Truncation of a mammalian myosin I results in loss of Ca2+-sensitive motility

MYR-1, a mammalian class I myosin, consisting of a heavy chain and 4-6 associated calmodulins, is represented by the 130-kDa myosin I (or MI(130)) from rat liver. MI(130) translocates actin filaments in vitro in a Ca(2+)-regulated manner. A decrease in motility observed at higher Ca(2+) concentrations has been attributed to calmodulin dissociation. To investigate mammalian myosin I regulation, we have coexpressed in baculovirus calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic "motor" domain and the first calmodulin-binding IQ domain of rat myr-1; we refer to this truncated molecule here as MI(1IQ). Association of calmodulin to MI(1IQ) is Ca(2+)-insensitive. MI(1IQ) translocates actin filaments in vitro at a rate resembling MI(130), but unlike MI(130), does not exhibit sensitivity to 0.1-100 micrometer Ca(2+). In addition to demonstrating successful expression of a functional truncated mammalian myosin I in vitro, these results indicate that: 1) Ca(2+)-induced calmodulin dissociation from MI(130) in the presence of actin is not from the first IQ domain, 2) velocity is not affected by the length of the IQ region, and 3) the Ca(2+) sensitivity of actin translocation exhibited by MI(130) involves 1 or more of the other 5 IQ domains and/or the carboxyl tail.     

Crystal Structure of Human Myosin 1c—The Motor in GLUT4 Exocytosis: Implications for Ca Regulation and 14-3-3 Binding

Myosin 1c (Myo1c) plays a key role in supporting motile events that underlie cell migration, vesicle trafficking, insulin-stimulated glucose uptake and hearing. Here, we present the crystal structure of the human Myo1c motor in complex with its light chain calmodulin. Our structure reveals tight interactions of the motor domain with calmodulin bound to the first IQ motif in the neck region. Several of the calmodulin residues contributing to this interaction are also involved in Ca^2 + binding. Contact residues in the motor domain are linked to the central β-sheet and the HO helix, suggesting a mechanism for communicating changes in Ca^2 + binding in the neck region to the actin and nucleotide binding regions of the motor domain. The structural context and the chemical environment of Myo1c mutations that are involved in sensorineural hearing loss in humans are described and their impact on motor function is discussed. We show that a construct consisting of the motor domain of Myo1c and the first IQ motif is sufficient to establish a tight interaction with 14-3-3β (K_D = 0.9 μM) and present the model of a double-headed Myo1c–14-3-3 complex. This complex has been implicated in the exocytosis of glucose transporter 4 storage vesicles during insulin-stimulated glucose uptake.     

Calcium regulation of calmodulin binding to and dissociation from the myo1c regulatory domain

Myo1c is an unconventional myosin involved in cell signaling and membrane dynamics. Calcium binding to the regulatory-domain-associated calmodulin affects myo1c motor properties, but the kinetic details of this regulation are not fully understood. We performed actin gliding assays, ATPase measurements, fluorescence spectroscopy, and stopped-flow kinetics to determine the biochemical parameters that define the calmodulin-regulatory-domain interaction. We found calcium moderately increases the actin-activated ATPase activity and completely inhibits actin gliding. Addition of exogenous calmodulin in the presence of calcium fully restores the actin gliding rate. A fluorescently labeled calmodulin mutant (N111C) binds to recombinant peptides containing the myo1c IQ motifs at a diffusion-limited rate in the presence and absence of calcium. Measurements of calmodulin dissociation from the IQ motifs in the absence of calcium show that the calmodulin bound to the IQ motif adjacent to the motor domain (IQ1) has the slowest dissociation rate (0.0007 s-1), and the IQ motif adjacent to the tail domain (IQ3) has the fastest dissociation rate (0.5 s-1). When the complex is equilibrated with calcium, calmodulin dissociates most rapidly from IQ1 (60 s-1). However, this increased rate of dissociation is limited by a slow calcium-induced conformational change (3 s-1). Fluorescence anisotropy decay of fluorescently labeled N111C bound to myo1c did not depend appreciably on Ca2+. Our data suggest that the calmodulin bound to the IQ motif adjacent to the motor domain is rapidly exchangeable in the presence of calcium and is responsible for regulation of myo1c ATPase and motile activity.     

