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INTRODUCTION |
G-actin, in the presence of Mg2+, slowly catalyzes the
hydrolysis of ATP that is bound at a high affinity site at the base of a deep cleft separating the two major domains of the protein. This
activity is greatly enhanced by polymerization of the actin (1). For
those actins studied to date, hydrolysis occurs shortly after the
incorporation of the actin monomer into the filament producing an
ADP-Pi species, which slowly loses its Pi to
yield an ADP-monomer. Because ATP and ADP-Pi F-actin are
both more stable than ADP-F-actin, this Pi release is
believed to play a major role in the treadmilling of actin filaments
that occurs within the cell.
Actin polymerization is a multiple-step process that includes a
salt-induced G to F conformational change, rate-determining nucleation,
and filament elongation until the monomer actin concentration reaches a
limiting value called the critical concentration. Prior to
incorporation of the actin monomer into the filament, it has been
hypothesized that the actin proceeds through an F-monomer state (2).
Evidence for such a species derives from experiments such as comparing
protease digestion patterns of the actin in the absence and presence of
polymerization-inducing salts at actin concentrations below the
critical concentration of actin needed for formation of filaments.
Using muscle actin, Shu et al. (3) determined that the
F-monomer exhibits a low ATPase activity similar to that observed with
G-monomer, suggesting that the enhanced ATPase activity associated with
actin polymerization requires the conformation and inter-monomer
contacts formed within the context of the actin filament.
We have previously created a mutant actin (GG-actin) in which we
simultaneously converted Val266 and Leu267 to
glycines (4, 5). These residues along with Leu269 reside in
the tip of a loop between actin subdomains 3 and 4. Holmes and
co-workers (6) proposed that one major interstrand force stabilizing
the actin filament was a "plug-pocket" interaction in which the tip
of this loop interacts with a hydrophobic surface formed by the
interface of two monomers on the opposing strand. In agreement with the
predictions of this model, we have demonstrated that, as assessed by a
change in light scattering, purified GG-actin alone fails to polymerize
under normal polymerization conditions. The actin binding
proteins tropomyosin (7) and fimbrin (8) can rescue GG-actin
polymerization. However, this rescue is temperature-sensitive. In
the presence of tropomyosin or fimbrin, polymerization of GG-actin occurs only at a temperature higher than 15 °C. Furthermore, when the temperature is lowered after polymerization reaches the steady state, depolymerization of actin filaments occurs.
The polymerization defect exhibited by GG-actin and the cold-sensitive
rescue of GG-actin polymerization are consistent with the Holmes model.
If the model is correct, removal of two hydrophobic residues at the tip
of the plug should greatly decrease the proposed hydrophobic
cross-strand interaction, leading to polymer instability of the mutant
actin. Tropomyosin or fimbrin binding may strengthen intermonomer
contacts along one strand partially compensating for the loss of
cross-strand stabilization to the point that polymerization at 25 °C
is restored. However, hydrophobic interactions are destabilized by
colder temperatures (9). Thus, at low temperatures, the residual
cross-strand interaction of the mutant actin is reduced to such an
extent that the extra stabilization can no longer compensate, and
filament formation cannot occur.
Although GG-actin has been used as a model system for examining the
filament-stabilizing abilities of actin-binding proteins (7, 8), the
specific mechanism responsible for the polymerization defect associated
with GG-actin is unclear. For example, it is not known whether GG-actin
remains in its G form under normal polymerization conditions, is
converted to an F-actin monomer, or actually forms transient unstable
F-actin oligomers. To understand the mechanisms by which this rescue of
GG-actin polymerization occurs, it is necessary to first understand the
nature of the polymerization defect. To explore these alternatives, we
have assessed in this paper the effect of F
buffer1 on the ATPase
activity exhibited by GG-actin and the dependence of this activity on
actin concentration and temperature. We have also determined the effect
of temperature on the ATPase activity of WT yeast actin under
polymerizing conditions.
