humanmyosinvcisalowdutyrationonprocessivemotor · 2008-05-30 · april 18, 2008•volume...

Human Myosin Vc Is a Low Duty Ratio Nonprocessive Motor*

Received for publication, September 12, 2007, and in revised form, December 3, 2007 Published, JBC Papers in Press, December 12, 2007, DOI 10.1074/jbc.M707657200

Shinya Watanabe‡1, Tomonobu M. Watanabe‡, Osamu Sato‡, Junya Awata‡, Kazuaki Homma‡, Nobuhisa Umeki‡,Hideo Higuchi§, Reiko Ikebe‡, and Mitsuo Ikebe‡2

From the ‡Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 and§Biomedical Research Organization, Tohoku University, Sendai 981, Japan

There are three distinct members of the myosin V family invertebrates, and each isoform is involved in differentmembranetrafficking pathways. Both myosin Va and Vb have demon-strated that they are high duty ratio motors that are consistentwith the processive nature of these motors. Here we report thatthe ATPase cycle mechanism of the single-headed construct ofmyosin Vc is quite different from those of other vertebratemyo-sinV isoforms.KATPase of the actin-activatedATPasewas 62�M,which is much higher than that of myosin Va (�1 �M). The rateof ADP release from actomyosin Vc was 12.7 s�1, which was 2times greater than the entire ATPase cycle rate, 6.5 s�1. Pi burstsizewas 0.31, indicating that the equilibriumof theATPhydrol-ysis step is shifted to the prehydrolysis form. Our kinetic model,based on all kinetic data we determined in this study, suggeststhat myosin Vc spends the majority of the ATPase cycle time inthe weak actin binding state in contrast to myosin Va and Vb.Consistently, the two-headedmyosin Vc construct did not showprocessive movement in total internal reflection fluorescencemicroscope analysis, demonstrating that myosin Vc is a non-processivemotor.Our findings suggest thatmyosinVc fulfills itsfunction as a cargo transporter by different mechanisms fromother myosin V isoforms.

Class V myosins function as actin-based motors for variouscargo transportations. Class Vmyosins have been widely foundin species from lower organisms, such as yeast, Caenorhabditiselegans, andDrosophila, to vertebrates. In vertebrates, there arethree distinct subclasses ofmyosin V. Among them,most of theresearch has focused onmyosinVa, which plays a critical role inmembrane trafficking, including melanosome transport (1, 2)and endoplasmic reticulum transport (3–6). In contrast tomyosin Va expressed mainly in brain and melanocytes, myosinVb and Vc are widely expressed in a variety of tissues, althoughthe relative expression level is distinct from each other. Forinstance, myosin Vb is most abundant in kidney, whereas myo-sin Vc is significantly expressed in epithelial and glandular tis-sues, including pancreas, colon, and stomach (7).

Evidence has shown that myosin V is a cargo transporter andserves as a membrane transporter in various biological pro-cesses. Myosin Va is involved in melanosome transportation inmelanocytes (1, 2) and synaptic vesicle movement in neuronalcells (3–6). On the other hand, myosin Vb is involved in theplasmamembrane recycling systems of transferrin receptor (8),chemokine receptor CXCR2 (9), andM4muscarinic acetylcho-line receptor (10). Although the tissue distribution of theexpression of myosin Vb and Vc is overlapped, it is thought thatthey have a distinct physiological relevance. Myosin Vb directlyinteractswith the smallGTP-bindingprotein,Rab11a (8),whereasmyosin Vc colocalizes with Rab8, but not Rab11a (7), suggestingthateachmyosinVisoformhasspecific targetcargomoleculesandis involved in different membrane trafficking pathways.The most intriguing finding was that myosin Va moves pro-

cessively on actin filaments for a long distance without dissoci-ating from actin (11, 12). Since myosin Va has a long neckdomain consisting of six IQ motifs and a coiled-coil domain toform a two-headed structure, it is thought that myosin Vamoves in a hand-over-hand fashion in which each head alter-natively steps on an actinmonomer at a half-helical pitch aheadon the filament. The enzyme kinetic studies have shown thatmyosin Va is a high duty ratio motor that spends the majority ofthe ATPase cycle in the strong actin binding state (13), which isthought to be required for processivemyosins to prevent the headdissociating away from the track during themovement on actin.Although the domain structure of myosin Va is common in

all class V myosins from lower eukaryotes to vertebrates,whether or not the processive nature of myosin Va is commonamong all class V myosins has been controversial. It has beenreported by multimolecule in vitro motility assays that twoyeast class V myosins, Myo2p and Myo4p, are nonprocessivemotors (14), although these myosins participate in membranetrafficking (15–17) and mRNA transport (18, 19), which sug-gests that they serve as cargo transporters. On the other hand, itwas shown recently by using singlemolecule assays thatMyo4pis a processive motor (20). Moreover, a recent kinetic studyshowed thatDrosophilamyosinV is a lowduty ratiomotor (21),suggesting that it is not a processive motor. Since there is noclear evidence thatDrosophilamyosin V is implicated in vesicletransport, myosin V may not be involved in the vesicle trans-porting process inDrosophila. The question is whether all threemyosin V isoforms expressed in vertebrates are processivemotors and support vesicular transportation in cells. Quiterecently, we reported that humanmyosinVb is a high duty ratioand processive motor like myosin Va (22). In the present study,we analyzed the detailed ATP hydrolysis mechanism of human

* This work was supported by National Institutes of Health Grants DC006103,AR 048526, AR 048898, and AR 41653. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.

1 Present address: Program in Molecular Medicine, University of Massachu-setts Medical School, Worcester, MA 01605.

2 To whom correspondence should be addressed: Dept. of Physiology, Uni-versity of Massachusetts Medical School, 55 Lake Ave. N., Worcester, MA01655. Tel.: 508-856-1954; Fax: 508-856-4600; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 16, pp. 10581–10592, April 18, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

APRIL 18, 2008 • VOLUME 283 • NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 10581

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myosin Vc for the first time. Our kinetic study demonstratedthat myosin Vc is a low duty ratio motor. Consistently, thesingle molecule assays of total internal reflection fluorescence(TIRF)3 microscopy revealed that myosin Vc does not moveprocessively on actin filaments. It is thought that myosin Vcserves as a membrane transporter in cells, but our results sug-gests that it would be achieved by a quite different mechanismfrom myosin Va and Vb.

