The ATPase Cross-bridge Cycle of the Kar3 Motor Domain
IMPLICATIONS FOR SINGLE HEAD MOTILITY*
Andrew T.
Mackey
and
Susan P.
Gilbert§
From the Department of Biological Sciences, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260
Received for publication, June 21, 2002, and in revised form, November 22, 2002
 |
ABSTRACT |
Kar3 is a minus-end directed microtubule motor
involved in meiosis and mitosis in Saccharomyces cerevisae.
Unlike Drosophila Ncd, the other well characterized
minus-end directed motor that is a homodimer, Kar3 is a heterodimer
with a single motor domain and either the associated polypeptides Cik1
or Vik1. Our mechanistic studies with Ncd showed that both motor
domains were required for ATP-dependent motor domain
detachment from the microtubule. We have initiated a series of
experiments to compare the mechanistic requirements for Kar3 motility
in direct comparison to Ncd. The results presented here show that the
single motor domain of Kar3 (Met383-Lys729)
exhibits characteristics similar to monomeric Ncd. The
microtubule-activated steady-state ATPase cycle of Kar3
(kcat = 0.5 s
1) is limited by ADP
release (0.4 s
1). Like monomeric Ncd, Kar3 does not
readily detach from the microtubule with the addition of MgATP. These
results show that the single motor domain of Kar3 is not sufficient for
ATP-dependent microtubule dissociation, suggesting that
structural elements outside of the catalytic core are required for the
cyclic interactions with the microtubule for force generation.
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INTRODUCTION |
Kar3 is a microtubule-activated ATPase of the kinesin superfamily
involved in spindle assembly and integrity in the yeast Saccharomyces cerevisiae (reviewed in Refs. 1-5).
KAR3 was originally identified in a screen for genes
essential for karyogamy, the nuclear fusion event during meiosis in
S. cerevisiae (6). In the absence of Kar3, meiosis will not
occur because of the failure to proceed beyond prophase. Once the gene
was cloned and sequenced, its relationship to conventional kinesin was
established. Kar3 is classified as a member of the Kin C subfamily
because its motor domain is located at the carboxyl terminus of the
polypeptide. Further experiments with Kar3 indicated that its cellular
localization was dependent upon a nonmotor polypeptide, Cik1 (7, 8). These results suggested a physical interaction between Kar3 and Cik1.
Detailed analysis of this interaction showed that these two proteins
heterodimerize along their respective
-helical coiled-coil domains
(9). Concomitant with this study, it was also demonstrated that Kar3
interacted with Vik1, another nonmotor polypeptide
-helical in
nature, very similar to Cik1 in both amino acid identity and predicted
secondary structure (10). The cellular localization and function of
Kar3 is dependent upon its associated polypeptide. When Kar3 is in
association with Cik1, its localization is in the spindle midzone
during mitosis and is thought to maintain and provide stability to the
mitotic spindle (10, 11). This function is in contrast to the Kar3-Vik1
complex, which localizes to the spindle pole and is thought to
depolymerize cytoplasmic microtubules during mitosis (11-14) as well
as provide opposing force to Cin8 and Kip1 (10, 15, 16). The roles of
both Cik1 and Vik1 during meiosis I and II are currently being explored (17).
This novel heterodimeric structure of Kar3 with Cik1 produces a
molecule with only one motor domain. This oligomer differs from other
Kin C subfamily members that are homodimers with two motor domains. At
the COOH terminus of both Cik1 and Vik1 is a globular domain that may
also interact with the motor domain of Kar3. This heterodimeric Kar3
complex could be analogous to the interactions of light chains and
calmodulin with myosin superfamily members. Alternatively, the
COOH-terminal globular domain of Cik1 and Vik1 may act to tether the
motor domain to the microtubule, and thereby modulate the Kar3 motor
domain for its cellular functions.
Monomeric motor domain constructs have been instructive in experiments
to define cooperative interactions. Both conventional kinesin monomeric
constructs (18-20) and Ncd monomeric constructs (21, 22) have been
studied. The kinesin monomeric constructs were observed to promote
motility, but it was thought that this motility arose from numerous
motors working together in a multiple motor motility assay as opposed
to a single motor promoting movement independently. Conversely, no
motility was reported with motor domain constructs of Ncd (23). Mackey
and Gilbert (21) observed that the Ncd motor domain did not detach from
the microtubule following ATP turnover, a stark difference from the
dimeric construct of Ncd that dissociated from the microtubule readily
upon the addition of ATP. The interpretation of this experiment was
that the second motor domain was required to weaken the affinity of the
first motor domain for the microtubule (21, 24).
Kar3 functions with only one catalytic domain; therefore, we assume
that its mechanism for force generation must differ from that of a
dimeric kinesin such as Ncd that exhibits cooperativity between the
motor domains. A glutathione S-transferase-Kar3 construct has been reported to be motile (16.7-33.3 nm/s), but the motility was
slower than dimeric Ncd motility (100-230 nm/s) and much slower than
conventional kinesin (500-800 nm/s) (23, 25-34). The motor domain of
Kar3 has been crystallized (35), and its structure is strikingly
similar to both kinesin (36) and Ncd (37). As the first step toward
understanding the mechanochemistry of Kar3, we have analyzed the Kar3
motor domain (Met383-Lys729) using kinetic and
thermodynamic approaches. These findings are compared directly to the
monomeric Ncd construct MC6 (Met333-Lys700), as
well as the Ncd dimeric construct MC1
(Leu209-Lys700).
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EXPERIMENTAL PROCEDURES |
Materials--
The N-methylanthraniloyl derivatives
of adenine nucleotides (mantATP and mantADP) were synthesized and
characterized as described previously (38-40).