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MATERIALS AND METHODS |
Actin Purification and Polymerization--
Purification of
wild-type and GG-actins from Saccharomyces cerevisiae was
done by a combination of DNase I affinity chromatography and
DEAE-cellulose chromatography as previously described (10). Affinity
columns were made with DNase I purchased from Worthington and Affi-Gel
10 obtained from Bio-Rad Laboratories. Purified
Ca2+/G-actin was stored at 4 °C in Ca2+/G
buffer (10 mM Tris-HCl, pH 7.5, 0.2 mM
CaCl2, 0.2 mM ATP, and 0.5 mM DTT)
and used within 4 days.
Actin polymerization was induced by the addition of 2 mM
MgCl2 and 50 mM KCl to a G-actin solution in G
buffer (F buffer). Reactions of a total volume of 120 µl were
followed in the thermostatted cuvette chamber of a fluorescence
spectrometer. The increase in light-scattering caused by polymerization
was monitored with the excitation and emission wavelengths set at 360 nm. For studies with the Mg2+ form of actin, purified
Ca2+-actin was converted into the Mg2+ form by
treatment with 0.2 mM EGTA in the presence of 0.1 mM MgCl2 at 4 °C for 10 min using a
modification of a previously published procedure (11).
Mg2+-G-actin was used immediately after the conversion from
Ca2+/G-actin.
Enzchek Phosphate Assay--
Release of inorganic phosphate from
actin was quantitated by the Enzchek phosphate assay. This assay
employs a kit obtained from Molecular Probes and is based on the
Pi-dependent liberation of
2-amino-6-mercapto-7-methylpurine from methyl-thioguanine, a
nucleoside derivative of this base, by a purine nucleoside
phosphorylase. The product base has a characteristic UV spectrum, not
evident when part of a nucleoside, which can be utilized to monitor its phosphate-dependent rate of release by the enzyme. Each
reaction mixture contained actin at the desired concentration as
specified by the manufacturer, 2 units/ml purine nucleoside
phosphorylase, and 200 µM methyl-thioguanine
riboside. Pi release from actin in F buffer (10 mM Tris-HCl, pH 7.5, 0.2 mM ATP, 0.5 mM DTT, 2 mM MgCl2, and 50 mM KCl) or in Mg2+-G buffer (10 mM
Tris-HCl, pH 7.5, 0.2 mM ATP, 0.5 mM DTT, and 0.1 mM MgCl2) was monitored continuously by the
increase in absorbance at 360 nm. Phosphate standard curves were
generated for each assay.
Tropomyosin Purification--
Bovine cardiac
tropomyosin was purified from cardiac acetone powder according to the
procedure of Butters et al. (12).
PDM Cross-linking--
G-actin in G buffer was centrifuged
through a micro Bio-Spin chromatography column (Bio-Rad)
pre-equilibrated with DTT-free G buffer to remove remaining DTT,
and the eluted G-actin was diluted to 20 µM with DTT-free
G buffer. 20 µl of 20 µM actin was then incubated at
25 °C for 10 min with or without the addition of 2 mM
MgCl2 and 50 mM KCl. A stock solution of 5 mM N,N'-1,4-phenylenedimaleimide (PDM) in dimethylformamide was freshly diluted 10 times with DTT-free G
buffer, and 1 µl of PDM was added into the 20-µl actin solution followed by an additional 8-min incubation. The final ratio of PDM to
actin was about 1:1. The cross-linking was quenched by the addition of
20 µl of 2× SDS sample buffer containing 20%
-mercaptoethanol. The presence of cross-linked actin oligomers was analyzed by 10% SDS-PAGE.
