Human Myosin Vc Is a Low Duty Ratio, Nonprocessive Molecular Motor*

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a Vmax of 1.8 ± 0.3 s-1 and a KATPase of 43 ± 11 μm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (∼1.5 s-1), rather than the ADP release step (∼12.0–16.0 s-1). Nevertheless, the ADP affinity of actomyosin Vc (Kd = 0.25 ± 0.02 μm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was ∼10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of ∼24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration.

Class V myosins are part of the myosin superfamily, currently composed of as many as 37 types of myosins (1). This diverse superfamily is constituted with proteins that have been identi-fied from conserved sequences in the catalytic domain (motor), which are needed for actin binding and ATP hydrolysis (2,3). Within this actin-based molecular motor superfamily, class V myosins are one of the most ancient and widely distributed forms (4). Myosin Vs have been implicated in actin-dependent organelle transport (5,6) and membrane trafficking (7,8). In recent years, various studies using assorted scientific techniques, including both in vitro single molecule/ensemble biochemical and biophysical assays, as well as cell biological assays, have contributed to our current understanding of the mechanisms used by myosin V family members (9 -11).
Within vertebrates, three genes of the class V myosin (Va, Vb, and Vc) have been discovered (12)(13)(14). Of these gene products, myosin Va (12,15) has been the focus of attention. Myosin Va is expressed at high levels in neurons and melanocytes (5, 16 -21). The lack of myosin Va in mouse can result in neurological seizures and critical defects in the transport of melanosomes (16,(22)(23)(24). Thus, studying this myosin at the molecular level may provide insights in human pathology. Myosin Vb is expressed in many tissue types, but Northern blot hybridization has shown that this protein is found primarily in the testes, kidney, liver, lung, and the heart (13). In vivo studies have shown that myosin Vb is associated with recycling of numerous receptors, including the transferrin receptor (25), muscarinic acetylcholine receptor (26), and the cystic fibrosis conductance regulator (27). Myosin Vc is expressed chiefly in epithelial cells in the pancreas, prostate, mammary, stomach, colon, and the lung (14). It has been suggested that one role of myosin Vc in vivo is membrane trafficking, specifically that of the transferrin receptor, where myosin Vb is also implicated. However, myosin Vc appears to be partially distinct with the same myosin Vbassociated membrane compartments (14).
The structure of myosin Va has been thoroughly studied (10, 12, 15, 28 -30). Myosin Va is an oligomeric protein, which is composed of a dimerized heavy chain, each containing a motor domain, a lever arm containing six IQ motifs that bind a total of six light chains (mostly or entirely calmodulins) per heavy chain, followed by a coiled-coil region and a C-terminal globular tail. The sequence alignment of the three vertebrate myosin V isoforms showed that throughout the class there is ϳ50% amino acid identity. Myosin Vc, for example, shares ϳ62% identity to the myosin Va and Vb motor domains, ϳ20 -30% identity in the coiled-coil region, and some differences in the globular tail region, such as the absence of the PEST site, found in myosin Va and Vb (14). The structure of myosin Vb (31) and Vc has not been studied as thoroughly as myosin Va; however, the predicted structures from the amino acid sequence analysis exhibited that three isoforms are comparatively similar. The lack of the PEST domain for the myosin Vc reduces its size to ϳ12 kDa smaller than the other myosin Vs.
To characterize the molecular mechanism of these vertebrate myosin V isoforms, steady-state and transient solution kinetic studies have been performed using both myosin Va (32)(33)(34)(35)(36)(37)(38) and myosin Vb (31). The enzymatic properties of myosin Vc have not yet been characterized. Ensemble solution kinetics is a powerful technique that can be used to elucidate the fundamental kinetic properties of the different types of myosin, and a detailed kinetic analysis can furthermore be used to gain insights into the chemo-mechanical coupling of the molecular motor, as done for myosin Va (34,35). One of the unique properties that distinguished the kinetic mechanism of myosin Va from the other previously studied myosins (2,3,39) is that it spends most of its kinetic cycle strongly bound to actin during steady-state ATPase cycling (Ͼ70%) and that the rate-limiting step of the actomyosin Va ATPase cycle is ADP release. These kinetic properties are consistent with this myosin being a processive motor, i.e. a protein that undergoes multiple enzymatic cycles while at least one of the motor domains of the molecule is attached to the filamentous actin, such that it can take multiple steps (40 -43). The kinetic mechanism of myosin Vb has been studied (31) and showed indications that also this molecule is processive; however, direct observations that the dimerized form of this molecule is processive have not been shown. The kinetic mechanism of myosin Vc has not yet been studied in detail, and a detailed kinetic mechanism may be needed to understand its physiological function in vivo. Furthermore, the question whether myosin Vc isoform is processive or not would be of interest to unravel, because Drosophila myosin V was recently determined as a low duty ratio motor (i.e. a myosin head that spends only a small fraction of the catalytic cycle strongly bound to actin) that is presumably nonprocessive (44).