EXPERIMENTAL PROCEDURES

Materials—Restriction enzymes and modifying enzymes werepurchased from New England Biolabs (Beverly, MA). Purinenucleoside phosphorylase, 7-methylguanosine, phosphoenol-pyruvate, pyruvate kinase, apyrase, ATP�S, and AMPPNP werepurchased from Sigma. Actin was prepared from rabbit skeletalmuscle according toSpudichandWatt (23). Pyrene-actinwaspre-pared as described (24). 7-Diethylamino-3-((((2-maleimidyl)-ethyl)amino)carbonyl)coumarin-labeled phosphate-bindingprotein (MDCC-PBP) was prepared as described (25, 26).Recombinant calmodulin was expressed in Escherichia coli andpurified as described previously (27). GFP-tagged myosin VaHMM was expressed in insect cells and purified as describedpreviously (28).Cloning, Expression, and Purification of Human Myosin Vc

Protein—Human myosin Vc cDNA was obtained from humankidney total RNAby reverse transcription-PCR.The nucleotidesequence was determined by direct DNA sequencing to con-firm the authenticity of the DNA sequence of the clone. ThecDNA fragments encoding Met1–Gln787 (subfragment 1 con-struct) and Met1–Glu1129 (HMM construct) were subclonedinto modified pFastBac1 baculovirus transfer vector (Invitro-gen) containing a FLAG tag sequence at the 5� end of thepolylinker region and GFP sequence for the HMM construct.The subfragment 1 construct (M5CIQ1) encodes the motordomain and the first IQ motif, and the HMM construct (GFP-M5CHMM) encodes the motor domain, all six IQ motifs, andthe first long coiled-coil region. To express the recombinantmyosin Vc proteins, Sf9 cells were co-infected with two virusesexpressing myosin Vc heavy chain and calmodulin. Theexpressed proteins were purified through an anti-FLAG M2affinity column (Sigma) as previously described (29). The puri-fied proteins were dialyzed against buffer A (25mMKCl, 20mMMOPS-KOH (pH 7.5), 2 mM MgCl2, 1 mM EGTA, and 1 mMDTT). The proteins were stored on ice and used within 2 days.Protein concentration was determined by the densitometryanalysis of SDS-PAGE using smooth muscle myosin heavychain as a standard. In addition, the active site concentration ofM5CIQ1 was determined by using the [3H]ADP�vanadate traptechnique as described previously (30).ATPase Assay—The steady-state ATPase activity was meas-

ured in the presence of the ATP regeneration system (20

units/ml pyruvate kinase and 2 mM phosphoenolpyruvate) at25 °C. The reaction was carried out in buffer A with 0.2 mg/mlcalmodulin. The liberated pyruvate was determined asdescribed (31).Stopped-flow Measurements—Kinetic measurements were

performed in bufferAwith 0.2mg/ml calmodulin at 25 °CusingaKinTek SF-2001 apparatus (KinTekCo., Clarence, PA).Mant-nucleotides (�ex � 280 nm) and pyrene-actin (�ex � 365 nm)were monitored through a 400 nm cut-off filter. Intrinsic tryp-tophan residues in M5CIQ1 were excited at 295 nm, and thefluorescence was monitored through a 340 nm cut-off filter.Light-scattering was monitored at 420 nm. MDCC-PBP(�ex � 465 nm) was monitored through a 450 nm cut-offfilter. To achieve nucleotide-free conditions, M5CIQ1 was incu-bated with 0.05 units/ml apyrase. In the measurement of phos-phate release, all solutions and syringes were preincubated with 3�M MDCC-PBP, 0.02 units/ml purine nucleoside phosphorylase,and 0.2 mM 7-methylguanosine. The volume ratio of the syringewas 1:1 in all singlemixing experiments and1:1:1 indoublemixingexperiments. Kinetic simulations were performed using STELLAsoftware (Isee Systems, Lebanon, NH).Quenched-flow Measurement—Quenched-flow measure-

ment was performed in buffer A with 0.2 mg/ml calmodulin at25 °C using a KinTek RQF-3 apparatus (KinTek Co., Clarence,PA) as described (32).Actin Cosedimentation Assay—Prior to the assay, M5CIQ1

was centrifuged at 300,000� g for 10min to remove any poten-tial aggregates, and the supernatant was used in the actincosedimentation assay. Various concentrations (2–60 �M) ofactin were mixed with 1 �M M5CIQ1 in buffer A with 0.2mg/ml calmodulin and incubated for 10 min at room tempera-ture. Immediately after adding 2.5 mM Mg-ATP�S or 5 mMMg-AMPPNP, the reaction mixtures were centrifuged at300,000 � g for 10 min. The supernatants and dissolved pelletswere subjected to the densitometry analysis of SDS-PAGE.Multimolecule in Vitro Motility Assay—The actin gliding

velocity was measured by an in vitro actin gliding assay asdescribed previously (22, 30). The experiment was done with abuffer containing 25mMKCl, 5mMMgCl2, 1mMEGTA, 25mMMOPS-KOH (pH 7.5), 0.2 mg/ml calmodulin, 1 mM ATP, 10mM DTT, and oxygen scavenger system (216 �g/ml glucoseoxidase, 36 �g/ml catalase, 4.5 mg/ml glucose).Single Molecule Assays—TIRFmicroscopy was set up as pre-

viously described (33). The fluorescently labeled actin filamentscontaining 0.5% biotinylated G-actin were adsorbed onto anavidin-coated quartz surface. GFP-tagged myosin Vc HMMand myosin Va HMM in buffer A containing 10 �M or 1 mMMgATP, 0.2 mg/ml calmodulin, and oxygen scavenger systemwere added to the actin filament-coated surface. The frame ratewas 20 ms, which was almost equal to the exposure time of theCCD camera. The movements of the individual fluorescentspots were analyzed using special software (G-Track, G-Ang-strom, Japan), which could find and track the position of thefluorescent spots automatically.

RESULTS

Expression and Purification of Myosin Vc Construct—In thekinetic study, we used a human myosin Vc single-headed con-

3 The abbreviations used are: TIRF, total internal reflection fluorescence;HMM, heavy meromyosin; AMPPNP, adenosine 5�-(�,�-imido)triphos-phate; ATP�S, adenosine 5�-[�-thio]triphosphate; MOPS, 4-morpho-linepropanesulfonic acid; MDCC, 7-diethylamino-3-((((2-maleimidyl)ethyl)-amino)carbonyl)coumarin; PBP, phosphate-binding protein; dmant-ATP,2�-deoxy, N-methylanthraniloyl-ATP; GFP, green fluorescent protein; DTT, di-thiothreitol; dmant-ADP, 2�-deoxy, N-methylanthraniloyl-ADP.