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RESULTS |
Pi Release by GG-actin in F Buffer Is Significantly
Faster Than in G Buffer--
We first determined whether increasing
the ionic strength to that normally used to induce actin polymerization
enhances the ATPase activity of GG-actin by comparing Pi
release from GG-actin under polymerization (F buffer) and monomeric (G
buffer) conditions. Fig. 1 shows the
time-dependent release of Pi from GG-actin with or without the addition of 2 mM MgCl2 and 50 mM KCl. Monomeric GG-actin exhibited a Pi
release curve similar to that of WT actin. Addition of 2 mM
MgCl2 and 50 mM KCl, which induces
polymerization of WT-actin, did not do so with GG-actin as judged by a
lack of change in light scattering and the absence of filaments in
samples examined by electron microscopy (5). Interestingly,
however, the rate of Pi release from GG-actin in F buffer
increased 4-fold compared with that in G buffer.

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Fig. 1.
Phosphate release from GG-actin in G buffer
and F buffer. Mg2+-GG-actin (10 µM) in G buffer ( ) or F buffer ( ), and 10 µM Mg2+ WT-actin in G buffer
(asterisks) were incubated at 25 °C. The rate of
Pi release was quantitated by the Enzchek phosphate assay.
This is a representative example of an experiment that was repeated
with three different actin preparations.
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Pi Release by GG-actin Is Greatly Increased Above a
Certain Actin Concentration--
To explore the mechanism behind this
enhanced GG-actin ATPase activity in the presence of F buffer, we first
measured the continuous release of Pi from WT-actin to
which F salts had been added. Our results demonstrate (see Fig. 6) that
Pi release from WT-actin closely mirrors its polymerization
curve reaching a plateau when F-actin formation reaches equilibrium. We
next measured Pi release by Mg2+-GG-actin at
different concentrations under polymerization conditions (Fig.
2A). At low concentrations, a continuous
slow monophasic Pi release occurred. However, at higher
actin concentrations, Pi release by GG-actin appeared to be
biphasic: a lag phase and a linear fast release phase with no plateau.
We observed the same behavior with Ca2+/GG-actin, showing
that this behavior, at least qualitatively, was independent of the
nature of the tightly bound divalent cation (Fig. 2B).

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Fig. 2.
Effect of actin concentration on GG-actin
ATPase activity. To different concentrations of GG-actin in G
buffer were added 2 mM MgCl2 and 50 mM KCl, and Pi release was monitored
continuously by the Enzchek phosphate assay. A,
Mg2+-GG-actin; B, Ca2+-GG-actin.
, 16 µM GG-actin; , 14 µM GG-actin;
×, 12 µM GG-actin; , 10 µM GG-actin; *,
8 µM GG-actin; +, 4 µM GG-actin. This
experiment was repeated with two different preparations of actin with
essentially similar results.
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This type of concentration-dependent Pi release
by GG-actin resembles a critical concentration curve determination for
actin polymerization, suggesting that GG-actin might form unstable
oligomers leading to abortive polymerization. For both cations, we thus plotted the rate of Pi release by determining the slopes of
the linear second phase of each curve as a function of actin
concentration (Fig. 3). This plot
exhibited two linear phases as a function of actin concentration, and
the intersection of the two lines yielded an apparent critical
concentration for enhancement of ATPase activity of ~8
µM for Mg2+-GG-actin. A duplicate experiment
with a second actin preparation yielded an apparent critical
characterization of 7 µM (data not shown). Both
determinations using Ca2+-actin yielded a critical
concentration value of 11 µM. This distinction in
apparent critical concentration for enhancement of Pi
release is consistent with the divalent cation-dependent
difference in critical concentration for polymerization determined
earlier for muscle actin (13), although the difference is smaller than
that reported in this previous study. Our results therefore indicate that the enhanced ATPase activity we observed for GG-actin in F buffer
results not merely from conversion of a G- to an F-monomer but derives
from the formation of unstable oligomers of F-actin.

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Fig. 3.
Relationship of the rate of Pi
release to GG-actin concentration. The slope of the linear portion
of each curve from Fig. 2 was determined and plotted as a function of
actin concentration. A, Mg2+-GG-actin;
B, Ca2+-GG-actin.