Rat myr 4 defines a novel subclass of myosin I: identification,distribution, localization,and mapping of calmodulin-binding sites with differential calcium sensitivity

We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I''s. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F- actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.     

Ca(2+)-dependent regulation of the motor activity of myosin V

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca(2+) concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca(2+) binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain.     

Regulatory implications of a novel mode of interaction of calmodulin with a double IQ-motif target sequence from murine dilute myosin V

Apo-Calmodulin acts as the light chain for unconventional myosin V, and treatment with Ca(2+) can cause dissociation of calmodulin from the 6IQ region of the myosin heavy chain. The effects of Ca(2+) on the stoichiometry and affinity of interactions of calmodulin and its two domains with two myosin-V peptides (IQ3 and IQ4) have therefore been quantified in vitro, using fluorescence and near- and far-UV CD. The results with separate domains show their differential affinity in interactions with the IQ motif, with the apo-N domain interacting surprisingly weakly. Contrary to expectations, the effect of Ca(2+) on the interactions of either peptide with either isolated domain is to increase affinity, reducing the K(d) at physiological ionic strengths by >200-fold to approximately 75 nM for the N domain, and approximately 10-fold to approximately 15 nM for the C domain. Under suitable conditions, intact (holo- or apo-) calmodulin can bind up to two IQ-target sequences. Interactions of apo- and holo-calmodulin with the double-length, concatenated sequence (IQ34) can result in complex stoichiometries. Strikingly, holo-calmodulin forms a high-affinity 1:1 complex with IQ34 in a novel mode of interaction, as a "bridged" structure wherein two calmodulin domains interact with adjacent IQ motifs. This apparently imposes a steric requirement for the alpha-helical target sequence to be discontinuous, possibly in the central region, and a model structure is illustrated. Such a mode of interaction could account for the Ca(2+)-dependent regulation of myosin V in vitro motility, by changing the structure of the regulatory complex, and paradoxically causing calmodulin dissociation through a change in stoichiometry, rather than a Ca(2+)-dependent reduction in affinity.     

Calmodulin bound to the first IQ motif is responsible for calcium-dependent regulation of myosin 5a

Myosin 5a is as yet the best-characterized unconventional myosin motor involved in transport of organelles along actin filaments. It is well-established that myosin 5a is regulated by its tail in a Ca(2+)-dependent manner. The fact that the actin-activated ATPase activity of myosin 5a is stimulated by micromolar concentrations of Ca(2+) and that calmodulin (CaM) binds to IQ motifs of the myosin 5a heavy chain indicates that Ca(2+) regulates myosin 5a function via bound CaM. However, it is not known which IQ motif and bound CaM are responsible for the Ca(2+)-dependent regulation and how the head-tail interaction is affected by Ca(2+). Here, we found that the CaM in the first IQ motif (IQ1) is responsible for Ca(2+) regulation of myosin 5a. In addition, we demonstrate that the C-lobe fragment of CaM in IQ1 is necessary for mediating Ca(2+) regulation of myosin 5a, suggesting that the C-lobe fragment of CaM in IQ1 participates in the interaction between the head and the tail. We propose that Ca(2+) induces a conformational change of the C-lobe of CaM in IQ1 and prevents interaction between the head and the tail, thus activating motor function.     

Interactions of Cdc4p, a myosin light chain, with IQ-domain containing proteins in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe undergoes cell division through a medially placed actomyosin-based contractile ring. One of the key components of this ring is the F-actin based motor protein myosin II. The myosin II heavy chain Myo2p has two light-chain-binding domains, IQl and IQ2, which bind the essential light chain, Cdc4p, and the regulatory light chain, Rlc1p. Previously, we have reported the characterization of cells expressing Myo2p lacking the IQ2 domain that facilitates Myo2p interaction with Rlc1p. In this study, we have created and characterized S. pombe strains carrying precise deletions of IQ1 and the entire neck region encompassing the IQ1 and IQ2 domains. Surprisingly, we found that the entire neck region of Myo2p is dispensable for Myo2p function. Cells deleted for IQ1, IQ2 and the entire neck region of Myo2p do not display any obvious cytoskeletal abnormalities. Immunofluorescence studies indicated that Cdc4p localizes at the ring in early and late mitotic cells in a strain in which interactions of Cdc4p with both the myosin II heavy chains (Myo2p and Myp2p) are abolished. Unlike mutations in Rlc1p that are suppressed by a simultaneous deletion of its binding site on Myo2p, mutations in the essential light chain Cdc4p are not suppressed by deletion of its binding sites on Myo2p, suggesting that Cdc4p may have additional partners essential for cytokinesis. Consistent with this, we provide evidence that two other IQ-domain containing actomyosin ring proteins, Rng2p (an IQGAP-related protein) and Myo51p (a type V myosin heavy chain), physically interact with Cdc4p. We concluded that Cdc4p, a novel myosin light chain, interacts with multiple actomyosin ring components to effect cytokinesis.     

