
R and
R subunits
(26) were refolded together as described under "Experimental Procedures." They did indeed assemble directly, but again only into
R1
R1 dimers with
no indication for the appearance of any larger complexes (Fig.
1A). This simple one-step
refolding/assembly procedure enabled the isolation of large amounts of
the pooled concentrated dimer peak, which remained very stable in TGN
buffer at protein concentrations above 1 mg/ml, even when incubated for 1 h at 22 °C (Fig. 1B). However, when diluted to the
10-20-fold lower protein concentrations used for ATPase activity
assays, they remained stable only in presence of either MgADP or MgATP or even CaAD(T)P (not shown).
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Fig. 1.
Refolding of the
RrF1- R and
R into
R1
R1 dimers and
their further association into stable
R3
R3 hexamers in
presence of aluminum fluoride. A, the urea-solubilized
R and
R were folded together, and 15 µg
of the concentrated product were loaded on a Superdex-200 column and
eluted as described under "Experimental Procedures";
B-D, 15 µg of the pooled
R1
R1 dimer peak,
concentrated to 1.5 mg of protein/ml as described under "Experimental
Procedures," were incubated in TGN buffer for 1 h at 22 °C
with the following additions: B, none; C, 10 mM NaF and 0.5 mM AlCl3;
D, as C plus 1 mM MgAD(T)P or
CaAD(T)P; E and F were incubated as in
D, diluted by 10-fold into TGN buffer, and further incubated
for 1 h at 22 °C without (E) or with (F)
10 mM NaF, 0.5 mM AlCl3, and 1 mM MgAD(T)P or CaAD(T)P. All incubated samples were loaded
on the column and eluted as described for A.
3
3 hexamer, resolved in the
x-ray crystallographic structure of bovine mitochondrial
MF1 (12), has all six F1 nucleotide-binding
sites arranged at alternating catalytic and noncatalytic
/
interfaces. The isolated
R1
R1 dimers could
therefore have either one of these interfaces. But the fact that their
MgATPase activity (26) is similar to that of
CF1-
3
3 (10, 34) indicates
that these dimers contain the catalytic nucleotide-binding site at
their
/
interface. Their inability to associate into an
R3
R3 hexamer
therefore seems to reflect a very specific lower stability of the
noncatalytic RrF1
/
interfaces. Indeed from the
R. rubrum chromatophore-bound
RrF0F1, only native dimers have been isolated
(35). From chloroplast, on the other hand, only unstable
C3
C3 hexamers
could be obtained, and they readily dissociated into mixtures of their
respective
and
monomers (10, 36).
/
interfaces and enable the
association of the
R1
R1 dimers into
hexamers has yielded the results demonstrated in Fig. 1, C
and D. Incubation of the concentrated stable dimers (Fig.
1B) with NaF and AlCl3 that form aluminum fluoride (AlFx),2 a
transition state analog of F1 nucleotide-binding sites
(37-40), resulted in their partial conversion into the closed
R3
R3 hexamers
(Fig. 1C). Full association of these dimers into hexamers
was obtained by their incubation with both NaF and AlCl3 in
the presence of either Mg2+ or Ca2+ and either
ADP or ATP at a cation/nucleotide ratio of 1 (Fig. 1D).
AlFx was reported earlier to inhibit various
F1-MgATPases but only after their very specific stepwise
preincubation first with low ADP and high MgCl2
concentrations, then with NaF, and finally with AlCl3. The
structure of bovine MF1, fully inhibited by this procedure,
has recently been resolved (41). It shows that both aluminum
trifluoride and Mg2+-ADP were bound, in a
quasi-irreversible manner, to the catalytic nucleotide-binding site of
the MF1-
DP subunit. However, under our very
different one-step incubation conditions, which associated practically
all the
R1
R1
dimers into
R3
R3
hexamers (compare Fig. 1, B and D), the
RrF1-MgATPase activity was hardly affected by AlFx
(see Fig. 5). Furthermore, these
R3
R3 hexamers
remained fully stable only in the presence of AlFx and Mg- or
CaAD(T)P at cation/AD(T)P ratios of 1 (Fig. 1, E and
F). So under our association conditions AlFx does
not bind irreversibly to the catalytic
/
interface of the dimers
and does not inhibit their MgATPase activity. It rather seems to bind
in a reversible manner to the open noncatalytic nucleotide-binding site
on the dimer
R subunit and facilitate its association
with the
R of another dimer, leading to their assembly
into the closed hexameric structure.