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It has previously been shown (14) that, for a polymerization process
like that of actin, a plot of ln (lag phase) versus ln
(actin) will produce a line with a slope equal to one-half of the
number of monomers in the nucleus. Using the data in Fig. 2 for
Mg2+-actin, we calculated the lag phase by determining the
point at which the second linear part of the curve intersected the
first section of the plot for the 10, 12, and 16 µM
curves. The plot of ln (lag phase) versus ln (actin) shown
in Fig. 4 is a straight line with a slope
of 2.1. This experiment was repeated with a second preparation of
actin, and a slope of 1.85 was obtained (data not shown). This result,
consistent with the data of Tobacman and Korn for muscle actin (14),
indicates that the increase in ATPase activity following addition of
salt depends on formation of a nucleus with three or four monomers,
further demonstrating that it results from abortive F-actin formation.

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Fig. 4.
Estimation of nucleus size for
Mg2+-GG-actin oligomerization. The lag times for the
fast release phase of 10, 12, and 16 µM
Mg2+-GG-actin were determined by the intersection of the
two phases. The ln (lag time) was plotted versus ln
[actin], yielding a straight line with a slope of 2.1 and a
correlation coefficient of 0.99. A repetition of this experiment with a
second preparation of actin yielded a slope of 1.85 with a correlation
coefficient of 1.
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Pi Release by GG-actin under Polymerization Conditions
Is Cold-sensitive--
Our previous work demonstrated that the ability
of beryllium fluoride, BeFx (5), tropomyosin (7), or Sac6p (8)
to rescue GG-actin polymerizability was cold-sensitive. We therefore determined whether or not the F-actin-like ATPase activity associated with GG-actin displayed a similar temperature sensitivity. Control experiments to determine the temperature sensitivity of the Enzchek assay system showed a peak activity at 20 °C, which decreased to
between 85% and 90% of this activity as the temperature was decreased
to 4 °C or increased to 25 °C. As an indication of ATPase activity, we assessed the release of Pi from 12 µM Mg2+-GG-actin at temperatures ranging from
15 °C to 25 °C following the addition of 2 mM
MgCl2 and 50 mM KCl. Results were then adjusted to reflect the small temperature-dependent differences in
the efficiency of the Enzchek system. As shown in Fig.
5A, at 15 °C, minimal
Pi release was observed. Increasing the temperature above this point resulted in a corresponding increase in the rate of Pi release. The biphasic nature of these plots is similar
to what is observed when one studies the polymerization of actin at
different temperatures and suggests that the lag phase is due to
nucleation whereas the faster second phase is associated with the
unstable addition of monomers to the nuclei. To further analyze the
dependence of Pi release on temperature, we plotted the
slopes of both the first and second phases of the plots shown
versus temperature in Fig. 5, B and C.
Fig. 5D shows that both sets of data points produce the same
line when the points in each curve are normalized to the slope at
25 °C for each curve. In each case, the intercept on the temperature
axis is about 15 °C. When the experiment was reproduced with another
preparation of actin, the onset of polymerization was 15.5 °C. Actin
polymerization follows nucleation/elongation kinetics, and if
nucleation is rate-limiting, the number of nuclei will dictate the rate
of oligomerization. Our data therefore suggest that a threshold for
nucleation for 12 µM Mg2+-GG-actin is
15 °C and that, over the temperature range tested, the increase
observed in Pi release results primarily from an increase
in the number of nuclei present as a function of temperature.

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Fig. 5.
Effect of temperature on the salt-induced
ATPase activity of Mg2+-GG-actin. A,
Pi release from 12 µM
Mg2+-GG-actin at various temperatures was monitored by the
Enzcheck assay following the addition of 2 mM
MgCl2 and 50 mM KCl. , 25 °C; ,
20 °C; ×, 17.5 °C; +, 15 °C. B, the slope for the
fast release phase at each temperature was replotted as a function of
temperature. C, the slope for the slow release phase at each
temperature was replotted as a function of temperature. D,
the release rate at each temperature relative to that at 25 °C for
each phase was plotted. , slow phase; , fast phase. This
experiment was repeated with two different actin preparations with same
results.
