Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal,smooth and non-muscle tropomyosins

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin.Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (K_app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (V_max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V_max.The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K_app as skeletal muscle tropomyosin but yields a value of V_max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin.On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.     

Quantitative determination of calcium-activated myosin adenosine triphosphatase activity in rat skeletal muscle fibres

Summary A quantitative histochemical technique was developed for determining the kinetics of the calcium-activated myosin ATPase (Ca²⁺-myosin ATPase) reaction in rat skeletal muscle fibres. Using this technique, the maximum velocity (V_max) and the apparent Michaelis-Menten rate constant for ATP (K_app) of the Ca²⁺-myosin ATPase reaction were measured in type-identified fibres of the rat medial gastrocnemius (MG) muscle. The V_max and the K_app of the Ca²⁺-myosin ATPase reaction were lowest in type I fibres and highest (i.e., approx. two times greater) in type IIb fibres. The K_app in type IIa fibres was similar to that in type I. However, the V_max was 1.5 times greater in type IIa fibres, compared to type I fibres. Evidence is presented to suggest that the type IIb fibre population in the MG does not represent a single myosin isozyme. In addition, the broad range of V_max and K_app values indicates that there is marked heterogeneity in the myosin heavy chain and myosin light chain composition of myosin isozymes among individual fibres.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

Mutating the Converter-Relay Interface of Drosophila Myosin Perturbs ATPase Activity, Actin Motility, Myofibril Stability and Flight Ability

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase (V_max) by ∼ 60% compared to wild-type myosin, but there is no change in apparent actin affinity (K_m). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by ∼ 15% or ∼ 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by ∼ 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional “cracking” of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.     

Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca²⁺ and incubated overnight at 4 °C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca²⁺-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.     

The reaction of myosin with N-ethylmaleimide in the presence of ADP

Myosin reacted with N-ethylmaleimide in the presence of ADP lost its ability to be activated by actin. Subfragment 1 behaved similarly. About 2 moles of N-ethylmaleimide per mole of Subfragment 1 were required to eliminate actin activation of the Mg²⁺-ATPase activity. At the point at which actin activation was lost the K⁺-EDTA-ATPase activity was also lost, but the Ca²⁺-activated ATPase activity was increased. Kinetic measurements indicated that the labelling with N-ethylmaleimide in the presence of ADP reduced V (the ATPase activity at infinite actin concentration) but did not effect K_app (which is related to the dissociation constant of the actin-Subfragment 1 complex). The Mg²⁺-activated activity of the reacted myosin alone remained unaltered and the ability to bind actin was retained. We propose that the N-ethylmaleimide labelling blocked the actin activation by preventing the accelerated release of hydrolysis products from the myosin.     

Mutational analysis of Ser14 and Asp157 in the nucleotide-binding site of beta-actin.

This paper compares wild-type and two mutant beta-actins, one in which Ser14 was replaced by a cysteine, and a second in which both Ser14 and Asp157 were exchanged (Ser14-->Cys and Ser14-->Cys, Asp157-->Ala, respectively). Both of these residues are part of invariant sequences in the loops, which bind the ATP phosphates, in the interdomain cleft of actin. The increased nucleotide exchange rate, and the decreased thermal stability and affinity for DNase I seen with the mutant actins indicated that the mutations disturbed the interdomain coupling. Despite this, the two mutant actins retained their ATPase activity. In fact, the mutated actins expressed a significant ATPase activity even in the presence of Ca2+ ions, conditions under which actin normally has a very low ATPase activity. In the presence of Mg2+ ions, the ATPase activity of actin was decreased slightly by the mutations. The mutant actins polymerized as the wild-type protein in the presence of Mg2+ ions, but slower than the wild-type in a K+/Ca2+ milieu. Profilin affected the lag phases and elongation rates during polymerization of the mutant and wild-type actins to the same extent, whereas at steady-state, the concentration of unpolymerized mutant actin appeared to be elevated. Decoration of mutant actin filaments with myosin subfragment 1 appeared to be normal, as did their movement in the low-load motility assay system. Our results show that Ser14 and Asp157 are key residues for interdomain communication, and that hydroxyl and carboxyl groups in positions 14 and 157, respectively, are not necessary for ATP hydrolysis in actin.     