The two IQ-motifs and Ca2+/calmodulin regulate the rat myosin 1d ATPase activity

The light chain binding domain of rat myosin 1d consists of two IQ-motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca2+/CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d-head, Myo1d-IQ1, Myo1d-IQ1.2, Myo1d-IQ2 and Myo1dDeltaLV-IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca2+. IQ2 had a lower affinity for CaM in the absence of Ca2+ than in the presence of Ca2+. The actin-activated ATPase activity of Myo1d was approximately 75% inhibited by Ca2+-binding to CaM. This inhibition was observed irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca2+-dependence, we propose that Ca2+-binding to the C-terminal pair of high affinity sites in CaM inhibits the Myo1d actin-activated ATPase activity. This inhibition was due to a conformational change of the C-terminal lobe of CaM remaining bound to the IQ-motif(s). Interestingly, a similar but Ca2+-independent inhibition of Myo1d actin-activated ATPase activity was observed when IQ2, fused directly to the Myo1d-head, was rotated through 200 degrees by the deletion of two amino acids in the lever arm alpha-helix N-terminal to the IQ-motif.     

Kinetic analyses of a truncated mammalian myosin I suggest a novel isomerization event preceding nucleotide binding

MI(1IQ) is a complex of calmodulin and an epitope-tagged 85-kDa fragment representing the amino-terminal catalytic motor domain and the first of 6 calmodulin-binding IQ domains of the mammalian myosin I gene, rat myr-1 (130-kDa myosin I or MI(130)). We have determined the transient kinetic parameters that dictate the ATP hydrolysis cycle of mammalian myosin I by examining the properties of MI(1IQ). Transient kinetics reveal that the affinity of MI(1IQ) for actin is 12 nm. The ATP-induced dissociation of actin-MI(1IQ) is biphasic. The fast phase is dependent upon [ATP], whereas the slow phase is not; both phases show a Ca(2+) sensitivity. The fast phase is eliminated by the addition of ADP, 10 micrometer being required for half-saturation of the effect in the presence of Ca(2+) and 3 micrometer ADP in the absence of Ca(2+). The slow phase shares the same rate constant as ADP release (8 and 3 s(-)(1) in the presence and absence of Ca(2+), respectively), but cannot be eliminated by decreasing [ADP]. We interpret these results to suggest that actin-myosin I exists in two forms in equilibrium, one of which is unable to bind nucleotide. These results also indicate that the absence of the COOH-terminal 5 calmodulin binding domains of myr-1 do not influence the kinetic properties of MI(130) and that the Ca(2+) sensitivity of the kinetics are in all likelihood due to Ca(2+) binding to the first IQ domain.     

Chlamydomonas outer arm dynein alters conformation in response to Ca2+

We have previously shown that Ca(2+) directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the beta and gamma heavy chains (HCs). The gamma HC-associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca(2+) with K(Ca) = 3 x 10(-5) M in vitro, suggesting it may act as a Ca(2+) sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and gamma HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the gamma heavy chain. In vitro experiments indicate that LC4 undergoes a Ca(2+)-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca(2+). The sedimentation profile of the gamma HC subunit changed subtly upon Ca(2+) addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated gamma subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca(2+) addition. We propose that Ca(2+)-dependent conformational change of LC4 has a direct effect on the stem domain of the gamma HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm.     

Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin

Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca²⁺, indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5.     