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Temperature Dependence of Pi Release by Polymerizing WT
Actin--
It is possible that the temperature sensitivity of
Pi release by GG-actin reflects an inherent sensitivity of
the ATPase reaction per se rather than temperature-sensitive
actin oligomerization. We thus assessed both the extent of
polymerization and the Pi release of WT yeast actin as a
function of temperature following induction of polymerization by the
addition of salts. Fig. 6 shows that
decreasing the temperature results in a concomitant decrease in
both the rate of polymerization and Pi release. ATPase
activity is present at all temperatures, and the extent of
Pi release from WT actin is governed solely by the extent
of actin polymerization. These results suggest that the decrease in
ATPase activity observed reflects a decrease in the rate of
polymerization and not an inherent temperature-sensitive decrease in
the ATPase activity per se. At 15 °C, where
Pi release from GG-actin had disappeared, WT actin exhibited significant Pi release with its rate dependent
solely on the rate of polymerization of the actin. Therefore, the lack of Pi release, and thus ATPase activity, seen with GG-actin
at 15 °C most likely results from the inability of this actin to nucleate and form transient F-actin oligomers.

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Fig. 6.
Effect of temperature on polymerization of
and Pi release from WT yeast actin. A,
polymerization of 4.5 µM Mg2+ WT-actin
induced by the addition of 2 mM MgCl2 and 50 mM KCl at different temperatures is shown using light
scattering as an assay. B, Pi release under the
same conditions, as monitored by the Enzcheck assay, is shown. ,
25 °C; ×, 20 °C; *, 15 °C; , 10 °C; +, 7 °C.
C, the data from light scattering and Pi release
at 25 °C were normalized and plotted together. , light
scattering; , Pi release. This experiment has been
repeated three times with different preparations of actin with
essentially the same results.
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Although it has been demonstrated that muscle actin slowly releases its
bound Pi after ATP hydrolysis (15), it is not known whether
or not yeast actin has the same behavior. To address this question, we
normalized the data from light scattering and Pi release at
25 °C and plotted them together (Fig. 6C). The
concentration of yeast actin we used, 4.5 µM, polymerized
as rapidly as 10 µM muscle actin (data not shown) under
the same conditions, in agreement with the results of Buzan and Frieden
(16) that yeast actin polymerizes substantially faster than muscle
actin. Although we observed a significant lag phase in the rate of
Pi release following polymerization for muscle actin, no
apparent lag phase was seen in the case of the yeast actin.
Tropomyosin Limits the Extent of Pi Release
by GG-actin--
We have previously shown that tropomyosin (7)
restores the polymerizability of GG-actin. If the continuous ATPase
activity observed with GG-actin alone in F salts is due to the
continuous turnover of oligomers, this activity should be greatly
curtailed in the presence of tropomyosin after polymerization reaches a steady state. We therefore determined the rate of Pi
release when polymerization-inducing salts were added to mixtures of
actin in the presence of tropomyosin. Fig.
7 demonstrates that, as predicted, the
linear increase in Pi is greatly inhibited when
polymerization approaches the steady state.

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Fig. 7.
Tropomyosin limits the extent of ATP
hydrolysis by GG-actin in F buffer. A,
Mg2+-GG-actin, 20 µM, was mixed with 6 µM cardiac tropomyosin, and 2 mM
MgCl2 and 50 mM KCl was added to the mixture.
This amount of tropomyosin is sufficient to saturate the tropomyosin
binding sites on the F-actin. Polymerization under such conditions was
monitored by the increase in light scattering. B,
Pi release under the same conditions as in A or
by GG-actin in the absence of tropomyosin was monitored by the Enzcheck
assay. , GG-actin with tropomyosin; , GG-actin by itself. This
experiment was performed twice with different preparations of actin
with essentially the same result.