In this study, we report a full biochemical kinetic characterization of the Homo sapiens myosin Vc subfragment-1 (MVc-S1) protein. Steady-state and transient solution kinetic characterizations were performed using a recombinant single-headed MVc-S1 construct, bound with a single calmodulin. Additionally, to directly investigate if the recombinant form of the H. sapiens myosin Vc translocates actin in an in vitro actin gliding assay and, furthermore, in an attempt to visualize processive motion of single molecules of H. sapiens myosin Vc, a heavy meromyosin (HMM) 3 form of the double-headed construct, intact with six IQ motifs and an enhanced green fluorescent protein (eGFP) attached to each motor domain (eGFP-MVc-HMM) on the N-terminal end, was expressed and purified from the Sf9 baculovirus system. The biochemical kinetic characterization revealed that MVc-S1 is a low duty ratio motor, which spends most of its time during the actomyosin ATPase cycle residing in the states weakly bound to actin. Unlike the other two vertebrate myosins, Va and Vb, the kinetic cycle of myosin Vc is not rate-limited by its ADP release but by the inorganic phosphate (P i ) release, which is biochemically closer in resemblance to myosins that form ensemble structures to function in physiological conditions, such as skeletal muscle myosin II.
We conclude that myosin Vc is a nonprocessive motor protein and therefore employs a different mechano-chemical mechanism to perform its tasks in the cytoskeleton compared with both myosin Va and Vb. Acto-MVc has a relatively high ADP affinity, and this unusual kinetic difference may possibly make the mechanical activity sensitive to ADP concentration, i.e. the duty ratio of this motor may increase at even the moderate ADP concentrations in an in vivo environment, consistent with our kinetic simulation results, as well as reported for nonmuscle myosin IIB (45).

EXPERIMENTAL PROCEDURES
Expression and Purification of the Myosin Vc-S1 and HMM Proteins-Using a cDNA clone of the H. sapiens myosin Vc encoding the 2930 amino acids fused with an N-terminally fused enhanced GFP, 4 we engineered a cDNA fragment that encodes the first 777 amino acids, containing the motor domain plus the first predicted light chain binding IQ motifs (myosin Vc subfragment-1; MVc-S1). In addition to the subfragment-1like construct, a heavy meromyosin-like myosin Vc construct with an N-terminally fused enhanced GFP (eGFP-MVc-HMM) was also engineered by encoding the first 1108 amino acids, containing the motor domain, the six predicted IQ motifs, and the predicted coiled-coil region. These amplified clones were subcloned into the pFastBac1 baculovirus transfer vector (Invitrogen) designed with a FLAG epitope tag (DYKDDDDK) fused C-terminally to aid in purification (46). Double-stranded DNA sequencing was performed to confirm the complete nucleotide sequencing from both engineered vectors.
Tetramethylrhodamine phalloidin (Invitrogen)-labeled filamentous actin and 10% biotinylated filamentous actin was prepared essentially as described by Ishijima et al. (50) and Takagi et al. (51). Labeled filaments lacking biotinylated globular actin were used for the in vitro actin gliding assay, and biotinylated filaments only for the single molecule TIRF assays.
MANT-ATP and MANT-ADP were purchased from Molecular Probes (Invitrogen) and stored at Ϫ20°C. MDCC-labeled, phosphate-binding protein (MDCC-PBP) (52) was generously provided by Dr. Howard D. White (Eastern Virginia Medical School, Norfolk, VA). All other reagents used in this study were from Sigma.
Steady-state ATPase Experiments-Steady-state ATPase measurements of myosin Vc, both in the presence and absence of filamentous actin, were measured using an NADH-coupled assay as described previously by Trentham et al. (53), De La Cruz et al. (35), and Wang et al. (54). The solutions used for these measurements included the following reagents: 10 mM MOPS (pH 7.2), 2 mM MgCl 2 , 0.15 mM EGTA; 2 mM ATP and 50 mM KCl, 40 units/ml lactate dehydrogenase, 200 units/ml pyruvate kinase, 1 mM phosphoenolpyruvate, and 200 M NADH. Changes in A 340 were monitored using a Beckman DU640 spectrophotometer and stored for further data analysis.
Stopped-flow Experiments-All measurements were performed using an SF-2001 stopped-flow apparatus (KinTek Corp., Austin, TX) at 25°C. For constancy, all stopped-flow experiments were performed using buffer (SF buffer) having the same contents. The SF buffer contained the following reagents: 20 mM MOPS (pH 7.0), 5 mM MgCl 2 , 0.05 mM EGTA, and 50 mM KCl. To determine the kinetic parameters for P i release, double-mixing stopped-flow experiments using MDCC-PBP (55) were performed as in Ref. 54. Only for the P i release experiment was 10 mM KCl used. MVc-S1 and acto-MVc-S1 were preincubated with 0.02 units/ml of apyrase for at least 30 min at 25°C to deplete any nucleotide in the sample when required. The optical setups of the apparatus are described in depth elsewhere (44,54,56). Post-mix concentrations of proteins and reagents are indicated throughout the text, unless stated otherwise.
Quenched-flow Experiments-Quenched-flow experiments were also performed at 25°C in SF buffer. Quenched-flow experiments were performed using a KinTek RQF-3 apparatus as described previously (54) except that 1 M HCl was used as a quench.
Data Analysis-Data analyses of the steady-state and transient solution kinetic measurements were performed using either SigmaPlot 8.0 (Systat Software, San Jose, CA), OriginLab 7.5 (Microcal Corp. Northampton, MA), or the KinTek SF-2004 data analysis software. The means Ϯ S.D. cited in this study are mostly averages of two to seven rounds of experiments performed using protein from different preparations.