Buffer Conditions--
The kinetic and equilibrium binding
experiments were performed at 25 °C in ATPase buffer (50 mM HEPES, pH 7.2, with KOH, 5 mM magnesium
acetate, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM potassium acetate, 1 mM dithiothreitol, and
5% sucrose).
Protein Purification--
The Kar3 motor domain was expressed
from the plasmid pMW/Kar3 (41), and this clone was generously provided
by Dr. Sharyn Endow, Duke University Medical Center. Kar3 was expressed
in Escherichia coli strain BL21(DE3)pLysS and purified as
described (35) with slight modifications. Microtubules were assembled
from soluble tubulin (cold depolymerized and clarified) and stabilized
with 20 µM taxol. Kar3 has been noted to depolymerize
microtubules in vitro (25). However, we saw no evidence of
Kar3-induced depolymerization of the taxol-stabilized microtubules in
sedimentation experiments as described under "Microtubule Equilibrium
Binding." The stability of the microtubules was evaluated in the
presence and absence of MgATP.
Steady-state ATPase Kinetics--
Steady-state ATPase
measurements were determined by following the hydrolysis of
[
-32P]ATP to form
[
-32P]ADP·Pi as described previously
(42). The rate of ATP hydrolysis as a function of microtubule
concentration (Fig. 1B) was fit to the quadratic
equation,
|
(Eq. 1)
|
where Rate is the amount of product formed per second
per site, kcat is the maximum rate constant of
steady-state ATP hydrolysis, E0 is the enzyme
concentration, K1/2,Mt is the
steady-state Michaelis constant, and S0 is the
tubulin concentration.
Microtubule Equilibrium Binding Experiments--
These
experiments were performed as described previously (21). All
concentrations reported are final after mixing; the total reaction
volume was 220 µl. Kar3 at 2 µM was incubated with
microtubules (0-12 µM) in the absence of any added
nucleotide for 30 min and then centrifuged as described previously. The
supernatant was removed, and 5× Laemmli sample buffer was added. The
microtubule pellet was rinsed with ATPase buffer plus 20 µM taxol to remove any remaining supernatant, and the
microtubule pellet was then resuspended in 5× Laemmli sample buffer
plus 220 µl of ATPase buffer to obtain supernatant and pellet samples
of equal volume for SDS-PAGE. The Coomassie Blue-stained gel was
analyzed by a Microtek ScanMakerTM X6EL scanner (Microtek,
Redondo Beach, CA) and quantified using NIH Image version 1.60 to
determine the concentration of Kar3 in the supernatant and pellet at
each microtubule concentration. Fractional binding, defined as the
ratio of sedimented Kar3 to total Kar3, is presented in Fig. 2 as a
function of microtubule concentration. The data were fit to the
quadratic equation,
|
(Eq. 2)
|
where Mt·K/K0 is the
fraction of Kar3 sedimenting with the microtubule pellet,
K0 is total Kar3, Mt0 is the total
tubulin concentration, and Kd is the dissociation constant.
Acid Quench Experiments--
The pre-steady state kinetic
experiments to determine the rate constant for ATP hydrolysis were
performed with a rapid chemical quench-flow instrument (RQF-3, Kintek
Corp., Austin, TX) at 25 °C in ATPase buffer. Kar3 and
taxol-stabilized microtubules were preincubated for 30 min to form the
Mt·Kar3 complex and reacted with [
-32P]ATP. The
reaction was then quenched with 5 M formic acid, expelled from the instrument, and aliquots of each reaction were spotted on TLC
plates and developed to separate radiolabeled ADP from ATP. The data
were fit to the burst equation,
|
(Eq. 3)
|
where A is the amplitude of the pre-steady state
exponential burst phase, which represents the formation of
[
-32P]ADP·Pi on the active site during
the first turnover; kb is the rate of the burst
phase; t is time in seconds; and kss is the rate constant of the linear phase (µM
product·sec
1) and corresponds to steady-state turnover.
The plot of the burst rate versus ATP concentration was fit
to the equation,
|
(Eq. 4)
|
where kburst is the rate of product
formation (s
1), Amax is the
maximum rate constant of pre-steady state ATP hydrolysis
(s
1), and Kd,ATP is the
dissociation constant (µM).
Pulse-Chase Experiments--
To investigate the decreased burst
amplitude in the rapid quench experiments, pulse-chase experiments were
employed. In these experiments, the time course of ATP turnover was
measured by chasing the Mt·Kar3·[
-32P]ATP
intermediate with a cold MgATP chase (16-250-fold dilution) for
10 s (7-10 half-lives of enzyme turnover,
kcat = 0.5 s
1) and then quenched
with 5 M formic acid. The data were fit to Equation 3.
Stopped-flow Experiments--
The pre-steady state kinetics of
mantATP binding, mantADP binding, mantADP release, Kar3 binding to
microtubules, and detachment of Kar3 from microtubules were measured
using a SF-2001 KinTek stopped-flow instrument at 25 °C in ATPase
buffer. For the experiments with the mantADP and mantATP, the
fluorescence emission was measured at 450 nm using a 400-nm cutoff long
wavepass filter with excitation at 360 nm (Hg arc lamp). The mantADP
and mantATP binding data in Fig. 4 were fit to the following
equation,
|
(Eq. 5)
|
where kobs is the rate constant obtained
from the exponential phase of the fluorescence change,
kon defines the second-order rate constant for
mantADP or mantATP binding, and koff corresponds to the observed rate constant of mantADP or mantATP release, as determined by the y intercept. The dissociation kinetics of
the Mt·Kar3 complex and the kinetics of microtubule association by Kar3 were determined by the change in turbidity monitored at 340 nm.