R3
R3
C
Complexes--
Incubation of the
R1
R1 dimers with
an F1-
subunit instead of AlFx resulted in their
assembly into a stable, highly active
3
3
complex (Fig. 2). A recombinant
CF1-
C subunit was used for these studies
since there is as yet no available native or recombinant
R. This
C was found to assemble with the
native unstable
CF1-
C3
C3
into a stable highly active
C3
C3
C
complex (11), and its incubation with the isolated
R1
R1 dimers
resulted in their assembly into a hybrid
R3
R3
C
(Fig. 2, A and B). This assembly could also be
followed by the dramatic 100-fold increase (Fig.
3) of the low 0.1-0.15 units/mg MgATPase
activity of the
R1
R1 dimers (26).
But unlike the fast assembly of the
R and
R monomers into dimers, which reached their maximal
MgATPase activity after a 5-min incubation at 22 °C (26), this
assembly was slow, requiring about 60 min for completion. A very
similar time dependence was reported for the assembly of a
CF1-(
)-complex from isolated native
CF1-(
) and a CF1-
C (10).
The increase in activity during the
R3
R3
C
assembly was also fully dependent on the amount of
C,
saturating at a molar ratio of 2
C/
1
1 (Fig. 3,
inset). The relatively high amount of
C
required for obtaining this saturated activity was due to the tendency
of both native and refolded
C to aggregate, since they
remained soluble only when stored in a buffer containing 0.3 M LiCl at pH 9.5 (10, 11). So when
C was
diluted into the incubation buffer with the
R1
R1, it
partially precipitated out during their slow assembly into the hybrid
R3
R3
C
complex. The time- and
C-dependent increase
in the MgATPase activity during the assembly is therefore presented in
units/mg
(Fig. 3).
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Fig. 2.
Size-exclusion HPLC demonstrates the assembly
of a hybrid
R3
R3
C
complex by two different procedures. In the first procedure
(A and B), the isolated
R1
R1
(A) was incubated at 9.2 µg of protein with 5 µg of
refolded
C for 1 h at 22 °C in 100 µl of TGN
buffer containing 1 mM MgADP (B). In the second
procedure (C and D), the individually expressed
urea-solubilized
R,
R, and
C subunits were refolded together as described under
"Experimental Procedures" (C). The pooled
R3
R3
C
peak of C was concentrated and re-run in D. Each
sample was loaded on the column and eluted as described in Fig. 1.
Inset, SDS-PAGE profile of the isolated RrF1 and
the pooled
R3
R3
C
peak.
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Fig. 3.
Time-dependent assembly of a
highly active hybrid
R3
R3
C-ATPase
complex. 4.6 µg of the isolated
R1
R1 dimers and
2.5 µg of a refolded
C were incubated at 22 °C in
50 µl of TGN buffer with 1 mM MgADP. The MgATPase
activity of separate samples was assayed at the indicated intervals
with the first point representing a sample mixed directly into the
ATPase assay mixture containing 4 mM ATP, 2 mM
MgCl2, and 50 mM sulfite. The ATPase activity
was measured for 5 min at 35 °C as described under "Experimental
Procedures." Inset, dependence of the increase in the
hybrid ATPase activity on the amount of
C. Samples
containing 9.2 µg of the isolated dimers were incubated for 1 h
at 22 °C with increasing amounts of
C and assayed for
their activity as described above.