Duty Ratio and Processivity of Myosin Vc

10582 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 16 • APRIL 18, 2008


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struct having the entire motor domain and the first IQ motif(M5CIQ1). M5CIQ1 and calmodulin were co-expressed in Sf9cells and purified with anti-FLAGM2 affinity chromatography.The isolated M5CIQ1 was co-purified with calmodulin (Fig.1A). Themobility change of the lowmolecular mass peptide onSDS-PAGE increased with Ca2�, suggesting that it is calmodu-lin (data not shown). It is known that the essential light chainwas co-purified with chicken brain myosin Va (34, 35) and isthought to be a part of the light chains ofmyosin Va. Therefore,we examined whether the essential light chain binds toM5CIQ1.M5CIQ1 was co-expressed with nonmuscle essentiallight chain (LC17b) that is more widely distributed among var-ious tissues than LC1sa and LC17a, and the purified M5CIQ1was subjected to SDS-PAGE analysis. As shown in Fig. 1B, thelow molecular mass peptide co-purified with M5CIQ1 showeda Ca2�-dependent change in the migration, a characteristic ofcalmodulin. The results suggest that endogenous calmodulin,but not LC17b, was co-purified withM5CIQ1. The results indi-cate that calmodulin has a much higher affinity for the first IQmotif ofmyosinVc thanLC17b. It should be noted that the lightchains ofmammalianmyosinVa andVb are calmodulin but notLC17 (22, 36). In the present study, we usedM5CIQ1 andGFP-M5CHMMwith calmodulin for all experiments.Steady-state ATPase Activity of M5CIQ1—The steady-state

ATPase activity of M5CIQ1 was markedly activated by actinwith a hyperbolic saturation curve (Fig. 2). The Vmax of thesteady-state ATPase activity was 6.5� 0.4 s�1. Surprisingly, theKATPase was 62 � 9 �M at 25 mM KCl, which was much higherthan that of myosin Va (1 �M at 50 mM KCl) (13). The KATPasevalue of myosin Vc is rather similar to that of conventionalmyosin IIs, such as cardiac muscle, smooth muscle, and non-muscle myosins (37–41).ATP Binding to M5CIQ1 and Acto-M5CIQ1—The fluores-

cent nucleotide, dmant-ATP, was used to measure the rate of

ATP binding to M5CIQ1 and acto-M5CIQ1 (Fig. 3). The timecourses of the fluorescence enhancement followed single expo-nential kinetics for both M5CIQ1 and acto-M5CIQ1 (Fig. 3,insets). The observed rate constants (kobs) showed a lineardependence on the dmant-ATP concentration. The secondorder rate constants, obtained from the slopes of the lines, were2.4 � 0.1 �M�1 s�1 for M5CIQ1 (K1k�2) and 1.6 � 0.1 �M�1

s�1 for acto-M5CIQ1 (K�1k��2).ATP-induced Acto-M5CIQ1 Dissociation—The kinetics of

the ATP-induced transition to the weak actin binding state andthe dissociation ofM5CIQ1 fromactinwasmonitored bymeas-uring the changes in the pyrene fluorescence and light scatter-ing, respectively (Fig. 4). The increase in the pyrene fluores-cence upon the formation of the weak actin-binding form ofM5CIQ1 and the decrease in light scattering upon dissociationof pyrene-acto-M5CIQ1 followed single exponential kinetics(Fig. 4, inset). The kobs of both signals showed almost identicalhyperbolic saturation curves on theATP concentration (Fig. 4).These results suggest that the process of ATP binding to acto-M5CIQ1 is composed of two steps (K�1 and K�2 in Fig. 13A), andthe dissociation of M5CIQ1 from actin occurs immediatelyafter the transition from a strong actin binding state to a weakactin binding state. The initial slopes of the curves represent asecond order rate constant of ATP binding to acto-M5CIQ1(K�1K��2). The K�1K��2 values, obtained from the change inpyrene fluorescence and light scattering, were 1.8 � 0.1 and1.6 � 0.1 �M�1 s�1, respectively. These values are similar tothat obtained fromdmant-ATPbinding (Fig. 3), suggesting thatthemantmoiety does not significantly influence the ATP bind-ing rate to M5CIQ1. The maximal rates of the hyperboliccurves gave k�2 of �300 s�1.Enhancement of Intrinsic Tryptophan Fluorescence—Human

myosin Vc contains a conserved tryptophan residue (Trp482)located at the rigid relay loop that is responsible for the fluores-cence change coupled with ATP-induced conformationalchange of myosin. The time courses of the intrinsic tryptophanfluorescence change, upon the addition of ATP, followed singleexponentials (Fig. 5, inset). The ATP dependence of the kobs

FIGURE 1. Purification of human myosin Vc construct. A, SDS-PAGE of thepurified human myosin Vc (M5CIQ1). M5CIQ1 expressed in Sf9 cells wasextracted and purified through an anti-FLAG affinity column, and the purifiedproteins were analyzed by SDS-PAGE. HC and CaM, heavy chain of M5CIQ1and calmodulin, respectively. A, M5CIQ1 co-expressed with calmodulin;B, M5CIQ1 co-expressed with LC17b. Lane 1, EGTA condition; lane 2, calciumcondition. Molecular mass markers are shown on the left.

FIGURE 2. Steady-state actin-activated ATPase activity of M5CIQ1. Theactin-activated ATPase activity of M5CIQ1 (20 nM) was measured in buffer Awith 2 mM phosphoenol pyruvate, 20 units/ml pyruvate kinase, 0.2 mg/mlcalmodulin, and 2 mM MgATP. The assay was done at 25 °C. The solid line is thehyperbola fit with Vmax of 6.5 � 0.4 s�1 and KATPase of 62 � 9 �M. The brokenline is simulated based on our kinetic model in this study (see “Discussion”),giving a Vmax value of 6.3 s�1 and KATPase of 50 �M.




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showed a hyperbolic saturation curve (Fig. 5), providing themaximum rate of 59 � 3 s�1. This value is much less than theATP binding rate at a high ATP concentration, suggesting thatthe rate constant represents the ATP hydrolysis step in theabsence of actin (k�3 � k�3). The rate for myosin Vc is 10-foldslower than that of myosin Va (13). The second order rate con-stant forATPbinding toM5CIQ1 (K1k�2) determined from theinitial slope of the curve was 2.5 � 0.1 �M�1 s�1, which wasconsistent with that obtained by direct ATP binding measure-ment using dmant-ATP (Fig. 3).ATP Hydrolysis—We measured the Pi burst of M5CIQ1 by

using a quenched-flow apparatus. Single turnover experimentswere carried out in which the active site concentrationexceeded the substrate concentration. Since all given ATP isbound to the ATP binding site of M5CIQ1, the equilibrium ofthe ATP hydrolysis step can be accurately determined bymeas-

uring the fraction of the fast Pi burst phase. The time course ofthe Pi burst followed double exponential kinetics (Fig. 6). Thefast phase corresponds to the initial rapid ATP hydrolysis inwhich ATP binding is rate-limiting. The slow phase (0.06 �0.03 s�1) represents the apparent phosphate release rate(k�4,obs). From the fractional amplitudes of the fast and slowphase, the Pi burst size was estimated to be 0.31 � 0.02. There-fore, the equilibrium constant of the ATP hydrolysis (K3) wascalculated to be 0.45 � 0.04. The results indicate that the equi-

FIGURE 3. Kinetics of dmant-ATP binding to M5CIQ1 and acto-M5CIQ1.The experiment was done in the same conditions as described in the legendto Fig. 2 except for the absence of the ATP regeneration system. A, in theabsence of actin. 0.3 �M M5CIQ1 was mixed with various concentrations ofdmant-ATP. The second order rate constant for dmant-ATP binding (K1k�2)was 2.4 � 0.1 �M

�1�s�1. Inset, a typical recording of the binding of 5 �M

dmant-ATP to M5CIQ1. The solid line is the best fit to single exponential kinet-ics with kobs of 12.6 s�1. B, in the presence of actin. 0.3 �M M5CIQ1 in thepresence of 0.4 �M actin was mixed with various concentrations of dmant-ATP. The second order rate constant (K�1k��2) of 1.6 � 0.1 �M

�1�s�1 wasobtained. Inset, a typical recording of the binding of 5 �M dmant-ATP to acto-M5CIQ1. The solid line is the best fit to single exponential kinetics with kobs of8.1 s�1. The error bars represent the S.E. from 3–5 independent experiments.