All concentrations reported are final after mixing.
ADP Equilibrium Binding Experiments--
These experiments were
designed to determine the dissociation constant that the Mt·Kar3
complex has for ADP at equilibrium. By incubating the Mt·Kar3 complex
with ATP and allowing the complex to come to equilibrium, any ADP still
bound to the Mt·Kar3 complex should pellet with the microtubules when
subjected to centrifugation. Kar3 at 2 µM was incubated
with microtubules (10 µM tubulin) and with varying
concentrations of radiolabeled MgATP (0.1-100 µM) for 30 min. The 30-min incubation allowed all radiolabeled ATP to be converted
to ADP + Pi, which was confirmed by thin layer chromatography. The reaction mixture was then centrifuged. The supernatant was removed, and the pellet was resuspended in 110 µl of
4 M NaOH, followed by addition of 110 µl of ATPase
buffer. The pellets were not rinsed with additional ATPase buffer.
Aliquots of the reaction mixture, the supernatant, and the pellet were evaluated by liquid scintillation counting. Multiple aliquots of 5 µl
were used to determine total counts for the calculation of ADP
concentration. Control reactions with microtubules and nucleotide in
the absence of Kar3 were performed to determine the amount of
radiolabeled nucleotide that partitioned in the pellet without any Kar3
active sites being present. The amount of nucleotide present in the
Mt·Kar3 pellet was corrected by subtracting the concentration of ADP
in the Mt control at each ATP concentration. The data were plotted and
fit to the quadratic equation,
|
(Eq. 6)
|
where Mt·K·ADP is the concentration of ADP that
partitioned with the Mt·K complex,
K0 is total Kar3, ADP is the total nucleotide present, and Kd is the dissociation constant.
Data Analysis--
The mechanism presented in Scheme
1 was refined by computer simulation
(KIMSIM (43)). The acid quench and pulse-chase experiments (Figs. 5-7)
indicated that ATP binding was weak, and the ADP equilibrium binding
experiment presented in Fig. 8 showed that the Mt·Kar3 complex
retained ADP at 37.5% of the Kar3 active sites. Therefore, for the
simulations, the Kar3 concentration was modeled as 62.5% of the sites
nucleotide-free with 37.5% sites as the Kar3·ADP intermediate. The
acid quench transients were modeled by the following equation.
|
(Eq. 7)
|
The partitioning factor X1 represents the fraction of
Mt·K·ATP that proceeds toward ATP hydrolysis with the following
equation.
|
(Eq. 8)
|
The mantADP release kinetics were modeled with the assumption
that mantADP would not rebind to the Mt·Kar3 complex upon release of
ADP because of the 1 mM MgATP chase in the microtubule
syringe (Fig. 3). However, the equilibrium binding experiments (Fig. 2) indicated that Kar3 bound microtubules weakly, suggesting that there
may be reversals at Step 6' in Scheme 1. We tested the hypothesis that
upon collision of the Kar3·mantADP intermediate with the microtubule,
mantADP release may not occur at each site. Therefore, the
equation,
|
(Eq. 9)
|
with the partitioning factor X1 representing the fraction of
Mt·K·mantADP that proceeds toward release of mantADP to
form the Mt·Kar3 intermediate with the following equation.
|
(Eq. 10)
|
 |
RESULTS |
Scheme 1 is an ATPase mechanism for the Kar3 motor domain based on
the observed kinetics presented in this article with refinement by
computer simulation (43). We have compared these results with those of
Ncd, another well characterized Kin C kinesin that is also involved in
spindle assembly and dynamics (21, 22, 24, 38, 44, 45). The constants
for Kar3, monomeric Ncd construct MC6, and dimeric Ncd MC1 are
presented in Table I for direct
comparison.
All steps in Scheme 1 were measured for the Kar3 motor domain except
inorganic phosphate release (k+5'). The
microtubule association kinetics were measured
(k+6'), but the signal to noise ratio made data
collection difficult to interpret because of the relatively small mass
of the motor domain in comparison to the microtubule. The data are
presented in Table I, but represent only an estimation of the rate constant.
Steady-state ATP Hydrolysis--
In the absence of microtubules,
the rate of ATP turnover by Kar3 is 0.004 s
1. For kinesin
superfamily members, this rate is dramatically activated by
microtubules (18-21, 44, 46, 47), and Kar3 also displays this property
(Fig. 1). For the experiments presented
in this paper, three preparations of the Kar3 motor domain were used. The steady-state parameters, based on 11 ATPase assays, are as follows:
kcat = 0.49 ± 0.02 s
1
(0.36-0.59 s
1), Km,ATP = 12.2 ± 2.8 µM (4.0-19.2 µM),
K1/2,Mt = 6.0 ± 0.7 µM
(3.9-8.7 µM).

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Fig. 1.
Steady-state ATP hydrolysis.
A, the Mt·Kar3 complex (1 µM Kar3, 50 µM tubulin, stabilized with taxol) was pre-formed and
incubated with varying concentrations of MgATP (1-1000
µM). The rate of turnover of [ -32P]ATP
is plotted as a function of ATP concentration, and the data were fit to
a hyperbola. The kcat was determined to be
0.56 ± 0.01 s 1 with the
Km,ATP of 15.3 ± 1.9 µM. B, the Mt·Kar3 complex (1 µM Kar3, 0-55 µM tubulin) was pre-formed
and incubated with 1 mM MgATP. The rate of ATP turnover in
the absence of microtubules is 0.004 s 1. The rate of
[ -32P]ATP hydrolysis is plotted as a function of
tubulin concentration, and the fit of the data to Equation 1 yields
kcat = 0.51 ± 0.02 s 1;
K1/2,Mt of 6.2 ± 1.0 µM.