In Vitro Actin Gliding Motility and Single Molecule TIRF Microscopy Assay-Both light microscopy assays were performed using an apparatus previously described by Sakamoto et al. (57,62) with some minor modifications. Motility assays were performed in buffer containing the following reagents: 20 mM MOPS (pH 7.4), 5 mM MgCl 2 , 0.1 mM EGTA, 50 mM KCl, 1 mM ATP, 5 M calmodulin, 25 g/ml glucose oxidase, 45 g/ml catalase, 2.5 mg/ml glucose, and 20 mM dithiothreitol (final concentrations listed). Experiments were performed at 25°C. Additionally, to determine whether ionic strength alters the motility of eGFP-MVc-HMM, single molecule motility assays were performed in the range varying from 0 to 250 mM KCl as shown previously for myosin Va (41). Observation chambers were also prepared from coverslips (Corning Inc., Corning. NY) using a protocol described previously (57). Data analysis of the in vitro actin gliding assay was performed using the ImageJ image analysis software (National Institutes of Health, Bethesda) with a data analysis algorithm described in Ref. 58 to track the leading ends of the labeled actin filaments.
Kinetic Simulation of the Actomyosin Vc ATPase Cycle-Kinetic simulations of the acto-MVc-S1 ATPase cycle were performed using Gepasi version 3.30 (Pedro Mendes, Virginia Bioinformatics Institute) based on Scheme 1 and the experimentally determined rate constants listed in Table 1.

Construct Design, Protein Expression, and Purification-Both
the MVc-S1 and eGFP-MVc-HMM constructs were purified using FLAG affinity chromatography (Fig. 1). The singleheaded subfragment-1 (S1) construct was used for the kinetic characterization, whereas the double-headed HMM construct was used to study its mechanical properties via in vitro actin gliding and single molecule TIRF assays. As described previously, the S1 construct only contained the motor domain and the first IQ motif, whereas the HMM construct contained the motor domain, the six (predicted) IQ-binding motifs, and the Ͼ200 amino acid stretch of the coiled-coil region to promote dimerization of the two heavy chains.
Protein expression was performed using the baculovirus expression system with the Sf9 insect cells, co-expressed with Xenopus calmodulin, at 27°C using shaken cultures. Optimum expression of the protein was reached at ϳ3-3.5 days. Both constructs showed reasonable levels of expression, generally yielding ϳ1 mg of protein purified from ϳ1 ϫ 10 9 cells for the single-headed S1 construct, and ϳ0.5-0.7 mg of protein for the double-headed HMM construct.
Actin Activation of the Steady-state ATPase Activity-The steady-state ATPase activity of MVc-S1 was activated by actin to a maximal rate of 1.8 Ϯ 0.3 s Ϫ1 (V max ), and half-maximal activation was achieved at 42.5 Ϯ 10.6 M (K ATPase ) ( Fig. 2A and Table 1). Furthermore, in the absence of actin, the basal steadystate activity of MVc-S1 was 0.05 Ϯ 0.01 s Ϫ1 . Association of MVc with actin therefore activates the steady-state activity of MVc-S1 by ϳ30-fold. Similarly to MVc-S1, steady-state ATPase activity of eGFP-MVc-HMM was activated by actin and yielded similar V max and K ATPase values (data not shown).
Association and Dissociation of MVc-S1 with Actin in the Absence of Nucleotide and in ADP-Pyrene-labeled filamentous actin has been used extensively to monitor the strong binding of myosin to actin in many myosins (59). Pyrene fluorescence is quenched on mixing of MVc-S1 or MVc-S1⅐ADP with pyrene-actin in the stopped flow, and the fluorescence decreases indicate the strong binding of MVc-S1 with pyrenelabeled actin. Fig. 3A shows the dependence of the k obs (observed rate constant) of the observed fluorescence decreases on pyrene-actin concentration. The k obs versus [pyrene-actin] plots were linear, and their slopes represent the second-order actin-binding rate constants of 0.66 Ϯ 0.02 and 1.17 Ϯ 0.03 M Ϫ1 ⅐s Ϫ1 in the absence of nucleotide (k Ϫ6 ) and in ADP (k Ϫ10 ), respectively.
The dissociation rates of MVc-S1 from pyrene-actin in the absence and presence of ADP were made using stopped-flow spectrophotometry chase experiments. The pyrene-acto-MVc-S1 rigor complex or the pyrene-acto-MVc-S1⅐ADP ternary complex was rapidly mixed with excess unlabeled filamentous actin in the stopped-flow apparatus. Pyrene fluorescence increases occur during dissociation of MVc-S1 or MVc-S1⅐ADP from the pyrene-actin, and the rebinding reaction is inhibited by the excess unlabeled filamentous actin. Single exponentials were fitted to the average transients, and the corresponding dissociation rate constants in the absence of nucleotide and in ADP were determined as 0.019 s Ϫ1 (k 6 ) and 0.051 s Ϫ1 (k 10 ), respectively.
The equilibrium constants in the absence (K 6 ) and presence of nucleotide (K 10 ) were also calculated using the determined second-order actin binding rate constants and the dissociation rate constants. The corresponding equilibrium constants in the absence of nucleotide and in ADP were determined as 0.029 M (K 6 ) and 0.044 M (K 10 ), respectively.