FIGURE 4. ATP-induced dissociation of acto-M5CIQ1. Pyrene-acto-M5CIQ1(0.3 �M M5CIQ1 plus 0.5 �M pyrene-actin) was mixed with various concentra-tions of MgATP, and the changes in the pyrene fluorescence or light scatter-ing were monitored. The second order rate constants for ATP binding to acto-M5CIQ1 (K�1k��2) estimated from the initial slopes of the linear fits were 1.8 �0.1 �M

�1�s�1 for pyrene fluorescence and 1.6 � 0.1 �M�1�s�1 for light scatter-

ing. Inset, time courses of the pyrene fluorescence and light-scatteringchanges after mixing acto-M5CIQ1 with 10 �M MgATP. The solid lines are thebest fits to single exponential kinetics, with kobs of 14.7 s�1 for pyrene fluo-rescence and 13.1 s�1 for light scattering. The experimental conditions are asdescribed in the legend to Fig. 3. The error bars represent the S.E. from 3–5independent experiments.

FIGURE 5. ATP-induced intrinsic tryptophan fluorescence change ofM5CIQ1. 0.8 �M M5CIQ1 was mixed with various concentrations of MgATP,and the tryptophan fluorescence change was monitored. The observed rates(kobs) were saturated at 59 � 3 s�1 (k�3 � k�3). The second order rate con-stant for ATP binding can be estimated from the initial slope of the linear fit,and the obtained value (K1k�2 � 2.5 � 0.1 �M

�1 s�1) was consistent with thatobtained in Fig. 3. The inset shows a typical recording of the intrinsic trypto-phan fluorescence change after mixing M5CIQ1 with 10 �M MgATP. The solidline is the best fit to single exponential kinetics, with kobs of 23.7 s�1. Theexperimental conditions are as described in the legend to Fig. 3. The error barsrepresent the S.E. from 3–5 independent experiments.




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librium of the ATP hydrolysis step is significantly shifted to theprehydrolyzed form (MT). The rate of the ATP hydrolysis wasalso measured at higher ATP concentration, where multipleturnovers of ATP hydrolysis take place. The Pi burst rate of55 � 18 s�1 was obtained with 100 �M ATP (date not shown).Since the ATP binding rate at this ATP concentration does notlimit the Pi burst rate, the obtained rate constant represents therate of ATP hydrolysis (k�3 � k�3). The observed value is con-sistent with the value obtained by the measurement of intrinsictryptophan fluorescence change and is 10-fold lower than thatof myosin Va, although it is significantly larger than the steady-state ATPase cycle rate, thus not limiting the overall ATPaserate.Phosphate Release Rate—The rate of the phosphate release

step was determined by measuring the fluorescence increase ofthe fluorescently labeled MDCC-PBP. Double-mixing, singleturnover experiments were carried out in which M5CIQ1 wasmixed with substoichometric amounts of ATP. The mixturewas aged for 5 s to advance the ATP binding and hydrolysis andthen mixed with various concentrations of actin. Upon therelease of the bound phosphate, MDCC-PBP rapidly binds tothe released phosphate, resulting in an increase in the fluores-cence intensity. The observed fluorescence increase followedsingle exponential kinetics in the absence of actin (Fig. 7A,inset). The observed rate (k�4,obs) of 0.11 � 0.01 s�1 was con-sistent with the rate of the slow phase obtained from thequenched-flow experiment (Fig. 6). In the presence of actin, thetime courses of the fluorescence increase were best fitted todouble exponential kinetics (Fig. 7A). The fast phase showedlinear actin dependence due to the actin rebinding step (K9)(Fig. 7B). From the slope of the line, k��4/K9,obs of 0.46 � 0.05�M�1 s�1 was obtained. The maximum phosphate release ratefrom acto-M5CIQ1�ADP�Pi (k��4) was estimated to be �60 s�1

based upon the lack of curvature on the actin dependence ofkobs. The rates of the slow phase were around 1 s�1 and actin-independent (data not shown). A similar slow phase was previ-ously observed formyosinX (30) andmyosinVIIa (29).Hommaet al. (30) found that the slow phase is due to ATP rebinding to

myosin X, because the rates of the slow phase were linearlyincreased with myosin concentration. In this study, it was dif-ficult to estimate the fraction of the slow phase from theobtained kinetic parameters, because the rate constant for ATPdissociation (k�2) could not be determined accurately. Sincethe ATP hydrolysis step (K3) is largely shifted toward the pre-hydrolyzed form (MT) and the rates of the slow phase wereclose to the ATP binding rate at the ATP concentration in thisexperiment (0.75 s�1 at 0.3 �MATP), it is possible that the slowphase is due to the ATP rebinding step of M5CIQ1. However,the ATP dissociation step seems to be slower (Fig. 3), and theorigin of the slow phase may not be the same as myosin X.Another possibility is that the slow phase is due to the ATPhydrolysis via the actin-associated pathway, as previously

FIGURE 6. Kinetics of ATP hydrolysis of M5CIQ1. A single turnover experi-ment was carried out by mixing 0.9 �M M5CIQ1 with 0.5 �M [�-32P]ATP, andthe fraction of hydrolyzed ATP was plotted against time. The time course wasfitted to double exponential kinetics. The rate constant of the burst phase(kobs � 4.0 s�1) with a fractional amplitude (equal to Afast/(Afast � Aslow)) of0.31 � 0.02 was obtained. The rate constant of the slow phase was kobs of0.064 � 0.025 s�1. The experimental conditions are as described in the legendto Fig. 3, except no actin was added.

FIGURE 7. Kinetics of phosphate release from M5CIQ1. The rate of phos-phate release from M5CIQ1 was measured by using MDCC-PBP. 1.2 �M

M5CIQ1 was mixed with 0.9 �M MgATP, aged for 5 s, and then mixed withvarious concentrations of actin. Other experimental conditions are asdescribed in the legend to Fig. 3. A, a typical recording of the MDCC-PBPfluorescence change at 50 �M actin. The time courses were fitted to doubleexponential kinetics. The fast and slow rates were 23.8 and 1.7 s�1, respec-tively. The inset shows a typical recording in the absence of actin. The fluores-cence change in the absence of actin was best fitted to single exponentialkinetics, with kobs of 0.11 s�1. B, actin concentration dependence of the rate ofphosphate release. The apparent rate of the fast phase was linearly increasedwith actin concentration. The apparent second order rate constant was0.46 � 0.05 �M

�1 s�1. The rates of the slow phase (0.2–1.7 s�1) were actin-independent. The error bars represent the S.E. from 4 – 6 independentexperiments.