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Equilibrium Binding of Kar3 to the Microtubule--
To ascertain
the relative affinity of the Kar3 motor domain construct for
microtubules, equilibrium binding experiments were employed. These
experiments were performed as a function of microtubule concentration
and in the absence of added nucleotide or nucleotide analogs. Fig.
2 shows that the Kar3 motor domain
partitioned with the microtubules as a function of microtubule
concentration with the Kd,Mt at 0.68 µM tubulin. Fractional binding reached 100%, suggesting
the Kar3 was fully active. This dissociation constant is 3-fold weaker
than observed for the monomeric construct of Ncd, MC6 at
Kd,Mt = 0.20 µM
tubulin (21).

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Fig. 2.
Equilibrium binding of Kar3 to the
microtubule. Kar3 (2 µM) was incubated with
microtubules (0-12 µM tubulin, 20 µM
taxol) in the absence of added nucleotide. The fraction of Kar3 that
sedimented with the microtubules was plotted as a function of tubulin
concentration. The fit of the data to Equation 2 provides the
Kd,Mt = 0.68 ± 0.13 µM with maximal fractional binding at 1.1 ± 0.05 µM.
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Pre-Steady State Kinetics of MantADP Release from the
Mt·Kar3·MantADP Complex--
The Kar3 motor domain was incubated
with a fluorescent analog of ADP, mantADP, at a 1:2 ratio to exchange
the ADP bound at the active site for mantADP. The Kar3·mantADP
complex was then rapidly mixed with microtubules or with microtubules
plus 1 mM MgAMP-PNP,1 1 mM
MgADP, or 1 mM MgATP in the stopped-flow instrument (Fig. 3A). Note that the amplitude
of the exponential quenching of fluorescence, monitored as a function
of time, is greatest in the presence of the MgATP or MgADP chase,
implying that the mantADP nucleotide, once released, may
rebind the active site of Kar3 in the absence of the chase.
These data may indicate that AMP-PNP binds weakly to the active site as
observed for Ncd (24). The observed differences in the fluorescence
amplitude based on nucleotide present suggest that the Kar3 motor
domain remains in a conformation after microtubule-activated ADP
release that permits rapid mantADP rebinding. Monomeric MC6 also
exhibited this characteristic (21).

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Fig. 3.
Pre-steady state kinetics of mantADP release
from the Mt·Kar3·MantADP complex. A, a pre-formed
Kar3·mantADP complex (2 µM Kar3, 4 µM
mantADP) was rapidly mixed in the stopped-flow with taxol-stabilized
microtubules (50 µM tubulin) containing either no added
nucleotide, 1 mM MgAMP-PNP, 1 mM MgADP, or 1 mM MgATP. B, the observed
exponential rate constants were plotted as a function of microtubule
concentration. All microtubule concentrations contained 1 mM MgATP to prevent rebinding of the mantADP. The data were
fit to a hyperbola where the maximum rate constant of
microtubule-stimulated mantADP release (k+7) was
0.40 ± 0.009 s 1;
K1/2,Mt = 3.9 ± 0.3 µM.
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|
The experiment was repeated as a function of microtubule concentration
with MgATP to provide a chase and prevent mantADP rebinding to the
active site. The fit of these data to a hyperbola (Fig. 3B)
yielded the maximum rate constant of mantADP release,
k+7 = 0.4 s
1. This rate constant
appears to be the rate-limiting step for the Mt·Kar3 ATPase as it is
the most similar to the steady-state kcat at 0.5 s
1. Rate-limiting ADP release was also observed for Ncd,
another Kin C kinesin (21, 22, 24, 45, 46, 48).
Pre-Steady State Kinetics of MantADP and MantATP Binding to the
Mt·Kar3 Complex--
Our previous work with MC6 suggested that ADP
is in a rapid equilibrium on and off the active site of the Mt·MC6
complex (21). In the case of MC6, this partitioning between ADP
rebinding and ATP binding (M·N·ADP
M·N
M·N·ATP) appeared to limit steady-state turnover for
monomeric Ncd. To investigate if this mechanism accounted for the Kar3
motor domain kinetics, as implied by the mantADP release data, we
explored the possibility that ADP could rebind the active site of the
Mt·Kar3 complex. In this experiment, a pre-formed Mt·Kar3 complex
was rapidly mixed in the stopped-flow with varying concentrations of
mantADP. The inset of Fig.
4A shows a typical experiment
that was performed at 25 µM mantADP. The kinetics are
biphasic with a rapid fluorescence enhancement as mantADP moves from
the hydrophilic buffer into the more hydrophobic active site of the
Mt·Kar3 complex. The slow, second phase of fluorescence enhancement
is attributed to a population of Kar3 sites with ADP bound initially
that subsequently became available for mantADP binding once ADP was
released. The experiment was repeated as a function of mantADP
concentration, and the initial exponential rates provided the apparent
second-order rate constant of mantADP binding,
k
7 = 1.12 µM
1
s
1. The k+7, obtained from the
y intercept, was 0.9 ± 0.3 s
1. This rate
constant is somewhat higher than the microtubule-dependent rate constant of mantADP release (Fig. 3B), but the rate
constant obtained from the y intercept is less accurate than
the direct measure. However, the mantADP association kinetics, as well
as the results presented in Fig. 3, provide evidence that step 7 in
Scheme 1 is reversible.

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Fig. 4.