Interaction of ATP with Acto-MVc-S1-We measured the binding of ATP to acto-MVc-S1 by monitoring the ATP-induced dissociation of the pyrene-acto-MVc-S1 complex using the stopped-flow apparatus. Pyrene-acto-MVc-S1 (0.5 M MVc-S1 and 1.0 M pyrene-actin) incubated with apyrase was rapidly mixed with increasing concentrations of ATP (0 -450 M) under pseudo first-order conditions. Pyrene fluorescence increases after MVc-S1 dissociates from actin, and this fluorescence change can be fitted by a single exponential curve. k obs increased hyperbolically with ATP concentration, approaching a maximal rate constant (k 1 Ј) of 287 s Ϫ1 with a half-maximum (K 1 ) at 288 M ATP (K 1 Ј) (Fig. 4A). At low ATP concentrations, the data sets of the observed rate constants increased linearly with ATP concentration, and the apparent second-order rate constant (K 1 Јk 1 Ј) was 0.75 Ϯ 0.03 M Ϫ1 ⅐s Ϫ1 (Fig. 4B), for this particular example. K 1 Јk 1 Ј determined from the average of multiple experiments yielded an apparent second-order rate constant of 0.82 Ϯ 0.03 M Ϫ1 ⅐s Ϫ1 ( Table 1).
Interaction of ADP with Acto-MVc-S1-To determine the ADP dissociation rate constant from acto-MVc-S1 (k 5 Ј), we monitored the decrease in fluorescence upon dissociation of MANT-ADP from acto-MVc-S1. 1 M MVc-S1 was incubated with 20 M filamentous actin and 50 M MANT-ADP (pre-mix concentrations). This ternary complex was mixed rapidly with excess ATP (1 mM; pre-mix concentration). Upon mixing, the decrease in the MANT-ADP fluorescence was recorded, and the transients were fitted by single exponential curves. Fig. 5 shows an example of these averaged transients. The fits yielded rate constants of 15.6 Ϯ 0.6 s Ϫ1 (ϭ k 5 Ј).
To measure the ADP affinity of acto-MVc-S1, 1.0 M pyrenelabeled actin was incubated with 0.5 M MVc-S1 and various concentrations of ADP and then rapidly mixed with 150 M ATP (pre-mixing concentrations). The transient of the pyrene fluorescence increases as the pyrene-actin dissociates from the MVc-S1. Fig. 6A shows an example of these transients, whereby pyrene-acto-MVc-S1 was incubated with 3 M ADP (pre-mixing concentrations). The fluorescence increase was fitted by a double exponential, whereby the fast phase (ϳ100 s Ϫ1 ) corresponded to the ATP-induced dissociation of the ADP-free actomyosin complex, and the relative amplitude of this phase reports the fraction of acto-MVc-S1 free of bound nucleotide. The slower phase of the transient at low ADP concentrations, 15.8 Ϯ 0.8 s Ϫ1 , reflects the kinetics of ADP dissociation from the actomyosin complex (k 5 Ј) (which must occur before ATP can bind and dissociate pyrene-acto-MVc-S1), and therefore the relative amplitude of this phase reports the fraction of acto- MVc-S1 bound with ADP. This ADP dissociation rate constant determined from the pyrene-actin fluorescence signal agrees well with the MANT-ADP fluorescence decrease upon dissociation from the acto-MVc-S1 (15.6 Ϯ 0.6 s Ϫ1 ) ( Fig. 5 and Table 1).
A hyperbolic fit to the plot of the percent amplitudes of the slow phase (i.e. 100% ϫ (A slow /(A slow ϩ A fast ))) versus ADP concentration allowed for the determination of the acto-MVc-S1 ADP dissociation constant (K 5 Ј) of 0.25 Ϯ 0.02 M (Fig. 6B and Table 1). Thus, the ADP affinity of MVc-S1, in the presence of actin, is high, ϳ3-fold greater than that of chicken myosin Va (34).
Transient Kinetics of ATP Hydrolysis by MVc-S1-The ATP hydrolysis kinetics of MVc-S1 was measured using the quenched flow technique. MVc-S1 or acto-MVc-S1 was rapidly mixed with [␥-32 P]ATP, and the time course of P i liberation was monitored over time. When MVc-S1 was mixed with ATP in single turnover conditions (1.5 M protein and 1.5 M nucleotide post-mix), the time course of the reaction consisted of two phases (Fig. 7A). The first phase had an amplitude of 0.49 M P i (corresponding to a fractional amplitude of 0.34) and a k obs of 1.1 s Ϫ1 , which is likely limited by ATP binding. The slower second phase had a k obs of 0.083 s Ϫ1 , in agreement with the basal steady-state activity of MVc-S1 (measured as 0.062 s Ϫ1 under the same reactant conditions in the NADH-linked assay).