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reported (26). The obtained results indicate that the phosphaterelease step (�60 s�1) is not the rate-limiting step of the acto-M5CIQ1 ATPase cycle.Kinetics of ADP Binding to and Dissociation from Acto-

M5CIQ1—We employed dmant-ADP as a probe to determinethe rate of ADP binding to acto-M5CIQ1. The time courses ofan increase in the fluorescence intensity followed single expo-nential kinetics (Figs. 8A and 9A, insets). In the absence of actin,the kobs showed a hyperbolic saturation curve on the dmant-ADP concentration (Fig. 8A), indicating that the ADP bindingis a two-step process (Fig. 13A). The maximum rate and theinitial slope of the curve represent k�5 � k�5 � 12.5 � 0.8 s�1

and k�5/K6 � 2.9 � 0.1 �M�1 s�1, respectively. From the yintercept of the curve, the rate of dmant-ADP dissociation(k�5) was estimated to be 3.6� 0.7 s�1. The rate of dmant-ADPdissociation was also determined by monitoring the fluores-cence change after mixingM5CIQ1�dmant-ADP complex with2 mM MgADP (Fig. 8B). The rate of dmant-ADP dissociation(k�5), obtained by this method, was 3.9 � 0.1 s�1, which is

FIGURE 8. Kinetics of dmant-ADP interaction with M5CIQ1. A, kinetics ofdmant-ADP binding to M5CIQ1. 0.3 �M M5CIQ1 was mixed with various con-centrations of dmant-ADP. The second order rate constant for dmant-ADPbinding (k�5/K6) was 2.9 � 0.1 �M

�1 s�1. From the y intercept, a k�5 value of3.6 � 0.7 s�1 was obtained. The inset shows a typical recording of the bindingof dmant-ADP (5 �M) to M5CIQ1. The solid line is the best fit to single expo-nential kinetics, with kobs of 9.2 s�1. B, kinetics of dmant-ADP dissociationfrom M5CIQ1. 0.5 �M M5CIQ1 in the presence of 5 �M dmant-ADP was mixedwith 2 mM MgADP. The solid line is the best fit to single exponential kinetics,with kobs of 3.9 � 0.1 s�1. Error bars, S.E. from 3–5 independent experiments.Other experimental conditions are as described in the legend to Fig. 3.

FIGURE 9. Kinetics of dmant-ADP interaction with acto-M5CIQ1. A, kineticsof dmant-ADP binding to acto-M5CIQ1. 0.3 �M M5CIQ1 in the presence of 0.4�M actin was mixed with various concentrations of dmant-ADP. The secondorder rate constant (k�5k�6) of 6.0 � 0.4 �M

�1�s�1 and k��5 of 17.1 � 1.3 s�1 fromthe y intercept were obtained. Inset, a typical recording of the binding ofdmant-ADP (5 �M) to acto-M5CIQ1. The solid line is the best fit to single expo-nential kinetics with kobs of 46.8 s�1. B, kinetics of dmant-ADP dissociationfrom acto-M5CIQ1. 0.5 �M M5CIQ1 in the presence of 5 �M dmant-ADP and0.6 �M actin was mixed with 2 mM MgADP. The solid line is the best fit tosingle exponential kinetics, with kobs of 17.7 � 0.6 s�1. C, the rate of ADPdissociation from acto-M5CIQ1 was determined by measuring the timecourse of change in the light-scattering intensity of acto-M5CIQ1. Acto-M5CIQ1 (0.3 �M M5CIQ1 and 0.4 �M actin) in the presence of 50 �M MgADPwas mixed with various concentrations of MgATP. The apparent dissocia-tion rates were saturated at 12.7 � 0.9 s�1 (k��5), which reflected the ADPdissociation rate from acto-M5CIQ1. The error bars represent the S.E. from3–5 independent experiments. Other experimental conditions are asdescribed in the legend to Fig. 3.




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consistent with that obtained from the dmant-ADP bindingexperiment (Fig. 8A).In the presence of actin, the kobs were linearly increased with

dmant-ADP concentration (Fig. 9A). A second order rate con-stant (k��5/K�6) for dmant-ADP binding was 6.0� 0.4�M�1 s�1.The y intercept of the line gave the rate constant of the dmant-ADP dissociation (k��5) of 17.1 � 1.3 s�1. This value was con-sistent with that determined by the direct measurement ofdmant-ADP dissociation upon the addition of 2mMMgADP toacto-M5CIQ1�dmant-ADP complex (17.7 � 0.6 s�1) (Fig. 9B).The rate of ADP dissociation from acto-M5CIQ1 was alsodetermined by monitoring the light-scattering change of theacto-M5CIQ1�ADP complex after the addition of ATP. In thepresence of ADP, the rate of ATP-induced dissociation of acto-M5CIQ1 is limited by ADP release from the acto-M5CIQ1�ADP complex. Fig. 9C shows the rate of ATP-induceddissociation of the acto-M5CIQ1�ADP complex as a function ofATP concentration. In contrast to the results obtained in theabsence of ADP (Fig. 4), the rate of change in the light-scatter-ing intensity of acto-M5CIQ1 plateaued to yield the maximumrate of 12.7 � 0.9 s�1. The observed rate of dissociation ofacto-M5CIQ1 is explained by the ADP dissociation step (k��5)from acto-M5CIQ1, since the ATP binding step does not limitthe dissociation rate at these ATP concentrations. Theobtained ADP dissociation rate from acto-M5CIQ1 was signif-icantly larger than the Vmax of the steady-state ATPase cyclerate of M5CIQ1, in contrast to myosin Va and Vb, whose ratesof ADP dissociation explain the overall actin-activated ATPasecycling rate (13, 22). These results suggest that the ADP disso-ciation step is not the rate-limiting step in the ATPase cycle ofmyosin Vc, which is quite different from myosin Va and Vb.Actin Binding to M5CIQ1—The rate of actin binding to

M5CIQ1 was measured using pyrene-actin. The fluorescenceintensity of pyrene-actin was decreased upon the binding ofM5CIQ1 to pyrene-actin, as is known for othermyosins (13, 42,43). The time courses of the change in the pyrene fluorescenceintensity followed single exponential kinetics (Fig. 10A, inset),and the kobs increased linearly with actin concentration (Fig.10A) to yield a second order rate constant (k�12) of 1.11 � 0.03�M�1 s�1. The dissociation rate of actin from acto-M5CIQ1was measured by mixing an excess amount of nonlabeled actinwith pyrene-acto-M5CIQ1. The time courses of the fluores-cence change followed single exponential kinetics, giving therate constant of k�12 � 0.011 � 0.001 s�1 (Fig. 10C). The affin-ity ofM5CIQ1 for actin (K12) was calculated to be 9.9 nM, whichis 2000-fold weaker than that of myosin Va (13).The rate of actin binding to M5CIQ1 was also measured in

the presence of ADP. The second order rate constant (k�10) of0.88� 0.02 �M�1 s�1 was obtained (Fig. 10B). The dissociationrate of actin from the pyrene-acto-M5CIQ1�ADP complex wasmeasured as described above to yield the rate constant of k�10