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Pi Release by GG-actin under F-monomeric
Conditions--
Although Shu et al. (3) reported that the
F-monomer did not exhibit enhanced ATPase activity over the G-monomer,
their study was performed for very short times following addition of salt to actin solutions above the critical concentration, which might
have led to inaccuracies in their measurements. Second, in a number of
instances dealing with polymerization rates and nucleotide exchange,
yeast and muscle actins have been shown to behave quantitatively quite
differently (4, 16). Fig. 3 shows that oligomerization of GG-actin only
occurs when its concentration is above the critical concentration. This
suggests that we could possibly gain a population representing
F-monomeric actin when we induce the conformational switch by F salts
with GG-actin at concentrations below the critical concentration. Thus,
we determined whether, below the critical concentration, GG-actin in F
buffer would display an enhanced ATPase activity over that seen in G buffer. We incubated 5 µM Mg2+-GG-actin at
25 °C with or without the addition of salts and determined the rate
of Pi release by the Enzchek phosphate assay. No difference could be seen between these two salt conditions in agreement with the
results of Shu et al.
Probing GG-actin Oligomer Formation by
Cross-linking--
N,N'-1,4-Phenylenedimaleimide
(PDM) has been used to probe the oligomeric state of actin
polymerization. Millonig and Aebi (17) have demonstrated the
time-dependent appearance of two cross-linked species
during polymerization, a lower dimer (LD) with an apparent molecular
mass of 86 kDa and an upper dimer (UD) with an apparent
molecular mass of 115 kDa by SDS-PAGE. The LD, a
Cys374-Cys374 cross-linked species (18),
appears immediately after salt induction and is not compatible with the
structure of the actin filament. The UD forms between
Lys191 on one subunit and Cys374 on another
subunit along the helical filament and reflects the structure of a
dimer in the actin filament. The LD is consumed during polymerization
probably via two pathways (19). In one path, the LD is in equilibrium
with a G-actin pool and indirectly involved in polymerization. In a
second pathway, during polymerization LD directly incorporates into
actin filaments to form LD-decorated F-actin. The unincorporated actin
subunits are then released during filament maturation probably due to
an F-conformational change resulting in smooth-looking filaments.
We monitored GG-actin oligomerization by PDM cross-linking. 20 µM Ca2+-GG-actin in G or F buffer was
incubated at 25 °C for 10 min during which phosphate release is
linear, and cross-linking by PDM was initiated as described under
"Materials and Methods." WT actin from rabbit skeletal muscle was
treated in the same way as a control. Fig.
8 shows that muscle actin yielded all
upper dimer, consistent with what was previously reported and what we
observed with yeast WT actin (20). In contrast, a significant amount of
LD was observed with GG-actin along with a fainter UD band
demonstrating that GG-actin could form F-actin like assemblies. The
Mg2+ form of GG-actin gave essentially the same result
(data not shown).

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Fig. 8.
PDM cross-linking demonstrates GG-actin
oligomerization. 20 µM Ca2+ actin in
DDT-free G or F buffer was incubated for 10 min at 25 °C. PDM was
added at a 1:1 ratio to actin and incubated at 25 °C for 8 more min.
The cross-linking reaction was stopped by SDS sample buffer containing
20% -mercaptoethanol, the products were analyzed by 10% SDS-PAGE,
and the bands were visualized by staining with Coomassie Blue.
UD, upper dimer with apparent molecular mass of 115 kDa;
LD, lower dimer with apparent molecular mass of 86 kDa. The
identity of these species was based on the migration of a series of
molecular weight markers not shown. This experiment was repeated with
two preparations.
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DISCUSSION |
We previously demonstrated that, in agreement with Holmes' model
of the actin filament, eliminating hydrophobicity in a subdomain 3/4
loop in yeast actin (GG) resulted in a nonpolymerizing protein (5).