The unconventional myosin, Myo2p, is a calmodulin target at sites of cell growth in Saccharomyces cerevisiae

Myo2p is an unconventional myosin required for polarized growth in Saccharomyces cerevisiae. Four lines of evidence suggest that (a) Myo2p is a target of calmodulin at sites of cell growth, and (b) the interaction between Myo2p and calmodulin is Ca2+ independent. First, as assessed by indirect immunofluorescence, the distributions of Myo2p and calmodulin are nearly indistinguishable throughout the cell cycle. Second, a genetic analysis indicates that mutations in CMD1 show allele- specific synthetic lethality with the myo2-66 conditional mutation. Mutations that inactivate the Ca(2+)-binding sites of calmodulin have little or no effect on strains carrying myo2-66, whereas an allele with a mutation outside the Ca(2+)-binding sites dramatically increases the severity of the phenotype conferred by myo2-66. Third, Myo2p coimmunoprecipitates with calmodulin in the presence of Ca2+ or EGTA. Finally, we used a modified gel overlay assay to demonstrate direct interaction between calmodulin and fusion proteins containing portions of Myo2p. Calmodulin binds specifically to the region of Myo2p containing six tandem repeats of a motif called an IQ site. Binding occurs in either Ca2+ or EGTA, and only two sites are required to observe binding.     

Motor function and regulation of myosin X.

Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, neck, and coiled-coil domain and purified bovine myosin X in Sf9 cells. The Mg(2+)-ATPase activity of myosin X was significantly activated by actin with low K(ATP). The actin-activated ATPase activity was reduced at Ca(2+) concentrations above pCa 5 in which 1 mol of calmodulin light chain dissociates from the heavy chain. Myosin X translocates F-actin filaments with the velocity of 0.3 microm/s with the direction toward the barbed end. The actin translocating activity was inhibited at concentrations of Ca(2+) at pCa 6 in which no calmodulin dissociation takes place, suggesting that the calmodulin dissociation is not required for the inhibition of the motility. Unlike class V myosin, which shows a high affinity for F-actin in the presence of ATP, the K(actin) of the myosin X ATPase was much higher than that of myosin V. Consistently nearly all actin dissociated from myosin X in the presence of ATP. ADP did not significantly inhibit the actin-activated ATPase activity of myosin X, suggesting that the ADP release step is not rate-limiting. These results suggest that myosin X is a nonprocessive motor. Consistently myosin X failed to support the actin translocation at low density in an in vitro motility assay where myosin V, a processive motor, supports the actin filament movement.     

Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.     

Mouse Myosin-19 Is a Plus-end-directed,High-duty Ratio Molecular Motor

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.     

Intermolecular Autophosphorylation Regulates Myosin IIIa Activity and Localization in Parallel Actin Bundles

Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 μm, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.     

Structural basis for the interaction of the myosin light chain Mlc1p with the myosin V Myo2p IQ motifs

Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.     

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

Mammalian myosin IXb (Myo9b) has been shown to exhibit unique motor properties in that it is a single-headed processive motor and the rate-limiting step in its chemical cycle is ATP hydrolysis. Furthermore, it has been reported to move toward the minus- and the plus-end of actin filaments. To analyze the contribution of the light chain-binding domain to the movement, processivity, and directionality of a single-headed processive myosin, we expressed constructs of Caenorhabditis elegans myosin IX (Myo9) containing either the head (Myo9-head) or the head and the light chain-binding domain (Myo9-head-4IQ). Both constructs supported actin filament gliding and moved toward the plus-end of actin filaments. We identified in the head of class IX myosins a calmodulin-binding site at the N terminus of loop 2 that is unique among the myosin superfamily members. Ca²⁺/calmodulin negatively regulated ATPase and motility of the Myo9-head. The Myo9-head demonstrated characteristics of a processive motor in that it supported actin filament gliding and pivoting at low motor densities. Quantum dot-labeled Myo9-head moved along actin filaments with a considerable run length and frequently paused without dissociating even in the presence of obstacles. We conclude that class IX myosins are plus-end-directed motors and that even a single head exhibits characteristics of a processive motor.     

Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites

Myosins‐I are conserved proteins that bear an N‐terminal motor head followed by a Tail Homology 1 (TH1) lipid‐binding domain. Some myosins‐I have an additional C‐terminal extension (C_ext) that promotes Arp2/3 complex‐dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin‐related light chains. Myosins‐I are known to participate in actin‐dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin‐I Myo5 and its C_ext. The TH1 domain prevented binding of the Myo5 C_ext to the yeast WIP homologue Vrp1, Myo5 C_ext‐induced actin polymerization and recruitment of the Myo5 C_ext to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C_ext. Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin‐I lifespan at endocytic sites and activated Myo5‐induced actin polymerization.