Pre-steady state kinetics of mantADP and
mantATP binding to the Mt·Kar3 complex. A, the
inset represents the pre-formed Mt·Kar3 complex (5 µM Kar3, 10 µM tubulin, 20 µM
taxol) being rapidly mixed in the stopped-flow instrument with 25 µM mantADP. The jagged line represents the
increase in fluorescence over time, and the smooth line is
the fit of the data to two exponential functions. The rate of the
initial rapid phase was 29.2 ± 0.3 s 1, and the rate
of the slower, second phase was 0.4 ± 0.005 s 1. The
initial exponential rate of the fluorescence enhancement increased as a
function of mantADP concentration, and these rate constants were
plotted as a function of mantADP concentration. The fit of these data
to Equation 5 gave an apparent second-order rate constant of mantADP
binding (k 7) of 1.12 ± 0.01 µM 1 s 1. B, a
pre-formed Mt·Kar3 complex (8 µM Kar3, 20 µM tubulin, 20 µM taxol) was rapidly mixed
with varying concentrations of mantATP. The inset represents
the experiments performed with 25 µM mantATP, and the fit
provides the rate constant of the initial phase at 30.8 ± 0.4 s 1, and the rate constant of the second phase at
0.24 ± 0.001 s 1. The exponential rate constants
from the initial rapid fluorescence enhancement were plotted as a
function of mantATP concentration. The fit of the data to Equation 5
yielded the apparent second-order rate constant of mantATP binding
(k+1) = 1.20 ± 0.02 µM 1 s 1.
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To investigate the rate of ATP binding to the Mt·Kar3 complex, a
pre-formed Mt·Kar3 complex was rapidly mixed in the stopped-flow with
varying concentrations of mantATP (Fig. 4B). The observed rate of the initial fluorescence enhancement increased as a function of
mantATP concentration with k+1 = 1.2 µM
1 s
1. The slow, second
phase of fluorescence enhancement is attributed to ADP being present on
a population of Kar3 active sites; therefore, for these sites, mantATP
binding was limited by the rate of ADP release (0.4 s
1).
Acid Quench Kinetics of the Mt·Kar3 Complex--
A pre-formed
Mt·Kar3 complex (10 µM Kar3, 30 µM
tubulin) was rapidly mixed in the rapid quench instrument with varying
concentrations of [
-32P]ATP, and the time dependence
of ATP hydrolysis was determined (Fig.
5). The results show that there was a
burst of product formation (ADP·Pi) at the active site
during the first ATP turnover, indicating that the rate-limiting step
occurs after the hydrolysis. The linear phase of the data correlated
well with the steady-state experiments for Kar3. The burst rates
increased as a function of ATP concentration, and the rate constant for
ATP hydrolysis (k+3) was 16 s
1
with the Kd,ATP at 319 µM.
The Kd,ATP implies that ATP binding was
very weak for the Mt·Kar3 complex, a result in agreement with the
mantATP binding data.

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Fig. 5.
Acid quench kinetics of the Mt·Kar3
complex. A, a pre-formed Mt·Kar3 complex (10 µM Kar3, 30 µM tubulin, 30 µM
taxol) was rapidly mixed in a rapid quenched-flow instrument with 800 µM [ -32P]MgATP. The data were fit to
Equation 3 which provided the burst amplitude at 2.53 ± 0.78 µM, the rate of the pre-steady state burst at 11.2 ± 7.8 s 1, and the rate of the linear phase at 4.47 ± 0.56 µM s 1. B, the
exponential rate constants of the burst phase (kb)
were plotted as a function of [ -32P]MgATP
concentration. The data were fit to Equation 4, and the maximum rate
constant of ATP hydrolysis (k+3) = 16.2 ± 1.9 s 1;
Kd,ATP = 319 ± 92 µM.
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Examination of the amplitude of the burst phase reveals that at even
very high ATP concentrations (800 µM ATP), the amplitude of the burst did not approach the enzyme concentration used in the
experiment. The burst amplitude represents formation of the Kar3·ADP·Pi intermediate; therefore, this constant can
be related to the active sites of Kar3 available to bind and hydrolyze
ATP during the first turnover. There are several possible explanations for the reduced burst amplitude. The first is that there is inactive enzyme present. We assume that the vast majority of Kar3 present in
these assays is active because of data presented in Fig. 2 as well as
active site assays not shown. Second, there could be ADP still bound at
the active site, preventing access to the active site for ATP binding.
The results presented in Figs. 3A and 4A indicate
that mantADP can rebind the active site; therefore this could be one
explanation for the decreased burst amplitude. A third explanation
could be a slow on-rate associated with ATP binding to the active site
of the Mt·Kar3 complex. Fig. 4B shows that the rate of
mantATP binding is 1.2 µM
1
s
1. This rate should not limit ATP hydrolysis. Finally,
there could be a significant off-rate (k
1),
implying that there is kinetic partitioning where ATP is in a rapid
equilibrium on and off the active site before moving forward in the
ATPase cycle to ATP hydrolysis.
Acid Quench Pulse-Chase Comparison of the Mt·Kar3
Complex--
To investigate the decreased burst amplitude, pulse-chase
experiments were pursued (Figs. 6 and
7). In these experiments, any
[
-32P]ATP bound at the active site would proceed
forward toward hydrolysis by the addition of an unlabeled ATP chase.
ATP weakly bound at the active site would be diluted by the high
concentration of unlabeled ATP. The experimental design is such that it
will reveal a stable Mt·Kar3·ATP intermediate if one were to
exist. Fig. 6 depicts two experiments performed by mixing the Mt·Kar3
complex (5 µM Kar3, 30 µM tubulin) at 10 and 25 µM MgATP. Note that at each ATP concentration, the
amplitude of the pulse-chase transient was significantly higher than
the acid quench amplitude. These results show that a stable
Mt·Kar3·ATP intermediate can be trapped.