However, we showed that polymerizability could be restored by
incubation with phalloidin (5), Sac6p (8), or tropomyosin (7). Whether
the polymerization defect lay in the nucleation or elongation phase
could not be determined. The demonstration that BeFx, a phosphate
analogue, also restored polymerization (5) suggested that the protein
could still hydrolyze ATP. However, BeFx-dependent rescue
of polymerization could have resulted from stabilization of labile
oligomers following nucleation or by generation of ADP-BeFx actin,
which then polymerized due to an altered conformation.
We have addressed these questions by employing a continuous phosphate
release assay to assess nucleotide hydrolysis by GG-actin. This assay
provides a combined estimate of the hydrolysis of ATP and the
subsequent release of the Pi from the actin, although it
does not allow us to examine the ATP hydrolysis step per se. However, the virtual coincidence of the polymerization and
Pi-release curves for yeast actin makes it very unlikely
that Pi release is rate-limiting in comparison to ATP
hydrolysis, thereby validating this approach.
Although GG-actin will not form stable filaments, our results
demonstrate a substantial F buffer-dependent increase in
GG-actin ATPase activity for both the Ca2+ and
Mg2+ forms of the actin. In contrast with the case of WT
actin, however, the ATPase activity of GG-actin is continuous.
Theoretically, the continuous nature of this activity could result from
an F-monomer ATPase, because previous work had presented evidence for
the generation of such a species in solution (2). Alternatively,
continuous activity could result from the formation and cycling of
small oligomers, undetectable by light scattering. Our results
demonstrate that the second alternative is the correct one. In the case
of WT actin polymerization, continuous low level linear ATPase activity due to treadmilling occurs at the steady state. In response to the
introduction of F salts into solution, GG-actin exhibits a biphasic
ATPase activity, an initial low linear ATPase activity followed by a
faster but still linear phase. The low ATPase activity is very likely
due to a process involving nucleation. The second faster phase could
not result from a typical net elongation phenomenon, because this would
require the attainment of a steady-state level of filament formation
that does not occur. Instead, the continuous activity probably reflects
a steady state of exchange of monomers from unstable oligomers driven
by the hydrolysis of ATP. In essence, the system is one in which, once
an apparent critical concentration for GG-actin is reached, nuclei or
short oligomers provide a surface catalyst for the hydrolysis of ATP by
cycling monomers. Because this is a steady-state situation governed by
the critical concentration of the system, as the actin concentration
increases, the filament ends, but not the free monomers, increase
concomitantly leading to the actin-dependent linear
increase in ATPase activity we observe.
The apparent critical concentration of GG-actin, about 11 µM for Ca2+ actin and 7 or 8 µM
for Mg2+ actin is ~50-60 times higher than it is for WT
actin. Wen (7) had previously determined a critical concentration of
5.5 µM for the tropomyosin-dependent rescue
of Mg2+-GG-actin polymerization by light scattering. The
small difference (0.086 mg/ml) between the critical concentration of
intrinsic oligomerization and tropomyosin-dependent
polymerization may be due to different preparations of actin or small
variations in carrying out the experiments. It is also possible that
tropomyosin slightly decreases the critical concentration of GG-actin
polymerization. However, the measurement of an intrinsic critical
concentration for GG-actin oligomerization allows for the first time an
estimate of the importance for filament stabilization of the
hydrophobicity normally associated with Val266 and
Leu267, which was eliminated in this mutant. Furthermore,
this system provides us with a way of actually assessing nucleation and
oligomerization of actin under conditions that do not permit formation
of stable filaments.
Demonstration of an oligomerization-dependent ATPase
coupled with our previous finding that BeFx allows formation of stable GG-actin filaments allows one to understand the underlying basis for
the inability of GG-actin to form stable filaments alone. There is
clearly an effect on the critical concentration of the ATP-actin. In
other words, in terms of the Holmes model, in the ATP monomer, the
residual hydrophobicity of the plug is oriented correctly enough to
provide sufficient cross-strand stabilization for initiation of actin
filament formation. However, ATP hydrolysis and subsequent release of
the phosphate alters the conformation of either the plug or other
monomer contacts enough so that this residual plug hydrophobicity is no
longer sufficient for maintenance of the filament structure. In other
words, the critical concentration of the ADP-actin monomer is so high
that, under the conditions of the experiment, filaments cannot form.