When mixed with excess ATP in the quenched flow, MVc-S1 produced a P i burst with an amplitude of 0.40 mol of P i /mol of MVc-S1 (Fig. 7B). The burst was followed by a linear steadystate phase of P i production characterized by a turnover rate of 0.064 s Ϫ1 , again in agreement with the results of the above quenched flow and steady-state measurements. When the same experiment was performed in the presence of 15 M actin (postmix), the steady-state rate was accelerated to 5-fold to 0.32 s Ϫ1 (in line with NADH-linked steady-state ATPase measurements (Fig. 2)), but the burst amplitude was not markedly affected (0.35 mol of P i /mol of MVc-S1) (Fig. 7C). Using these burst amplitudes (B), the hydrolysis equilibrium constant (K 3 ) can be estimated by the following equation: B ϭ K 3 /(1 ϩ K 3 ); K 3 therefore is ϳ0.54. ATP hydrolysis for MVc-S1 is therefore reversible.
By following the time course of the reaction until near-complete exhaustion of ATP, we detected signs of weak product inhibition by ADP (Fig. 7C). As expected from the kinetic parameters in Table 1, the ADP inhibition was much less pronounced in acto-MVc-S1 than in acto-MVa-S1, where ADP release is rate-limiting (34,35).

Kinetics of the P i Release
Step-We monitored the time course of P i release from MVc-S1⅐ADP⅐P i and acto-MVc-S1⅐ADP⅐P i using MDCC-PBP, a fluorescently labeled P i -binding protein (55,60). In double-mixing stopped-flow experiments, MVc-S1 was first mixed with ATP under single turnover conditions (5.2 M MVc-S1 and 4 M ATP after the first mix), incubated for 3 s so that ATP binding and hydrolysis can occur, and then rapidly mixed with actin (0 -17.5 M after the second mix) to monitor basal and actin-activated P i release. Time courses of P i release were best fit with single exponentials. Fig. 8A shows examples of transients, including their curve fits. The fitted k obs values depended hyperbolically on actin concentration, starting from 0.16 s Ϫ1 in the absence of actin (k 4 ) and delineating a maximal P i release rate constant of 1.5 s Ϫ1 (k 4 Ј) reaching half-saturation at 14 M actin (K 9 ) (Fig. 8B).
In Vitro Actin Gliding Assay-In vitro actin gliding assays were performed to measure the velocity (v) at which the eGFP-MVc-HMM translocates tetramethylrhodamine-phalloidin-labeled filamentous actin filaments using a TIRF microscope setup equipped with a shutter to illuminate the field briefly every 10 or 20 s, to minimize photobleaching of the labeled actin filaments. Actin filaments more than 5 m in length were tracked for more than 3 min. Time-lapse images were collected digitally and analysis of the leading edge of the actin filaments in the direction of translocation was performed by tracking the actin filaments over sequential images using the ImageJ image analysis software. The velocity distribution of actin filaments gliding over eGFP-MVc-HMM at saturating ATP concentration (1 mM) was 24.1 Ϯ 7.8 nm⅐s Ϫ1 (means Ϯ S.D.) (Fig. 9). A Gaussian fit to the velocity distribution histogram was used to determine the means Ϯ S.D.
Single Molecule Motility Assay-Single molecule TIRF microscopy assay (43) was performed as reported previously from our laboratory (61,62). The single molecule TIRF microscopy assay is a direct indication of whether or not the myosin is processive in vitro. TIRF microscopy experiments were performed using the recombinant heavy meromyosin-like myosin Vc construct encoding the first 1108 amino acids, containing the motor domain, the six predicted IQ motifs, and the coiled-coil region with an N-terminally fused enhanced GFP (eGFP-MVc-HMM). The assay was performed over a range of KCl concentrations (0 -300 mM) to determine whether KCl affects the single molecule processivity of eGFP-MVc-HMM, as reported previously for myosin Va (41). The images recorded did not show any processive motion of the eGFP-MVc-HMM over the range of KCl or ATP concentrations (1 M to 1 mM) . Traces were offset for clarity. The transients were fitted using a single exponential curve fit. B, pyrene fluorescence transients to determine dissociation rates of MVc-S1 from pyrene-actin, both in the absence of nucleotide and in ADP. Chase experiments where a pre-mixture of the pyrene-acto-MVc-S1 rigor complex or the pyrene-acto-MVc-S1⅐ADP ternary complex (0.3 M MVc-S1 and 0.5 M pyrene-actin, in the absence of nucleotide (solid symbols) or 20 M ADP (solid symbols)) were rapidly mixed with excess unlabeled 20 M actin in the stopped-flow apparatus. Averaged transients were fitted to a single exponential curve. Corresponding dissociation rates in the absence of nucleotide and 20 M ADP were 0.019 and 0.051 s Ϫ1 , respectively. FIGURE 4. Interaction of ATP with acto-MVc-S1. A, binding of ATP to pyrene-acto-MVc-S1 was monitored to observe the ATP-induced dissociation of the pyrene-actomyosin complex. A preincubated pyrene-acto-MVc-S1 complex (0.5 M MVc-S1 and 1.0 M pyrene-actin) was mixed with increasing concentrations of ATP (0 -450 M). Pyrene fluorescence increase was observed because of MVc-S1 dissociation from actin. Fluorescence transients were fitted to single exponential curves. The k obs increased with increasing ATP concentration. The maximal observed rate constant was k 1 Ј ϭ 287 s Ϫ1 with a half-saturation ( whereby the experiments were performed (data not shown), even though the eGFP-MVc-HMM does bind to and detach from actin filaments. This nonprocessive behavior is expected of single molecules of eGFP-MVc-HMM from the transient kinetic rates, i.e. specifically the P i release limitation of the ATPase cycle, determined using the MVc-S1 in this study. We conclude that the recombinant double-headed myosin Vc construct, eGFP-MVc-HMM, under unloaded in vitro conditions, is not processive as a single molecule.