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Fig. 6.
Acid quench pulse-chase comparison of the
Mt·Kar3 complex. A pre-formed Mt·Kar3 complex (5 µM Kar3, 30 µM tubulin, 30 µM
taxol) was rapidly mixed with varying concentrations of
[ -32P]MgATP and either quenched with 5 M formic acid ( ) or chased with 5 mM MgATP
( ). A, 10 µM
[ -32P]MgATP. B, 25 µM
[ -32P]MgATP.
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Fig. 7.
Pulse-chase kinetics of the Mt·Kar3
complex. A, a pre-formed Mt·Kar3 complex (5 µM Kar3, 30 µM tubulin, 30 µM
taxol) was incubated with varying concentrations of
[ -32P]MgATP (10 µM, ; 25 µM, ; 100 µM, ; and 150 µM, ), followed by the MgATP chase. Each time
course was fit to Equation 3. B, the rate constants of the
exponential burst phase of each transient were plotted as a function of
[ -32P]MgATP concentration. The fit of the data to
a hyperbola yielded the maximum rate constant
(k+2) = 498 ± 76 s 1,
and the Kd,ATP = 82.8 ± 33.1 µM.
|
|
This experiment was repeated at higher ATP concentrations, ranging from
10 to 300 µM (Fig. 7). The results show that the
first-order rate of the burst is very rapid and saturates at >500
s
1. The fact that the burst rate in the pulse-chase
experiments is not linear and saturates at increasing concentrations of
ATP indicates that there is a rate-limiting conformational change (k+2, Scheme 1) that occurs prior to ATP
hydrolysis. The plot of the burst amplitude versus
MgATP concentration (data not shown) never reached saturation and
exceeded the enzyme concentration, implying that the Kar3 motor
domain remains associated with the microtubule for multiple cycles of
ATP turnover as observed for monomeric kinesin and Ncd constructs
(18-22).
ADP Equilibrium Binding to the Mt·Kar3 Complex--
To determine
whether the Mt·Kar3 complex could sequester ADP at the active site of
Kar3, equilibrium experiments using radiolabeled MgATP were employed
(Fig. 8). In these experiments, the
Mt·Kar3 complex was incubated with [
-32P]ATP for a
period of time sufficient for all the ATP to be hydrolyzed. Any
[
-32P]ADP still bound at the active site of Kar3
should partition to the microtubule pellet upon centrifugation. Fig. 8
shows that the concentration of ADP partitioning with the Mt·Kar3
complex reached 0.75 µM, implying that 37.5% of the
Mt·Kar3 sites (0.75 µM ADP/2 µM Kar3
sites) have ADP tightly bound to them. The
Kd,ADP for the Mt·Kar3 complex was
1.67 µM, and this experiment provides additional evidence
that ADP can remain bound to the Mt·Kar3 complex.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 8.
ADP equilibrium binding to the Mt·Kar3
complex. A pre-formed Mt·Kar3 complex (2 µM Kar3,
10 µM tubulin, 20 µM taxol) was incubated
with varying concentrations of [ -32P]MgATP. The
concentration of [ -32P]ADP that sedimented with the
Mt·Kar3 complex was plotted as a function of
[ -32P]MgATP concentration. The fit of the data to
Equation 6 provided the Kd,ADP = 1.67 ± 0.58 µM with maximal ADP binding at
0.75 ± 0.06 µM.
|
|
ATP-induced Dissociation Kinetics of the Mt·Kar3
Complex--
Our previous analysis of Ncd showed that the monomeric
construct, MC6, failed to dissociate from the microtubule upon the addition of MgATP. Rather, additional salt and MgATP were necessary to
weaken the interaction of the motor with the microtubule (21). Similarly, it has been proposed that monomeric kinesin constructs cannot easily dissociate from the microtubule at low salt conditions (18-20). There is evidence from the pulse-chase experiments (data not
shown) that the Kar3 motor domain hydrolyzes multiple ATP molecules per
encounter with the microtubule, implying that the Kar3 motor domain may
not readily dissociate from the microtubule. To explore if this were
truly the case, we employed the same experiment that was used to
determine whether monomeric Ncd dissociated from the microtubule
(21).
In this experiment (Fig. 9), a pre-formed
Mt·Kar3 complex was reacted with buffer, buffer with 50 mM KCl, and 1 mM MgATP with 50 mM
KCl. The 50 mM KCl was in addition to the 50 mM
potassium acetate present in the ATPase buffer. The additional salt
weakened the affinity of the motor domain for the microtubule and acted to increase the ATP-dependent signal to noise ratio.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 9.
ATP-promoted dissociation kinetics of the
Mt·Kar3 complex. A, a Mt·Kar3 complex (5 µM Kar3, 6 µM tubulin, 20 µM
taxol) was rapidly mixed in the stopped-flow apparatus with buffer,
buffer with 50 mM KCl, or 1 mM MgATP with 50 mM KCl, and turbidity was monitored. The additional salt
was used to weaken the motor affinity to the microtubule (57) and acted
to increase the signal to noise ratio. B, the same
experiment was repeated as in panel A, but the Mt·Kar3
complex was mixed with varying concentrations of ATP and KCl. ATPase
buffer contains 50 mM potassium acetate such that the final
salt concentrations, after the contribution of KCl and potassium
acetate, ranged from 50 to 300 mM. C, the rates
of the observed exponential decrease in turbidity were plotted as a
function of MgATP concentration. The inset represents the
experiments where the Mt·Kar3 complex is rapidly mixed in the
stopped-flow instrument with, from top to bottom
trace, 5, 8, 10, 25, and 50 µM MgATP. All reactions
contained 50 mM KCl. The data were fit to a double
exponential function. The maximum rate constant of ATP-promoted
dissociation (k+4') was 6.7 ± 0.1 s 1 with the K1/2,ATP = 5.7 ± 0.3 µM.
|
|
Fig. 9B shows the dissociation of Kar3 from the microtubule
as a function of ATP and KCl concentrations. Either ATP or 250 mM KCl did lead to a decrease in turbidity, but the
amplitude associated with the turbidity signal was small relative to
the amplitude of ATP plus KCl dissociation. The dissociation transients at 200 and 250 mM KCl display aberrant exponential curves,
suggesting that the high salt was required to drive all Kar3 motors off
the microtubule.