R3
R3
C
was also assembled by refolding together all three urea-solubilized
subunits under the conditions developed for refolding the
R1
R1 dimers (see
Fig. 1A). This much simpler one-step refolding procedure,
which resulted in direct assembly of the
R3
R3
C
complex (Fig. 2C), enabled the isolation of large amounts of a pure, fully stable hybrid WT complex (Fig. 2D and
inset). It was also used for the assembly of a hybrid mutant
complex containing the
R-T159S catalytic site mutant
(27). This mutant
R subunit was shown to bind in the
presence of small amounts of monomeric
R into a
-less
chromatophore membrane-bound RrF0F1, which also lacked about 20% of its
subunit and lost all ATP synthesis and hydrolysis activities. The reconstituted chromatophores regained all
their Mg2+-dependent but none of the
Ca2+-dependent activities (27).
R3
R3
C-ATPase
Activities by Their
C Oxidation/Reduction--
A
unique feature of the chloroplast CF1-ATPase activity is
its high regulation by the reduction/oxidation of the disulfide bond
formed between Cys199 and Cys205 in its
C subunit (24). The region containing these cysteine
residues is completely missing from respiratory F1-
subunits, as well as from the
subunit of cyanobacteria and purple
photosynthetic bacteria, including the RrF1-
(25). All
ATPase activities of RrF1 showed indeed no response to
either reduction by DTT or oxidation by CuCl2 (Fig.
4A). However, both
C-containing hybrid WT and mutant
F1-
3R
3R3
C
complexes showed clear redox regulation of their Mg2+-,
Mn2+-, as well as Ca2+-dependent ATPase
activities, which were about 2-fold higher in the reduced as compared
with the oxidized states (Fig. 4, B and C). The
MgATPase activity of a hybrid
3
3
complex containing TF1-
and
subunits and the
CF1-
C was also shown to be regulated by the
C redox state (42). These results indicate that the
specific thiol modulation (24) of
C can be transferred
to various hybrid
3
3
complexes.
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Fig. 4.
Modulation of the ATPase activities of
RrF1 (A), and the assembled hybrid WT
R3
R3
C
(B), and mutant
R3(
R-T159S)3
C
(C) complexes by reduction and oxidation. All
complexes were incubated for 1 h at 35 °C in TGN buffer without
(
) or with either 10 mM DTT (
) or 100 µM CuCl2 (
), and their ATPase activities
were measured with 4 mM ATP, 2 mM
MgCl2 or MnCl2, or 4 mM
CaCl2. The much lower RrF1 ATPase activities
are presented in a 4-fold lower scale.
R3
R3
C
Complexes--
An additional and more general tight regulation of the
MgATPase activity of both chloroplasts and chromatophores is their sensitivity to inhibition by excess free Mg2+ ions (22,
23). Both isolated hybrid complexes showed the same optimal dependence
on MgCl2 concentrations as the native RrF1,
reaching maximal levels at a Mg/ATP ratio of 0.5 (Fig.
5 and inset). However, their
much higher MgATPase activities showed a lower sensitivity than
RrF1 to inhibition by excess free Mg2+ ions,
being only 50% inhibited as compared with the RrF1 80% at
an Mg/ATP ratio of 2.5. On the other hand, the similar, very low
MgATPase activities of the dimers and the AlFx-stabilized hexamers were not subject to any regulation by excess free
Mg2+ ions. They showed only a simple saturation curve, with
no inhibition even at a Mg/ATP ratio of 2.5 (Fig. 5, inset).
The MgATPase activity of RrF1 (+AlFx), which was
incubated and assayed under the conditions used for assembly of the
R3
R3 hexamers in
presence of AlFx, was only slightly lower than that of native
RrF1 and retained its full pattern of inhibition by excess
free Mg2+ ions (Fig. 5, inset). These results
indicate that the completely different response of both dimers and
hexamers, as compared with RrF1, to increasing
MgCl2 concentrations is due to absence of the
subunit
and not to the presence of AlFx.