FIGURE 10. Kinetics of pyrene-actin interaction with M5CIQ1. A, kinetics ofpyrene-actin binding to M5CIQ1 in the absence of ADP. The rates of pyrene-actin binding to M5CIQ1 in the absence of ADP were measured as a functionof pyrene-actin concentration. M5CIQ1 was mixed with various concentra-tions of pyrene-actin, and time courses of the decrease in the pyrene fluores-cence were monitored. The apparent rate constant obtained by single expo-nential fitting increased with pyrene-actin concentration to give a secondorder rate constant (k�12) of 1.11 � 0.03 �M

�1 s�1. Inset, a typical recording ofthe binding of 4 �M pyrene-actin to M5CIQ1. The solid line is the best fit tosingle exponential kinetics, with kobs of 4.5 s�1. B, kinetics of pyrene-actinbinding to M5CIQ1 in the presence of ADP. The rates of pyrene-actin bindingto M5CIQ1 in the presence of 50 �M MgADP were measured as a function ofpyrene-actin concentration. The apparent rate constant obtained by singleexponential fitting increased linearly with pyrene-actin concentration to givea second order rate constant (k�6) of 0.88 � 0.02 �M

�1 s�1. The inset shows atypical recording of the binding of 4 �M pyrene-actin to M5CIQ1�ADP. Thesolid line is the best fit to single exponential kinetics, with kobs of 3.5 s�1.C, kinetics of pyrene-actin dissociation from acto-M5CIQ1. The rates of pyrene-actin dissociation from acto-M5CIQ1 or acto-M5CIQ1�ADP were measured by

mixing acto-M5CIQ1 (0.5 �M M5CIQ1 and 0.7 �M pyrene-actin) in the pres-ence or absence of 50 �M MgADP with an excess amount of nonlabeled actin(25 �M). The apparent rate constants obtained by single exponential fittingwere 0.011 � 0.001 s�1 (k�12) and 0.0099 � 0.0015 s�1 (k�10), in the absenceand presence of ADP, respectively. The error bars represent the S.E. from 3–5independent experiments. Other experimental conditions are as described inthe legend to Fig. 3.




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of 0.0099 � 0.0015 s�1 (Fig. 10C). The calculated affinity ofM5CIQ1�ADP for actin (K10), 11.3 nM, is almost identical tothat of myosin Va (13).

Actin Affinity of M5CIQ1 inWeak Binding State—Studies onmyosin Va using nonhydrolyzed nucleotide analogues haveshown that the actin affinity of myosin Va in the weak bindingstate is much tighter (10–20-fold) than that of nonprocessiveconventional myosin II (44). We examined the affinity of myo-sin Vc for actin in the weak binding state by using nonhydro-lyzed nucleotide analogues, ATP�S and AMPPNP. Actincosedimentation assays in the presence of ATP�S or AMPPNPwere performed in 25 mM KCl (Fig. 11). In the presence ofATP�S, the actin-bound fraction ofM5CIQ1was less than 50%at 70�M actin, suggesting that the apparent affinity ofM5CIQ1for actin in the presence ofATP�S (Kd(ATP�S)) was�70�M.Theapparent affinity of M5CIQ1 for actin in the presence of AMP-PNP (Kd(AMPPNP)) was 6.5 � 0.6 �M. These values of M5CIQ1are 6–20-fold weaker than those of myosin Va in 50 mM KCl(44). These results clearly showed that the affinity of myosin Vcfor actin in the weak binding state is much weaker than that ofmyosin Va and rather similar to that of conventional myosin II.Multimolecule and Single Molecule in Vitro Motility Assays—

To test the motility activity of myosin Vc, we first performed aconventional multimolecule in vitro motility assay using thetwo-headedmyosin Vc (GFP-M5CHMM) construct (Fig. 12A).

A majority of actin filaments weremoved, and the velocity was 0.16 �0.03 �m s�1 (Fig. 12A).

To address whether or not myo-sin Vc is a processive motor, weexamined the processivity of myo-sin Vc using TIRF microscopy. As acontrol, we used GFP-tagged myo-sin VaHMM, which is a typical pro-cessive motor. We observed theprocessive movement of GFP-tagged myosin Va HMM with amean travel length of 710 � 25 nmand velocity of 370 � 93 nm s�1 at20 °C in the presence of 1 mM ATP.30 of 59 total analyzedmoving spotsshowedmore than 1mmrun length.We observed that many moleculestraveled over 2 �m. The represent-ative movements of GFP-taggedmyosin Va HMM on actin areshown in Fig. 12B. The result wasconsistentwith previous studies (20,45). Next, we examined GFP-M5CHMM in the same conditionsas myosin Va. However, any proces-sivemovements of GFP-M5CHMMwere not observed. In the absence ofATP, the actin filaments decoratedwith GFP-M5CHMM wereobserved under the TIRF micro-scope. Upon the addition of ATP,the GFP-M5CHMM dissociatedfrom the actin filaments and did nottravel on the actin filaments, unlikemyosin VaHMM(Fig. 12C). If GFP-

FIGURE 11. Apparent binding affinities of M5CIQ1 for actin in the pres-ence of ATP analogues. Actin cosedimentation assays were performed inthe presence of 2.5 mM Mg-ATP�S or 5 mM Mg-AMPPNP at 25 °C. The experi-mental conditions are as described in the legend to Fig. 3. Apparent bindingaffinities of M5CIQ1 for actin were �70 �M in the presence of ATP�S (Kd(ATP�S))and 6.5 � 0.6 �M in the presence of AMPPNP (Kd(AMPPNP)).

FIGURE 12. Multimolecule and single molecule motility assay of myosin Vc. A, histogram of multimoleculeactin gliding velocity of GFP-M5CHMM. GFP-M5CHMM was attached to a coverslip, and the movement ofrhodamine-labeled actin filaments was observed with a fluorescent microscope. The solid line shows a fit to asingle Gaussian curve. The mean � S.D. of the actin sliding velocity was 0.16 � 0.03 �m s�1 (n � 51). B, singlemolecule movement of GFP-myosin Va HMM monitored by TIRF microscope. Almost all fluorescent spotsmoved in a continuous line and dissociated from actin filament until the photobleaching. Typical time courseof the distance from the binding position of individual GFP-myosin Va HMM. Mean velocity was 370 � 93 nms�1, and mean travel distance was 710 � 25 nm at 20 °C. C, single molecule movement of GFP-M5C HMMmonitored by TIRF microscope. The fluorescent spots appeared on the actin filament but disappeared withoutany movements. Typical time course of the distance from the binding position of individual GFP-M5C HMM.Inset, an expanded view of the lowest trace. D, histogram of dwell time of GFP-M5C HMM on the actin filamentmonitored by TIRF microscope. The dwell time was defined as the period during binding on the actin filament.We did not count the spots on the actin for less than 5 frames (100 ms). The distribution was followed to thesingle exponential function (solid line), and the time constant was 0.51 s in 10 �M ATP.