We next performed the dissociation experiment as a function of ATP
concentration (Fig. 9C) in the presence of 50 mM
KCl to obtain a more robust ATP-dependent signal and to
avoid the higher concentrations of KCl that produced stopped-flow
transients that were not easily interpretable. The apparent rate
constant of dissociation was 6.7 s
1 with the
K1/2,ATP at 5.7 µM.
These data (Fig. 9), taken together, imply that MgATP is not sufficient
to dissociate the Mt·Kar3 complex; salt must be added to weaken the
affinity with the microtubule. For our studies with monomeric Ncd
(MC6), we proposed that there was a small population of monomers that
can dissociate from the microtubule in the presence of ATP as reflected
by the very small change in turbidity that can be detected (21). The
addition of salt weakened the interaction of MC6 with the microtubule,
creating a turbidity signal with greater amplitude. The results for
ATP-induced dissociation for the Kar3 motor domain mirror the kinetics
we saw with MC6 (21). Therefore, the hypothesis that a small population
of motor molecules is causing a turbidity signal with less amplitude
may also be true for the Kar3 motor domain.
 |
DISCUSSION |
This study presents the ATPase mechanism of a motor domain
construct of the naturally occurring monomeric motor, Kar3. In yeast,
Kar3 heterodimerizes with either Cik1 or Vik1 (9, 10), but in each
case, there is only a single motor domain present. We have used
steady-state and pre-steady state kinetics to investigate the turnover
of ATP by the motor domain itself to determine what is the minimal
functional unit of Kar3 necessary for movement. Table I includes the
kinetic constants derived from the experimental data and refined by
computer simulation, and the mechanism is presented in Scheme 1.
Kar3 and Ncd--
Our initial observations were that the Kar3
motor domain was very similar to the single Ncd motor domain constructs
reported (21, 22). These motor domains are 46% identical, and their crystal structures are practically superimposable (35). The sequence
and structural similarity between these two motor domains makes
comparisons between these two motors more relevant because both are Kin
C motors that promote minus-end directed microtubule motility and both
function in the spindle. As seen with MC6, the steady-state ATPase was
very slow when compared with monomeric kinesin constructs (18, 20,
49-51). This observation implies that there are mechanistic
similarities between the Kin C family members that are distinct from
other kinesin superfamily members.
The analysis of the Kar3 motor domain revealed that like monomeric MC6
(21), Kar3 when bound to the microtubule did rebind ADP. This
partitioning may affect the overall steady-state rate of ATP turnover.
Furthermore, the rapid quench kinetics show a decreased burst
amplitude, indicative of enzyme sites being unavailable for ATP binding
and subsequent turnover. This behavior was seen with the monomeric Ncd
construct, although full burst amplitude was achieved at high ATP
concentrations with MC6 (21), unlike Kar3. For Ncd, we proposed that
the partner motor domain was required to return the motor to the
conformation more competent to bind ATP and less accessible for ADP
binding. The hypothesis we are presently testing is that for Kar3, the
associated polypeptides Cik1 and Vik1 provide this function.
There do appear to be several differences between the monomeric Ncd MC6
and the Kar3 motor domain. The fast ATP off-rate
(k
1) that was modeled for the pulse-chase and
acid-quench experiments (Figs. 5-7) was not observed for monomeric Ncd
(21, 22). Why this difference occurs is not readily apparent,
especially given the sequence and structure similarities. The rapid
quench burst experiments are very different as well. MC6 was able to
achieve a burst amplitude equal to its enzyme concentration (21). Even though both Kar3 and MC6 rebind ADP quite readily, the difference between the bursts may be rooted in the fast off-rate of ATP binding, as well as the weak Kd,ATP observed for
Kar3. Also, MC6 has a 3-fold tighter affinity for the microtubule (0.2 µM) than does the Kar3 motor domain (0.67 µM). Experiments to compare directly the dissociation of
both motor domains from the microtubule seem to indicate that MC6 does
not release from the microtubule as readily in the presence of salt as
does the Kar3 motor domain (data not shown). This is consistent with
the findings presented here and leads to the hypothesis that the Kar3
motor domain is better able to dissociate from the microtubule than
MC6, but both require something else structurally to detach from the
microtubule for ATP-dependent force generation. For MC6, it
is the other head of the dimer that is necessary for detachment. For
Kar3, it is unclear what is necessary for microtubule dissociation. A
key step in the ATPase cycle of any molecular motor is the detachment from the filament to take a step to the next binding site on the filament. Monomeric kinesin and Ncd motors stay attached to the microtubule, yet they continue to turnover ATP (18-22). However, this
ATP turnover is not coupled to movement as in the case of dimeric
kinesin and Ncd. Because Kar3 is a naturally occurring monomeric motor,
one hypothesis was that the elements necessary for movement would be
contained in the Kar3 motor domain. We have shown that this is not the
case for the Kar3 motor domain. However, some if not all of the
necessary components for movement must be contained in the GST-Kar3
construct because it generates microtubule sliding in vitro
(25).