Distinct interactions between actin and essential myosin light chain isoforms

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein–protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin^ala3) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (K_D = 575 nM) was significantly (p < 0.01) lower compared with the affinity of hVLC-1 to α-actin (K_D = 186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p < 0.01) lower association rate (k_on: 1018 M⁻¹ s⁻¹) compared with k_on of the hVLC-1/α-actin complex interaction (2908 M⁻¹ s⁻¹). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.     

Calponin-like protein from mussel smooth muscle is a competitive inhibitor of actomyosin ATPase

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters V_max and K_ATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in K_ATPase value and to a lesser extent the decrease in V_max, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of V_max and K_ATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.     

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492–505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin·gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca²⁺. Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca²⁺ sensitivity in the presence of tropomyosin-troponin.     

Implications of oxidovanadium(IV) binding to actin

Oxidovanadium(IV), a cationic species (VO²⁺) of vanadium(IV), binds to several proteins, including actin. Upon titration with oxidovanadium(IV), approximately 100% quenching of the intrinsic fluorescence of monomeric actin purified from rabbit skeletal muscle (G-actin) was observed, with a V₅₀ of 131 μM, whereas for the polymerized form of actin (F-actin) 75% of quenching was obtained and a V₅₀ value of 320 μM. Stern-Volmer plots were used to estimate an oxidovanadium(IV)-actin dissociation constant, with K_d of 8.2 μM and 64.1 μM VOSO₄, for G-actin and F-actin, respectively. These studies reveal the presence of a high affinity binding site for oxidovanadium(IV) in actin, producing local conformational changes near the tryptophans most accessible to water in the three-dimensional structure of actin. The actin conformational changes, also confirmed by ¹H NMR, are accompanied by changes in G-actin hydrophobic surface, but not in F-actin. The ¹H NMR spectra of G-actin treated with oxidovanadium(IV) clearly indicates changes in the resonances ascribed to methyl group and aliphatic regions as well as to aromatics and peptide-bond amide region. In parallel, it was verified that oxidovanadium(IV) prevents the G-actin polymerization into F-actin. In the 0-200 μM range, VOSO₄ inhibits 40% of the extent of polymerization with an IC₅₀ of 15.1 μM, whereas 500 μM VOSO₄ totally suppresses actin polymerization. The data strongly suggest that oxidovanadium(IV) binds to actin at specific binding sites preventing actin polymerization. By affecting actin structure and function, oxidovanadium(IV) might be responsible for many cellular effects described for vanadium.     

Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin,ATM1

Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg²⁺-ATPase activity (V_max = 4 s⁻¹), although their affinities for actin were high (K_actin = 4 μm). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s⁻¹, respectively). Physiological concentrations of free Mg²⁺ modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis.     

Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction

Ca2+-calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK-actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228-232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.     

Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg²⁺) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg²⁺- or Mn²⁺-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn²⁺, providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn²⁺ rescues this effect to near wild-type activity. 2′(3′)-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region.     

Velocity of myosin-based actin sliding depends on attachment and detachment kinetics and reaches a maximum when myosin-binding sites on actin saturate

Molecular motors such as kinesin and myosin often work in groups to generate the directed movements and forces critical for many biological processes. Although much is known about how individual motors generate force and movement, surprisingly, little is known about the mechanisms underlying the macroscopic mechanics generated by multiple motors. For example, the observation that a saturating number, N, of myosin heads move an actin filament at a rate that is influenced by actin–myosin attachment and detachment kinetics is accounted for neither experimentally nor theoretically. To better understand the emergent mechanics of actin–myosin mechanochemistry, we use an in vitro motility assay to measure and correlate the N-dependence of actin sliding velocities, actin-activated ATPase activity, force generation against a mechanical load, and the calcium sensitivity of thin filament velocities. Our results show that both velocity and ATPase activity are strain dependent and that velocity becomes maximized with the saturation of myosin-binding sites on actin at a value that is 40% dependent on attachment kinetics and 60% dependent on detachment kinetics. These results support a chemical thermodynamic model for ensemble motor mechanochemistry and imply molecularly explicit mechanisms within this framework, challenging the assumption of independent force generation.     