Oligomerization of GG-actin was directly demonstrated by PDM
cross-linking. Under conditions where steady ATPase activity is
observed, GG-actin forms a significant amount of LD along with much
less UD. Because the UD is the precursor for nucleus and filament
formation, presumably it is the conformation responsible for the F-like
ATPase activity. Taking into account the differences between the
critical concentrations of WT- and GG-actin, there can be no more than
half as much GG F-actin species as WT species at any point. The fact
that the ratio for UD between these two actins is much greater than 2:1
can be attributed to the continuous cycling of the GG F species and a
resulting reduction in the frequency with which they can react with the
cross-linker and perhaps to an inherently less reactive conformation
assumed by the GG-actin per se. This result substantiates
our previous hypothesis that tropomyosin-dependent rescue
of GG-actin polymerization depends initially on the capture of
pre-formed actin oligomers and not on tropomyosin-dependent
nucleation of filament formation.
Our temperature studies not only provide additional information
concerning the effect of the GG mutation on actin function, but they
also provide new insight into the ATPase of WT actin as well. The
extent of polymerization of WT actin is minimally affected by cold
temperatures, although, as the temperature drops, the rate of filament
formation slows. Our demonstration that the rate and extent of
Pi release during this process closely parallels the rate
of formation of F-actin indicates, for the first time, that the F-actin
ATPase activity per se is not cold-sensitive. This situation
is contrary to what is found with many enzyme-substrate systems in
which cold temperatures do not inhibit substrate binding but do inhibit catalysis.
With 12 µM Mg2+-GG-actin,
F-actin-dependent ATPase activity was not observed unless
the temperature was >15 °C. This cold-sensitive behavior of
GG-actin alone allows us for the first time to compare the effects of
this double mutation quantitatively with the less severe polymerization
defects observed with other alterations in the hydrophobic plug. The
L267D mutant, in which a negative charge was placed between the two
remaining hydrophobic groups required >6 °C for polymerization,
whereas little if any effect was observed when either
Val266 or Leu267 was altered singly to G or
when Val266 was substituted with D (21).
Pi release is the slow step in the nucleotide cycle that
occurs during the polymerization of muscle actin (1), and the lag
between ATP hydrolysis and Pi release has been proposed to be an important factor in the rate of actin filament turnover (22). In
this light, our observation that such a lag did not exist with yeast
actin was very surprising and suggests the possibility of a
comparatively greater degree of instability of yeast actin filaments
compared with those of higher eukaryotic actins. We previously observed
that nucleotide exchange in yeast actin is faster than in higher
eukaryotic actins (4, 23). Furthermore, when we compared the rate of
release of Pi by monomeric yeast and muscle actins (data
not shown), the rate constant for the reaction in yeast G-actin
(2.8 × 10
4 s
1) is faster than that
exhibited by muscle actin (9 × 10
5
s
1). These findings reinforce the suggestion of an
inherently less stable yeast actin filament in comparison with that of
higher eukaryotic actins.
Systematically studying the ATPase activity that accompanies actin
polymerization is inherently difficult because of the self-limiting nature of the reaction. Its rate slows drastically as polymerization reaches the steady state requiring that large amounts of actin be
employed if reasonable signals are to be obtained. This problem can be
circumvented somewhat by employing continuous sonication to make the
ATPase reaction continuous. However, the awkwardness of this approach
coupled with artifacts arising from the heat generated by the sonic
probe makes this approach less than desirable. The continuous ATPase
activity exhibited by GG-actin in F buffer potentially provides us with
a means for experimentally addressing the mechanism of the
actin-dependent hydrolysis of ATP under polymerizing conditions.