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M5CHMM moves more than 50 nm, we should be able to seesuch a movement, but as shown in Fig. 12C, such continuousmovements of myosin Vc were not observed. Fig. 12D showsdwell time distribution of GFP-M5CHMM in 10 �M ATP. TheATP hydrolysis turnover rate of acto-M5CHMM at this ATPconcentration was 2.5 s�1, which provides the calculated singlecycle dwell time of 0.4 s. The observed dwell time from thesingle molecule TIRF assay (0.5 s) agrees well with the calcu-lated value. If GFP-M5CHMM moves on actin with multiplecycles, the dwell time determined by the TIRF assay should besignificantly longer than that shown in Fig. 12D. The trackingaccuracy of a single GFP molecule in our optical system wasabout 30 nm/33 ms, which is calculated from the S.D. value ofthe tracking traces fixed on the glass surface. Since the kcat ofmyosin Vc was 6 s�1, GFP-M5CHMMwould produce one stepper 5 frames (165 ms) on average. Therefore, we should be ableto detect the stepwise movements if GFP-M5CHMM gener-ated the step with more than 15 nm, which can be calculatedby dividing the tracking accuracy (30 nm) by the route of thenumber of frames minus 1 (30/(5 � 1)1⁄2). However the step-wise movement of GFP-M5CHMM was not detected. Theresult suggests that while GFP-M5CHMM has the ATP-de-pendent actin interaction, it cannot remain on the actin fil-ament; thus, it cannot processively travel on the filament.From these results, we concluded that myosin Vc is a non-processive motor, which is consistent with the kinetic anal-yses in this study.

DISCUSSION

Although the overall structures of the various class V myo-sins resemble each other, the physiological function is thought

to be different due to the difference in the target proteins thatspecify the isoform-specific cargos. In addition, recent studieshave raised the question of whether class V myosins have sim-ilar motor characteristics, such as processivity. There are threeisoforms of myosin V in vertebrates. Although myosin Va andmyosin Vb are similar to each other in their motor characteris-tics that are consistent with their high sequence homology ofthe motor domain, the mechanoenzymatic characteristics of aless conserved myosin Vc have not been studied. In the presentstudy, we analyzed the ATP hydrolysis mechanism of actomy-osin Vc for the first time. All of the rate constants and equilib-rium constants obtained in this study are summarized in Table1.We found several unique features for the actomyosinVcATPhydrolysis cycle. First, the ADP release rate from actomyosinVc was significantly larger than the overall ATPase cycle rate;therefore, it does not solely determine the rate of the ATPhydrolysis cycle. This is quite different from myosin Va andmyosin Vb, in which the ADP release step explains 80–90%of the entire cycle rate (13, 22). Second, unlike myosin Va,the Pi burst size is only 0.3, suggesting that the ATP hydrol-ysis step (K3) is largely shifted to the prehydrolyzed form(MT). Third, the apparent affinity of myosin Vc for actin wasmuch lower than other vertebrate myosin V isoforms. TheKATPase of the steady-state ATPase of myosin Vc was 62 �M,which is 44-fold higher than that of myosin Va and 7-foldhigher than that of myosin Vb (13, 22) (Table 1). The lowaffinity of the weak binding state of myosin Vc for actin wasalso supported by the actin cosedimentation assays in thepresence of nonhydrolyzed ATP analogues (Table 1). Sincethe actin affinity of myosin Vc in the rigor state (K12) was also2000-fold lower than that of myosin Va (Table 1), the actin-myosin interface participating in the rigor interaction may,in part, contribute to the low affinity of myosin Vc for actinduring the ATPase cycle. Although the amino acidsequences in the motor domain among the myosin V familyshow high homology, a significant difference among the ver-tebrate myosin V isoforms is found in loop 2. Loop 2 isknown to affect the actin binding affinity of myosin, and ithas been reported that the charged residues in loop 2 influ-ence the KATPase of myosin Va (46). Therefore, it is plausiblethat the difference in the loop 2 sequences among themyosinV isoforms is in part responsible for the unique actin affinityof each myosin V isoform. In addition to loop 2, we found thatthere is a significant sequence difference at the C-loop adjacent tothemyopathy loopbetweentheprocessivemyosin (myosinVaandVb) and myosin Vc. Since it has been shown that the C-loop andthe myopathy loop are important for the interaction with actin(41), it is plausible that the sequence difference in the C-loop inaddition to thedifference in the loop2causes thedecreasedaffinityof myosin Vc for actin.Based on the obtained rate constants and equilibrium con-

stants of each elementary kinetic step of the actomyosin VcATPase cycle, we carried out the simulation of the steady-stateATPase activity under a saturating ATP concentration with anATP regeneration system as a function of actin concentration.It should be noted that, since K8, K9, and k��4 were not deter-mined directly in this study, we assumed the values of theseparameters to fit the experimentally obtained ATPase activity.

FIGURE 13. A, reaction scheme of myosin Vc ATPase cycle. A, actin; M, myosin;T, ATP; D, ADP; P, phosphate. The major kinetic pathway is shown in grayshading. B, actin concentration dependence of the duty ratio of M5CIQ1. Theduty ratio was calculated based on our kinetic model using experimentallyobtained parameters as described under “Discussion.” The duty ratio was 0.33at saturating actin concentration.




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The result is shown by the broken line in Fig. 2. The contribu-tion of k�4, [M], and [MD] to the overall ATPase rate wasignored for the simplicity of the simulation. The initial values,rates, and equilibrium constants employed in the simulationwere as follows. [AM]0 � 20 nM, [AMT]0 � [MT]0 � [MDP]0 �[AMDP]0 � [AMD]0 � 0 nM,K�1k��2[ATP]� 500 s�1,K8 � 190�M (rapid equilibrium), k�3 � 19 s�1, k�3 � 41 s�1 (K3 � 0.46),K9 � 340 �M (rapid equilibrium), k��4 � 155 s�1, k��5 � 12.7s�1.Vmax andKATPase calculated by this simulationwere 6.3 s�1

and 50 �M, respectively. These values were in agreement withthe experimentally obtained values, supporting the validity ofthe kinetic model (Fig. 13A).The basal steady-state ATPase activity of myosin Vc is also

well explained by the kinetic constants obtained in the presentstudy. The basal steady-state ATPase activity (v0 � 0.10 � 0.01s�1) is in agreement with the observed phosphate release rate(k�4,obs) of 0.11� 0.01 s�1, which is explained by a combinationof the slow phosphate release rate (k�4) and the unfavorableequilibrium of the ATP hydrolysis step (K3).