Kinetic Simulations-Kinetic simulations of the acto-MVc-S1 ATPase cycle were performed based on Scheme 1 and the experimentally determined rate constants listed in Table 1.
Steps K 1 , K 1 , and K 7 (ATP binding to M5c-S1 detached from actin) were omitted from the simulations, because their parameters are unknown and the flux through these steps is negligible. Parameters used in the simulations but not listed in Table 1 were as follows: k 9 ϭ 1400 s Ϫ1 , k -9 ϭ 100 M Ϫ1 s Ϫ1 (to yield a rapid step with the measured K 9 ϭ 14 M); k 3 ϭ 31.6 s Ϫ1 , k Ϫ3 ϭ 58.4 s Ϫ1 (calculated from K 3 and (k 3 ϩ k Ϫ3 ) in Table 1); k 5 Ј ϭ 14.7 s Ϫ1 (average of k 5 Ј values listed in Table  1 First, we performed a global fit of the mechanism to attain the measured actin concentration dependence of the steady-state ATPase activity. In this fit, we floated the parameters of the experimentally inaccessible steps K 8 and K 3 Ј as well as the k 4 Ј rate constant, which might not precisely equal the maximal observed P i release rate constant of the MDCC-PBP measurements (Fig. 7).
The [actin] dependence of the partitioning between the weak binding MVc⅐ATP, MVc⅐ADP⅐P i , and acto-MVc⅐ADP⅐P i states (and acto-MVc⅐ATP, if relevant) is dictated by the reversible K 3 and K 9 equilibria, with possible contributions from K 8 and/or K 3 Ј. The fact that K 9 ϫ (1 ϩ K 3 )/K 3 (ϭ 40 M) nearly equals K ATPase (ϭ 42.5 M) caused the fits to produce nearly complete dissociation of MT from actin following ATP binding (i.e. in the well fitting models K 8 was Ն1 mM). The presence or absence of actin-attached hydrolysis (K 3 Ј) did not affect the outcome of the steady-state parameters (ATPase activity and duty ratio) at the experimentally applied actin concentrations. Therefore, in further simulations we used two extremes of the adequately fitting parameter sets to calculate the dependence of the ATPase activity and the duty ratio on actin and ADP concentration. Importantly, these two sets of parameters (see Fig. 10, A and B) yielded similar ATPase activity and duty ratio values because of the same maximal effective rate of P i release from acto-MVc (around 1.5 s Ϫ1 ). Set 1 represented a mechanism in which actin-attached ATP hydrolysis does not occur (k 3 Ј ϭ 0, k Ϫ3 Ј ϭ 0, K 8 ϭ 1 mM (rapid step), k 4 Ј ϭ 1.618 s Ϫ1 ), whereas set 2 contained a rapid and favorable actin-attached ATP hydrolysis step (k 3 Ј ϭ 100 s Ϫ1 , k Ϫ3 Ј ϭ 3 s Ϫ1 , K 8 ϭ 1 mM (rapid step), k 4 Ј ϭ 1.508 s Ϫ1 ). In both conditions, the duty ratio of MVc-S1 depended hyperbolically on actin concentration, yielding a maximum value of 0.095 (reaching half-saturation at 31

DISCUSSION
The kinetic characterization of the MVc-S1 and the in vitro motility assays performed using the eGFP-MVc-HMM revealed that the biochemistry of the vertebrate myosin Vc actomyosin ATPase cycle is fun-FIGURE 5. MANT-ADP release from acto-MVc-S1. Average time course of the MANT-ADP fluorescence decrease as MANT-ADP was displaced from MVc-S1. Initially, 50 M MANT-ADP, 1 M MVc-S1, and 20 M filamentous actin were mixed, and this mixture was subsequently mixed with 1 mM ATP (premixing concentrations). The solid line is the best single exponential fit, whereby the observed rate constant (k 5 Ј) was 16.0 Ϯ 0.6 s Ϫ1 . FIGURE 6. Acto-MVc-S1 ADP affinity. A, 1.0 M pyrene-labeled actin was initially incubated with 0.5 M MVc-S1 and various concentrations of ADP. This preincubated mixture was then mixed with 150 M ATP (pre-mixing concentrations). Shown is an averaged time course of the pyrene fluorescence increase because of dissociation of the MVc-S1 from the pyrene-labeled actin. In this particular example, 3 M ADP was used in the preincubated mixture. Solid line is the best double exponential fit to the data. The observed rate constant of the slow phase (k 5 Ј) in this case was 15.1 Ϯ 0.6 s Ϫ1 . B, shown is the ADP dependence of the relative amplitude of the slower phase of the double exponential fit to the averaged time course of the pyrene fluorescence increase (i.e. 100% ϫ (A slow /(A slow ϩ A fast ))). Fitting the data with a quadratic equation yielded a K d value 0.25 Ϯ 0.02 M (ϭK 5 Ј). damentally different from the other vertebrate myosin V isoforms, as well as the invertebrate Drosophila myosin V. We discuss these differences and the implications of these unique characteristics of myosin Vc.