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[in a new window]
Fig. 5.
Effect of increasing MgCl2
concentrations on the ATPase activities of RrF1, its
assembled
R1
R1
dimers,
R3
R3hexamers,
and hybrid WT
R3
R3
C
and mutant
R3(
R-T159S)3
C
complexes. The MgATPase activities of RrF1 (
), and
the dimers (
), hybrid WT (
), and mutant (
) complexes were
measured as described under "Experimental Procedures" using 4 mM ATP and the indicated concentrations of
MgCl2. For comparing the activities of RrF1
(
) and the hexamers (
), the dimers were first assembled into the
hexamers by incubation in the presence of 10 mM NaF, 0.5 mM AlCl3, and 1 mM MgADP as
described under "Experimental Procedures," and RrF1
underwent the same treatment. Both complexes were diluted into the
assay mixtures described above, except that all of them contained also
10 mM NaF and 0.5 mM AlCl3. The
highly active hybrid WT (
) and mutant (
) complexes are presented
in the main figure. Inset presents the much lower activities
of RrF1 (
,
), the dimers (
), and hexamers
(
).
View larger version (19K):
[in a new window]
Fig. 6.
Effect of increasing CaCl2
concentrations on the ATPase activity of RrF1 ( ) and the
hybrid WT
R3
R3
C
(
) and mutant
R3(
R-T159S)3
C
(
) complex. The CaATPase activities were measured as described
under "Experimental Procedures" with 4 mM ATP and the
indicated concentrations of CaCl2.
Ca2+- and Mg2+-dependent ATPase activities
of RrF1 and the assembled RrF1-() dimers,
hexamers, and hybrid (
) complexes containing the chloroplast
CF1
R3
R3 hexamers were assembled and
assayed in the presence AlCl3 and NaF, which form AlFx,
and the RrF1(+AlFx) was incubated and assayed under
identical conditions. The ATPase activities were measured as described
under "Experimental Procedures" with 4 mM ATP and 2 mM MgCl2 or 4 mM CaCl2, either
with no additions (None) or with 50 mM sulfite or 2 mM azide.
-containing
complexes show much higher Ca- and MgATPase activities as well as clear differences between the functional properties of these two ATPase activities. Their MgATPases are tightly regulated by excess free Mg2+ ions (Fig. 5), highly stimulated by sulfite and
methanol (not shown) and inhibited by azide (Table I). But their
3-10-fold higher CaATPase activities are not regulated (Fig. 6) and,
as both dimer and hexamer MgATPase activities, do not respond to any
tested MgATPase effectors (Table I).
3R
R3 hexamers
might be misleading, because their low CaATPase is the
AlFx-inhibited activity. There is no possible way to confirm
this suggestion in the RrF1 hexamers since they can
assemble only in presence of AlFx (Fig. 1). But in the
TF1-
3
3 hexamers, which were
assembled without AlFx, the CaATPase activity is 5-fold higher
than their MgATPase, although the whole native TF1 has a
10-fold lower Ca- than MgATPase activity (43). The specific inhibition
of the RrF1-CaATPase by AlFx, which has not been
tested on any other F1-CaATPase activity, provides an
additional clear difference of functional properties of the
RrF1-CaATPase and MgATPase activities.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
3
complexes were assembled by refolding the recombinant R. rubrum RrF1-
R and WT
R
(26) or mutant
R-T159S subunits (27) together with the
spinach CF1-
C (11). All ATPase activities of
both isolated hybrid complexes showed, unlike those of
RrF1, the specific thiol modulation (24) of their unique
C disulfide bond. Also all ATPase activities of the
hybrid WT
R3
R3
C,
which were between 9- and 30-fold higher than those of the
-containing RrF1 (Table I), retained the catalytic
properties of both RrF1 and CF1-ATPases. This
includes the specific regulation of the photosynthetic
F1-ATPases by excess free Mg2+ ions (Refs. 22
and 23 and Fig. 5), which is released by sulfite resulting in a
2-3-fold stimulation of activity. (This MgATPase activity is also
fully inhibited by azide (Table I). This also includes a much higher
Ca2+- than Mg2+-dependent ATPase
activity that is not inhibited by increasing Ca2+
concentrations nor by azide and is not stimulated by sulfite.