Mechanism of formation of actomyosin interface

Force generation in muscle results from binding of myosin to F-actin. ATP binding to myosin provides energy to dissociate actomyosin complex while the hydrolysis of ATP is needed for re-binding of myosin to F-actin. At the end of each cycle myosin and actin form a tight complex with a substantial interface area. We investigated the dynamics of formation of actomyosin interface in presence and absence of nucleotides by quenched flow cross-linking technique. We showed previously that myosin head (subfragment 1, S1) directly interacts with at least two monomers in the actin filament. The quenched flow cross-linking experiments revealed that the initial contact (in presence or absence of nucleotides) occurs between loop 635-647 of S1 and 1-12 N-terminal residues of one actin and, then, the second contact forms between loop 567-574 of S1 and the N terminus of the second actin. The distance between these two loops in S1 corresponds to the distance between N termini of two actins in the same strand (53 A) but is smaller than that between two actins from the different strands (102 A). The formation of the actomyosin complex proceeds in ordered sequence: S1 initially binds to one actin then binds with the second actin located in the same strand but probably closer to the barbed end of F-actin. The presence of nucleotides slows down the interaction of S1 with the second actin, which correlates with recently proposed cleft movement in a 50 kDa domain of S1. The sequential mechanism of formation of actomyosin interface starting from one end and developing towards the barbed end might be involved in force generation and directional movement in actin-myosin system.     

Organization and nucleotide sequence of ten ribosomal protein genes from the region equivalent to the spectinomycin operon in the archaebacterium Halobacterium marismortui.

Summary We have created missense mutations in the indirect flight muscle (IFM)-specific Act88F actin gene of Drosophila melanogaster by random in vitro mutagenesis. Following P element-mediated transformation into wild-type flies and subsequent transfer of the inserts into Act88F null strains, the effects of the actin mutants on the structure and function of the IFMs were examined. All of the mutants were antimorphic for flight ability. E316K and G368E formed muscle with only relatively small defects in structure whilst the others produced IFMs with large amounts of disruption. E334K formed filaments but lacked Z discs. V339I formed no muscle structure in null flies and did not accumulate actin. E364K and G366D both had relatively stable actin but did not form myofibrils. Using an in vitro polymerisation assay we found no significant effects on the ability of the mutant actins to polymerise. E364K and G366D also caused a strong induction of heat shock protein (hsp) synthesis at normal temperatures and accumulated large amounts of hsp22 which, together with the mutant actin, was resistant to detergent extraction. Both E316K and E334K caused a weak induction of hsp synthesis. We discuss how the stability, structure and function of the different mutant actins affects myofibril assembly and function, and the induction of hsps.     

Site-specific mutations in the myosin binding sites of actin affect structural transitions that control myosin binding.

We have examined the effects of actin mutations on myosin binding, detected by cosedimentation, and actin structural dynamics, detected by spectroscopic probes. Specific mutations were chosen that have been shown to affect the functional interactions of actin and myosin, two mutations (4Ac and E99A/E100A) in the proposed region of weak binding to myosin and one mutation (I341A) in the proposed region of strong binding. In the absence of nucleotide and salt, S1 bound to both wild-type and mutant actins with high affinity (K(d) < microM), but either ADP or increased ionic strength decreased this affinity. This decrease was more pronounced for actins with mutations that inhibit functional interaction with myosin (E99A/E100A and I341A) than for a mutation that enhances the interaction (4Ac). The mutations E99A/E100A and I341A affected the microsecond time scale dynamics of actin in the absence of myosin, but the 4Ac mutation did not have any effect. The binding of myosin eliminated these effects of mutations on structural dynamics; i.e., the spectroscopic signals from mutant actins bound to S1 were the same as those from wild-type actin. These results indicate that mutations in the myosin binding sites affect structural transitions within actin that control strong myosin binding, without affecting the structural dynamics of the strongly bound actomyosin complex.     

A kinetic model that explains the effect of inorganic phosphate on the mechanics and energetics of isometric contraction of fast skeletal muscle

A conventional five-step chemo-mechanical cycle of the myosin–actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding of a new ATP molecule, is able to fit the [Pi] dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in [Pi] much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with [Pi]. The large dissociation constant of the product release in the branched pathway allows the isometric myosin–actin reaction to fit the equilibrium constant of the ATPase.     

Involvement of an Actomyosin Contractile Ring in Saccharomyces cerevisiae Cytokinesis

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother–bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother–bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Δ strains divide nearly as efficiently as wild type; other myo1Δ strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell–wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.