Hum-MVc-S1 Exhibits a Low Duty Ratio-Kinetic analysis of MVc-S1 demonstrated that the rate-limiting step in the acto-MVc-S1 ATPase cycle is the P i release step (k 4 Ј ϭ 1.5 s Ϫ1 ), which is close to the maximal steady-state ATPase rate (V max ϭ 1.8 Ϯ 0.3 s Ϫ1 ) and ϳ10 times slower than the ADP release rate. The rate limitation of myosin Vc by the P i release step in the actomyosin ATPase cycle contrasts to other vertebrate myosin Vs (Va and Vb) whose ADP dissociation rate constant (k 5 Ј) is rate-limiting (31,34,36). It has been shown that the rate-limiting P i release is a common feature of ensemble myosins such as skeletal muscle myosin II (63), non-muscle myosin IIA (45), and some myosin Is (59,64,65). The duty ratios of ensemble myosins are generally known to be small (i.e. Յ10%).
By assuming a very simplified cycle in which only two states alternate, a strong and a weak actin-binding one, which the ADP release rate constant (k 5 Ј ϭ 12-16 s Ϫ1 ) is the exit rate from the strong binding state and the P i release rate constant (k 4 Ј ϭ 1.5 s Ϫ1 ) is the exit rate from the weak binding state, we can estimate the duty ratio of MVc-S1 by using the equation k 4 Ј/ (k 4 Ј ϩ k 5 Ј) (34,39). This yields a duty ratio within the range of 6 -13% for MVc-S1, which is dramatically lower compared with some other high duty ratio motors such as myosin Va and VI (34,66) but quite comparable with the low duty ratio myosins as mentioned.
To calculate the duty ratio under different conditions, we performed global kinetic simulations using the parameters determined from the biochemically observed rates in this study. The simulations generated a duty ratio in line with the above simple calculations (around 10%), and an increase of this duty ratio by ϳ7-fold to more than 60% in the presence of moderate background levels of [ADP] (Fig. 10). Simulations were performed for ADP concentrations from 0 to 200 M, which has Open symbols show all the data obtained in this experiment. Closed symbols show the initial phase (i.e. time/50) of the data. C, similar burst phase was observed in the presence of 15 M actin (amplitude, 0.35 mol of P i /mol of MVc-S1, k obs ϭ 50 s Ϫ1 ), but the steady-state turnover rate was accelerated to 0.32 s Ϫ1 . The time course of ATP hydrolysis was followed until quasi-complete exhaustion of the substrate to monitor product inhibition by ADP. The solid line in the main panel is a simulated progress curve of P i liberation resulting from a global fitting procedure using the kinetic parameters of Table 1 and leaving the ADP binding rate constant of actomyosin (k Ϫ5 Ј) as a floating parameter. The best fit was obtained at k Ϫ5 Ј ϭ 18 M Ϫ1 ⅐s Ϫ1 . The dashed line is a progress curve with a fixed k Ϫ5 Ј ϭ 5 M Ϫ1 ⅐s Ϫ1 . Inset shows the data points from the initial period (i.e. time 0 -1 s) of this burst phase with the fit.  been suggested as physiological concentrations, for example in muscle at rest and during work (67)(68)(69). This increase in duty ratio because of increased [ADP] may be a regulatory mechanism in vivo to modulate the intracellular function of myosin Vc in the cell, perhaps as a tension generating, or anchoring, myosin.
Furthermore, the estimated low duty ratio of the MVc-S1 is consistent with our observations that single molecules of eGFP-MVc-HMM exhibit nonprocessive movements in the single molecule TIRF microscopy assays. Thus, the biochemical rates obtained from the steady-state and transient kinetic solution experiments provided a satisfactory basis for its mechanochemical behavior.
Biochemical and Mechanical Implications of Human Myosin Vc as a Cytoskeletal Cargo Transporter-The duty ratio, discussed above, captures the essence of how different myosin Vc is compared with myosins Va and Vb. However, a number of unique details of the kinetic mechanism provide insights on the functional role of the myosin Vc in vivo. In this section we will discuss these different kinetic features.
The maximal steady-state ATPase rate of MVc-S1 (1.8 s Ϫ1 ) is markedly lower than that of chicken myosin Va (12 s Ϫ1 ) (34) and H. sapiens myosin Vb (9.7 Ϯ 0.4 s Ϫ1 ) (31). Moreover, the apparent affinity of MVc-S1 to actin filaments (K ATPase ) is ϳ30fold lower than chicken myosin Va (34) and 1.5-fold higher than that of myosin Vb (see Table 1). This lower actin affinity of MVc may be due in part to the difference in the loop 2 region, which has been shown to affect the K ATPase of chicken myosin Va (27). Comparing the loop 2 of H. sapiens MVc to that of the H. sapiens myosin Va, the overall charge of MVc in this region is approximately half that of myosin Va (14). The in vitro actin gliding assays also reflected this phenomenon, whereby the velocity of filamentous actin translocation by eGFP-MVc-HMM was approximately an order of magnitude slower (v Average ϳ 24 nm⅐s Ϫ1 ) than myosin Va (v Average ϳ 320 nm⅐s Ϫ1 (70) or myosin Vb (v Average ϳ 220 nm⅐s Ϫ1 (31))). The in vitro actin gliding assays were also performed using the creatine phosphate/creatine phosphokinase ATP regeneration system to determine whether the levels of ADP contamination account for this slow velocity of actin filaments. Using the ATP regeneration system, the velocity of the actin filaments increased by approximately a factor of 2 (data not shown), to which is still reporting actin filaments velocity an order of magnitude slower than both myosins Va and Vb. Based on our transient kinetic measurements, we expected the actin gliding velocity of myosin Vc would be closer to that of myosins Va and Vb. The slower actin filament velocity may be accounted for by the hindrance because of weakly actinbound heads of the eGFP-MVc-HMM.