R3
R3
C
complex provides a most suitable candidate for studies aimed at
elucidating the molecular mechanism involved in the
C
thiol modulation of its high, ~40 units/mg, Mg2+- and
Ca2+-dependent ATPase activities. Two other hybrid
3
3
subcomplexes, exhibiting the
regulatory thiol modulation of
C, were constructed with
the TF1-
and
subunits. In the first report (42) the
TF1 subunits were mixed with a recombinant
C, but the isolated hybrid showed at least a 10-fold
lower MgATPase activity than the hybrid WT
R3
R3
C
complex. In a more recent report (44) a mutant
TF1-
3
3
complex was
constructed by replacing 111 amino acid residues from the central
region of the TF1-
with 148 residues of the homologous region from spinach
C, including the regulatory stretch
with Cys199 and Cys205. This mutant complex was
expressed and purified in large amounts and responded to the
C thiol modulation, but even its DTT-reduced MgATPase
activity reached at the most 5 units/mg. Furthermore, no CaATPase
activity has been reported in this mutant
TF1-
3
3
complex, probably
because in the native TF1, unlike in RrF1
(Table I) and CF1 (30), the CaATPase activity is 10-fold
lower than its MgATPase activity (43).
3R
3R3
C
complex provides also a promising system for following the possible
CaATPase as well as MgATPase-induced
C rotation, which
has not been measured as yet in any photosynthetic F1
complex. The CaATPase activity has not been tested as an inducer of any
F1-
rotation. It is, however, a most important candidate for such assays because, although it reaches in both RrF1
and the hybrid WT complexes even higher rates than those of the
MgATPase, it has very different catalytic properties. CaATPase, unlike
the MgATPase, appears only in the AlFx-stabilized, closed
R3
R3 hexameric
structure (Table I). These results suggest that the Ca2+
binding affinity to the RrF1 catalytic nucleotide-binding
sites is lower than that of Mg2+, which induces a similar
MgATPase activity in the open dimers as in the closed hexamers. A lower
binding affinity of Ca2+ could lead to its lower catalytic
cooperativity. Indeed, the RrF1-CaATPase, unlike its
MgATPase, is not inhibited at all by azide (Table I). Furthermore, the
similar low MgATPase activity of the
RrF1-
R1
R1
dimers and
R3
R3
hexamers, which in the open dimers has certainly no catalytic
cooperativity, is also fully resistant to inhibition by azide (Table
I). Azide, which was used as the inhibitor of
rotation inside the
3
3 hexamer cavity (17, 18), has recently been suggested to block the signal transmission between catalytic sites, which leads to positive catalytic cooperativity in all F1-MgATPases (45).
R3
R3
C
the catalytic nucleotide-binding site in presence of Ca2+
has no clear catalytic cooperativity and is decoupled from any proton
translocation. In a recent study on
rotation in a genetically engineered EcF1-
3
3
containing an uncoupled mutation of
Met23 to Lys (18),
the mutant
was found to rotate rather similarly to the WT
. This
unexpected capacity of the uncoupled
subunit to rotate was
explained by suggesting that its defective coupling might be after
rotation, possibly in the interactions between the F1 and
F0 sectors (46). This explanation cannot hold for a
catalytic site bound CaATP that cannot induce any proton translocation. The highly active CaATPase of the hybrid WT
R3
R3
C
is therefore a very interesting target for comparing the capacity of
its Ca2+ versus Mg2+-occupied
catalytic nucleotide-binding sites to induce catalytic cooperativity
and/or rotational catalysis.