Monomeric Kinesins--
This study addresses the question of
how a monomeric motor can promote motility. Okada and Hirokawa (52)
propose a mechanism for the processive monomeric kinesin Kif1A that
involves the use of the carboxyl-terminal tail of tubulin to allow the
motor to essentially swing to the next microtubule binding site. This
mechanism is not applicable to Kar3 because Kar3 does not contain the K loop motif necessary for Kif1A motility. Also, Kar3 probably functions in a cooperative manner with multiple Kar3 motors, either in the spindle itself or the array of microtubules that forms when two yeast
nuclei fuse during karyogamy.
Our analysis of Kar3 motor domain emphasizes the need to understand the
role of dimerization with either Cik1 or Vik1 for Kar3 function. Why
have two separate polypeptide partners? Manning and Snyder (10) have
shown that the localization of the Kar3 motor is dependent upon the
polypeptide that is bound. Kar3 when bound to Cik1 localizes to the
spindle midzone during mitosis and to the microtubule bundle between
nuclei during karyogamy. Mitosis in the yeast S. cerevisiae
takes place within the nucleus as there is no nuclear membrane
breakdown. During karyogamy, some other factor must mask the nuclear
localization signals of both Kar3 and Cik1 for the motor to be exported
to the cytoplasm. It is not known whether the mechanochemistry of
Kar3-Cik1 is differentially regulated for its function during mitosis
as compared with meiosis. Also, the role of the Kar3-Vik1 heterodimer
is largely unknown. When in a complex with Vik1, Kar3 localizes to the
spindle poles where it may be involved in regulating microtubule number
as proposed by Manning et al. (10). One testable hypothesis
is that Cik1 and Vik1 modulate Kar3 mechanochemistry such that the
Kar3-Vik1 heterodimer behaves differently than the Kar3-Cik1
heterodimer. If they were mechanistically comparable, then the results
would argue for differential targeting of Vik1 in comparison to Cik1.
Microtubule-destabilizing Activity--
Kar3 also possesses a
microtubule destabilizing activity. This activity has been documented
both in vitro (25) and in vivo (12).
Overexpression of KAR3 leads to a shortening of nuclear microtubules in vivo (15). How this activity relates to the ATP-dependent force generation of the Kar3 motor domain is
unknown. One hypothesis is that Kar3 motor activity translocates the
protein to the minus-end of the microtubule where it can cause
microtubule instability. Endow et al. (25) documented
microtubule shortening from the minus-end in vitro with the
GST-Kar3 construct; however, we did not observe depolymerization of
taxol-stabilized microtubules by the Kar3 motor domain.
Another question to be addressed is whether the
microtubule-destabilizing activity of Kar3 relates to the
microtubule-destabilizing activity of another subfamily of kinesins,
the Kin I kinesins (53), and specifically XCKM1 and MCAK (54-56). Kin
I kinesins have not been documented to promote microtubule
movement although they do contain the conserved kinesin motor domain,
albeit in the interior of the polypeptide. All kinesins that are known
to promote motility have either an amino-terminal motor domain or a
carboxyl-terminal motor domain. How does the microtubule-destabilizing activity of Kar3 compare with the destabilizing activity of XKCM1 and
MCAK? All bind and hydrolyze ATP to drive conformational changes, but
XKCM1 and MCAK partition with the tubulin subunit rather than remaining
associated with the ends of microtubules as observed for Kar3. In the
studies reported here, the added taxol was sufficient to prevent
microtubule depolymerization. Therefore, our analysis evaluated
microtubule-dependent force generation for motility separate from nucleotide-dependent microtubule depolymerization activity.
The kinetic study of the Kar3 motor domain presented in this article
lays the foundation for our study of the Kar3 motor in more detail. The
GST-Kar3 construct will be interesting to study as it does promote
motility (25). Future experiments involving Kar3-Cik1 and Kar3-Vik1
heterodimers will probe the cellular roles for each associated
polypeptide as well as provide us with the first example of a kinesin
superfamily member whose mechanochemistry may be affected by an
associated polypeptide, analogous to myosin and dynein family members.
Our results with Kar3 in comparison to cytoplasmic dynein, Ncd, and
kinesin, will provide insight to understand the mechanistic diversity
that nature has evolved to promote microtubule-based motility.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Sharyn Endow (Duke University
Medical Center) for the generous gift of the Kar3 clone, and Drs.
William Saunders (University of Pittsburgh) and Timothy Lohman
(Washington University School of Medicine) for thoughtful review of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by NIGMS National Institutes
of Health Grant GM54141 and NIAMS National Institutes of Health Department of Health and Human Services Career Development Award K02-AR47841.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 an Andrew Mellon Predoctoral Fellowship. Present
address: Dept. of Molecular, Cellular, and Developmental Biology, Yale
University, P. O. Box 208103, New Haven, CT 06520-8103.
§
Recipient of an American Cancer Society Junior Faculty Research
Award JFRA-618. To whom correspondence should be addressed: Dept. of
Biological Sciences, 518 Langley Hall, University of Pittsburgh,
Pittsburgh, PA 15260. Tel.: 412-624-5842; Fax: 412-624-4759; E-mail: spg1+@pitt.edu.
Published, JBC Papers in Press, November 24, 2002, DOI 10.1074/jbc.M206219200
 |
ABBREVIATIONS |
The abbreviation used is:
AMP-PNP, 5'-adenylyl-
,
-imidodiphosphate.
 |
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