Another possible reason for the slower in vitro actin gliding velocity of MVc may be due to the structure of the MVc lever arm or IQ motifs. The IQ motifs assembling the myosin Va lever have been shown to be separated by a repetition of 23 and 25 amino acid residues. However, the first two IQ motifs assembling the MVc lever seem to be separated by 23 and 28 amino acid residues (14). The different spacing of IQ motifs in the myosin Vc lever may contribute to the slow in vitro actin gliding velocities reported here as found previously with a mutant myosin Va construct where two alanine residues were inserted between the third and fourth IQ motifs (57). Both ensemble and single molecule studies of this myosin Va chimera showed slower velocity and smaller single displacements compared with the wild type construct without the additional alanines.
We note that the unexpectedly low in vitro actin gliding velocity of eGFP-MVc-HMM might possibly be due to a slow, rate-limiting isomerization between two actomyosin⅐ADP states that could occur after P i release and precede ADP release. Such a step might remain undetected in the ADP release stopped-flow experiments if the "starting" state of the isomerization is an energetically unfavorable state that does not become populated upon ADP addition. However, the inability  of eGFP-MVc-HMM to perform processive movement implies that its duty ratio is low, and thus the low actin gliding velocity results from hindrance by weakly actin-bound heads rather than an undetected strong actin-binding state. Furthermore, both in the presence of nucleotide, as well as in the absence of nucleotide, myosin Vc seems to have a lower affinity to actin than myosin Va. The binding rate of MVc-S1 to actin in the absence of nucleotide is ϳ100-fold slower as compared with chicken myosin Va (ϭ 3 M Ϫ1 ⅐s Ϫ1 (34)). These rigor and nucleotide-bound on-rate constants may not be directly related to single molecule processivity. However, it may indirectly have implications on the actin on-rate constant in the weak actin-binding states, which parameter indeed is an important factor in processivity (e.g. a common factor determining strong and weak binding on-rate constants may be surface charge distribution). These implications may suggest that myosin Vc is inadequate for a single molecule cargo transporter.
Thus, for myosin Vc to function as a cargo transporter motor protein in vivo, it may function collectively as an ensemble of low duty ratio motors that can become a processive cargo transporter. However, myosin Vc may be useful for other cellular processes, such as budding, tethering, or fusion within the membrane trafficking system to which myosin Vc has been implicated (6,14). The high affinity of myosin Vc for ADP may allow myosin Vc to have long lived, nonmoving attached states that may be a useful feature for a tension-generating, or anchoring, myosin in vivo.
Phylogenetic Analysis of H. sapiens Myosin Vc-A consensus tree of the H. sapiens myosin V isoforms (Va, Vb, and Vc) and Drosophila myosin V was reported recently (44), which indicated that the motor domain of the H. sapiens myosin Vc had a closer resemblance to the Drosophila myosin V than the other motor domains of the H. sapiens myosin V isoforms. Both Drosophila myosin V and the H. sapiens myosin Vc, as characterized in this study, are low duty ratio motors; however, the kinetic mechanisms of these two myosins are quite different. We therefore performed a more extensive phylogenetic analysis of the motor domain of myosin Vs from different organisms (Fig. 11). The result of the analysis indicated that the alignment of vertebrate myosin Vc in the consensus tree is separate from those of vertebrate myosin Va and Vb. Furthermore, all the vertebrate myosin Vs were located far from the Drosophila myosin V too. This result implies that the H. sapiens myosin Vc, even with the general dissimilarity of the kinetic cycle with its vertebrate myosin V counterparts, is closer to these isoforms than the Drosophila myosin V, which has a closer kinetic property to the H. sapiens myosin Vc. Perhaps evolution had evolved other vertebrate myosin V isoforms, Va and Vb, to become processive molecular motors.
High ADP Affinity of Myosin Vc-One unusual and intriguing aspect of the MVc-S1 actomyosin ATPase cycle is its high affinity for ADP. The ADP affinities were measured using the pyrene-actin fluorescence signal and the MANT-ADP signal, and independent experiments indicated similar results, suggesting that the ADP affinity (K 5 Ј) is higher for MVc-S1 (0.25-0.4 M) than myosin Va (0.8 M (34)) but not as high as the non-muscle myosin IIB (0.15 M (54)). It may be that the myosin Vc is sensitive to ADP concentration changes and increases the duty ratio at high, or even at moderate, [ADP] as hypothesized previously for non-muscle myosin IIB (54). This unique feature may enhance the function of myosin Vc as a possible tensiongenerating molecular motor inside of a cell.
Future experiments are required to reveal if this is a plausible mechanism; however, this could be a novel in vivo process to alter a naturally occurring low duty ratio motor into a high duty ratio motor by functionally adapting to the physical environment they encounter, and it also may be a form of regulation for this molecular motor.