R3(
R-T159S)3
C.
The
R-Thr159 is equivalent to the
MF1
-Thr163, which was identified as a
ligand to Mg2+ in the catalytic nucleotide-binding sites of
the bovine heart crystal structure (12). This fully conserved residue
has been mutated to serine in several respiratory
F1-ATPases (47-49) as well as in Chlamydomonas
reinhardtii CF1 (50). All of them showed, as our
hybrid mutant
R3(
R-T159S)
C
(Table I), a very high MgATPase activity, with a much lower sensitivity
to stimulation by sulfite or methanol and to inhibition by azide (Table
I). But our hybrid mutant also has a 3-4-fold lower CaATPase activity
that has not been followed in the other mutants. Since the hydroxyl
group of serine is less nucleophilic than that of threonine, it would
lower the bond energy between serine and both divalent cations and
decrease their binding affinity. In the hybrid mutant, the
Mg2+-occupied binding site would thus become rather similar
to the lower affinity Ca2+-occupied catalytic site of our
hybrid WT complex, which shows very high rates but lower or no
catalytic cooperativity. But the mutant Ca2+-occupied
catalytic sites would reach an even lower affinity that does already
drastically reduce its overall activity (Table I).
R-T159S mutant was, however, as effective as the WT
R in restoring the proton-translocating
Mg2+-dependent ATP synthesis and hydrolysis.
But it could not restore any membrane-bound CaATPase activity (27).
These results demonstrate that there must be two sets of clear
differences in the geometry of either the WT or mutant
R
catalytic sites when occupied by Ca2+ as compared with
Mg2+. One is operating in the soluble state and a different
one in the membrane-bound state, whose
R-T159S-containing RrF0F1 shows
the maximal difference between the fully operative proton-coupled
Mg2+-occupied sites and the complete absence of any active
Ca2+-occupied catalytic sites. The similar catalytic
properties of the highly active hybrid mutant MgATPase and the hybrid
WT CaATPase make them very promising tools for obtaining information on
the as yet unknown factors that couple ATP binding and hydrolysis to
rotational motion of the
subunit of the catalytic
F1-ATPase. Since azide does not inhibit the CaATPase
activity, another inhibitor will be required for such comparative
studies. A very suitable candidate is the specific CF1
effector tentoxin, which at low concentrations inhibits but at high
concentrations stimulates both CF1 Ca- and MgATPase
activities (30). RrF1 is, however, completely resistant to
tentoxin. We have therefore assembled another set of hybrids composed
of WT and mutated
R together with
C and
C.3 They
provide the possibility of assaying both
C thiol
modulation and rotational catalysis in the presence of inhibitory as
well as stimulating tentoxin concentrations.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the United States-Israel Binational Science Foundation, Jerusalem, and from the Avron-Wilstätter Minerva Center for Research in Photosynthesis, Rehovot, Israel.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of National Institutes of Health Predoctoral Training Grant GM08545.
To whom correspondence should be addressed. Tel.:
972-8-9342729; Fax: 972-8-9344118; E-mail:
z.gromet-elhanan@weizmann.ac.il.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M007568200
2
The exact species of aluminum fluoride in the
incubated mixture of AlCl3 and NaF that exerts the
stabilizing effect on the noncatalytic RrF1 /
interfaces is as yet unknown. We therefore refer to it here as
AlFx.
3 W. C. Tucker, Z. Du, Z. Gromet-Elhanan, and M. L. Richter, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CF1, EcF1, MF1, RrF1, and
TF1, F1-ATPases from chloroplasts, E. coli, mitochondria, R. rubrum, and thermophilic
Bacillus PS3, respectively;
R and
R, the
and
subunits of the
RrF1-ATPase;
C, the
subunit of spinach
chloroplast CF1-ATPase;
DTT, dithiothreitol;
HPLC, high
performance liquid chromatography;
PAGE, polyacrylamide gel
electrophoresis;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
WT, wild
type.
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