On the other hand, in the presence of actin, the steady-state ATPase activity was markedly activated by actin. TheATP hydrolysis rate (k�3 � k�3), as well as the phosphate

release rate from AMDP complex (k��4), is much faster thanthe entire ATPase cycle rate, thus not the rate-limiting steps.Since the ADP release rate is 2-fold faster than the Vmax, itcannot explain the overall cycle rate, although it partiallylimits the ATPase cycle. The overall ATPase cycle rate ofactomyosin Vc can be primarily explained by the unfavor-able equilibrium of the ATP hydrolysis step (K3), the rela-tively low actin rebinding rate constant (k��4/K9), and theADP release rate from AMD (k��5). The major kinetic path-way is shown in gray shading in Fig 13A. These results sug-gest that myosin Vc spends a significant time during theATPase cycle in the “weak” actin binding forms. We calcu-lated the duty ratio of myosin Vc based on our kinetic model(Fig. 13B). In contrast to myosin Va, the duty ratio was sat-urated at a high actin concentration that is consistent withthe weak affinity of myosin Vc for actin. With a saturatingactin concentration, the duty ratio of myosin Vc was esti-mated to be �0.33, which is much lower than those of myo-sin Va (0.7) (13) and myosin Vb (�0.8) (22). This result sup-ports the notion described above that myosin Vc spends themajority of the ATP cycle in the weak actin-binding state, incontrast to myosin Va and Vb, whose major steady-state

TABLE 1Kinetic parameters of M5CIQ1 ATPase cycle and comparison with myosin Va and Vb

Signal Myosin Vca Myosin Vab Myosin Vbc

Steady statev0 (s�1) 0.10 � 0.01 0.03 0.09Vmax (s�1) 6.5 � 0.4 15 9.7KATPase (�M) 62 � 9 1.4 8.5Kd(ATP�S) (�M) 70 13dKd(AMPPNP) (�M) 6.5 � 0.6 0.3d

ATP bindingK1k�2 (�M�1 s�1) Mant-ATP 2.4 � 0.1 1.6 0.78

Tryptophan 2.5 � 0.1 1.5K�1k��2 (�M�1 s�1) Mant-ATP 1.6 � 0.1 0.9 0.42

Light scattering 1.6 � 0.1 0.31Pyrene-actin 1.8 � 0.1 0.9

k��2 (s�1) Pyrene-actin �300 870ATP hydrolysisk�3 � k�3 (s�1) Tryptophan 59 � 3 750

Quenched flow 55 � 18K3 Quenched flow 0.45 � 0.04 5.3

Phosphate releasek�4,obs (s�1) MDCC-PBP 0.11 � 0.01k�4,obs (s�1) Quenched flow 0.064 � 0.025k�4 (s�1) k�4,obs(1 � K3)/K3 0.34 � 0.04k��4 (s�1) MDCC-PBP �60 �250

ADP bindingk�5 (s�1) Mant-ADP cold chase 3.9 � 0.1 1.2

Mant-ADP binding 3.6 � 0.7k�5/K6 (�M1 s�1) Mant-ADP 2.9 � 0.1 4.6K5K6 (�M) k�5/(k�5/K6) 1.3 0.27k��5 (s�1) Light scattering (ADP) 12.7 � 0.9 16e 12.2

Mant-ADP cold chase 17.7 � 0.6Mant-ADP binding 17.1 � 1.3

k��5/K�6 (�M1 s�1) Mant-ADP 6.0 � 0.4 12.6K�5K�6 (�M) k��5/(k��5/K�6) 2.1 0.93

Actin bindingk�12 (�M 1 s�1) Pyrene-actin 1.11 � 0.03 73k�12 (s�1) Pyrene-actin 0.011 � 0.001 0.00036K12 (�M) k�12/k�12 0.0099 4.9 � 10�6

k�10 (�M1 s�1) Pyrene-actin 0.88 � 0.02 4.2k�10 (s�1) Pyrene-actin 0.0099 � 0.0015 0.0032K10 (�M) k�10/k�10 0.0113 0.0076

a 25 mM KCl, 20 mM MOPS (pH 7.5), 2 mM MgCl2, 1 mM EGTA, 1 mM DTT.b Data from Ref. 13; 50 mM KCl, 10 mM imidazole (pH 7.0), 1 mM MgCl2, 1 mM EGTA, 1 mM DTT.c Data from Ref. 22; 50 mM KCl, 20 mM MOPS (pH 7.0), 3 mM MgCl2, 1 mM EGTA, 1 mM DTT.d Data from Ref. 44; 50 mM KCl, 10 mM imidazole (pH 7.0), 1 mM MgCl2, 1 mM EGTA, 1 mM DTT.e Pyrene actin.




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intermediates in the ATPase cycle are in the strong actinbinding state. It has been suggested previously that the equi-librium constant of the hydrolysis step (K3) may be increasedat a higher temperature in cells (35, 47), and this could influ-ence the duty ratio. However, the Pi burst size of M5CIQ1 at30 °C was virtually the same as that at 25 °C (data not shown).Therefore, the possible increase in the Pi burst size at thephysiological temperature would be minimal, and it isunlikely that myosin Vc becomes a high duty ratio motoreven at body temperature.One of the most important issues to evaluate the physio-

logical role of the motor proteins is the processivity, becauseit is thought that the processive motors are suitable for thecargo transporter in cells, whereas the nonprocessivemyosinmolecules function better as a force producer by simulta-neously interacting with a single actin filament, thus produc-ing a large force. It has been thought that a high duty ratio(�0.5) is required for the processive motor; therefore, ourkinetic study that shows myosin Vc to be a low duty ratiomotor suggests that this myosin V is a nonprocessive motor.Consistently, the single molecule assays in this study usingGFP-M5CHMM indicate that myosin Vc is a nonprocessivemotor. Recently, it was reported that Drosophilamyosin V isa low duty ratio motor (21), suggesting the presence of anonprocessive motor in the myosin V family. The presentresults are consistent with this notion and further suggestthe presence of the nonprocessive type of myosin V invertebrates.The cell biological studies onmyosin Vc showed that myosin

Vc colocalizes with Rab8, suggesting the function of myosin Vcin the membrane trafficking (7). How does myosin Vc functionas a cargo transporter despite a low duty ratio? One possibilityis that myosin Vc forms clusters on the surface of the cargoand transports it processively, although this is not a prefer-able mechanism for stable cargo transportation, because ifthe number of molecules in a cluster of myosin Vc is toolarge, the molecules may interfere with the movement ofeach other, and if too few, they do not serve continuousmovement. Another possibility is that there is an unknownprotein that associates with myosin Vc and tethers it to theactin filament, thus acquiring the processive movement.Further biochemical, biophysical, and cell biological studiesare required for the function of myosin Vc to provide a clueto this question.

Acknowledgment—We thank Dr. H. D.White (Eastern VirginiaMed-ical School) for providing the PBP cDNA clone.

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Homma, Nobuhisa Umeki, Hideo Higuchi, Reiko Ikebe and Mitsuo IkebeShinya Watanabe, Tomonobu M. Watanabe, Osamu Sato, Junya Awata, Kazuaki

Human Myosin Vc Is a Low Duty Ratio Nonprocessive Motor

doi: 10.1074/jbc.M707657200 originally published online December 12, 20072008, 283:10581-10592.J. Biol. Chem.

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humanmyosinvcisalowdutyrationonprocessivemotor · 2008-05-30 · april 18, 2008•volume...

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