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Myosin VI is involved in a wide range of endocytic and exocytic membrane trafficking pathways; clathrin-mediated endocytosis, intracellular transport of clathrin-coated and -uncoated vesicles, AP-1B-dependent basolateral sorting in polarized epithelial cells and secretion from the Golgi complex to the cell surface. In this study, using a yeast two-hybrid screen, we identified brain-enriched kinase/lemur tyrosine kinase 2 (BREK/LMTK2), a transmembrane serine/threonine kinase with previously unknown cellular functions, as a myosin VI-interacting protein. Several binding experiments confirmed the interaction of myosin VI with BREK in vivo and in vitro. Immunocytochemical analyses revealed that BREK localizes to cytoplasmic membrane vesicles and to perinuclear recycling endosomes. Notably, cells in which BREK was depleted by siRNA were still able to internalize transferrin molecules and to transport them to early endosomes, but were unable to transport them to perinuclear recycling endosomes. Our results show that BREK is critical for the transition of endocytosed membrane vesicles from early endosomes to recycling endosomes and also suggest an involvement of myosin VI in this pathway.

MYO18B is a class XVIII myosin, cloned as a tumor suppressor gene candidate. To investigate the mechanisms of MYO18B-dependent tumor suppression, MYO18B-interacting proteins were searched for by a yeast two-hybrid screen. HOMER2, a Homer/Ves1 family protein, was identified as a binding partner of MYO18B. These proteins co-localized in the regions of membrane protrusion and stress fiber, which are known as ones with filamentous actin-rich structures. Expression of HOMER2 enhanced the ability of MYO18B to suppress anchorage-independent growth. These results indicate that HOMER2 and MYO18B cooperate together in tumor suppression.

Cytoplasmic dynein is an AAA(+)-type molecular motor whose major components are two identical heavy chains containing six AAA(+) modules in tandem. It moves along a single microtubule in multiple steps which are accompanied with multiple ATP hydrolysis. This processive sliding is crucial for cargo transports in vivo. To examine how cytoplasmic dynein exhibits this processivity, we performed in vitro motility assays of two-headed full-length or truncated single-headed heavy chains. The results indicated that four to five molecules of the single-headed heavy chain were required for continuous microtubule sliding, while approximately one molecule of the two-headed full-length heavy chain was enough for the continuous sliding. The ratio of the stroking time to the total ATPase cycle time, which is a quantitative indicator of the processivity, was approximately 0.2 for the single-headed heavy chain, while it was approximately 0.6 for the full-length molecule. When two single-headed heavy chains were artificially linked by a coiled-coil of myosin, the processivity was restored. These results suggest that the two heads of a single cytoplasmic dynein communicate with each other to take processive steps along a microtubule.

MYO18B is a class XVIIIB unconventional myosin encoded by a candidate tumor suppressor gene. To gain insights into the cellular function of this protein, we searched for MYO18B-interacting proteins by a yeast two-hybrid screen. Sug1, a 19S regulator subunit of the 26S proteasome, was identified as a binding partner of the C-terminal tail region of MYO18B. The association of MYO18B with Sug1 was further confirmed by GST pull-down, co-immunoprecipitation, and immunocytochemistry. Furthermore, proteasome dysfunction by a proteasome inhibitor or siRNA-mediated knock-down of Sug1 caused the up-regulation of MYO18B protein and MYO18B was polyubiquitinated in vivo. Collectively, these results suggested that MYO18B is a substrate for proteasomal degradation.

Myosin XVIII is the recently identified 18th class of myosins, and its members are composed of a unique N-terminal domain, a motor domain with an unusual sequence around the ATPase site, one IQ motif, a segmented coiled-coil region for dimerization, and a C-terminal globular tail. To gain insight into the functions of this unique myosin, we characterized its human homologue, MYO18A, focusing on the functional roles of the characteristic N-terminal domain that contains a PDZ module known to mediate protein-protein interaction. GFP-tagged full-length and C-terminally truncated MYO18A molecules that were expressed in HeLa cells exhibited colocalization with actin filaments. Chemical cross-linking of these molecules showed that they form stable dimers as expected from their putative coiled-coil tails. Cosedimentation of the various types of truncated MYO18A constructs with actin filaments indicated the presence of an ATP-insensitive actin-binding site in the N-terminal domain. Further studies on truncated constructs of the N-terminal domain indicated that this actin-binding site is located outside the PDZ module, but within the middle region of this domain, which does not show any homology with the known actin-binding motifs. These results imply that this dimeric myosin might stably cross-link actin filaments by two ATP-insensitive actin-binding sites at the N-terminal domains for higher-order organization of the actin cytoskeleton.

During steady-state ATP hydrolysis by actomyosin, myosin cyclically passes through strong actin-binding states and weak actin-binding states, depending on the nature of a nucleotide in the ATPase site. This cyclic change of actin-myosin affinity is coupled with the lever-arm swing and is critical for the sliding motion and force generation of actomyosin. To understand the structure-function relationship of this ATPase-dependent actin-myosin interaction, Dictyostelium myosin II has been extensively used for site-directed mutagenesis. By generating a large number of mutant myosins, two hydrophobic actin-binding sites have been revealed, located at the tip of the upper and lower 50 K subdomains of Dictyostelium myosin, one of which is the 'cardiomyopathy loop'. Furthermore, the slight change in relative orientation of these two hydrophobic sites around the 'strut loop' has been shown to work as a switch to turn on and off the strong binding to actin. Once the switch is turned off, myosin enters in the weak-binding state, where ionic interactions between actin and the 'loop 2' of myosin become the dominant force to maintain the actin-myosin association. The details of actin-myosin interactions revealed by the Dictyostelium system can serve as a framework for further examinations of the myosin superfamily proteins.

The strut loop, one of the three loops that connects the upper and lower 50K subdomains of myosin, plays a role as a “strut” to keep the relative disposition of the two subdomains. A single residue was either inserted into or deleted from this loop. The insertion or deletion mutation abolished the in vivo motor functions of myosin, as revealed by the fact that the mutant myosins did not complement the phenotypic defects of the myosin-null cells. In vitro studies of purified full-length myosins and their subfragment-1s (S1s) revealed that the insertion mutants virtually lost the strong binding to actin although their motor functions in the absence of actin remained almost normal, showing that only the hydrophobic actin-myosin association was selectively affected by the insertion mutations. Unlike the insertion mutants, the deletion mutant showed defects both in the strong-binding state and the rate-limiting step of ATPase cycle. These results indicate the functional importance of the strut loop in establishing the strong-binding state of myosin and thereby achieving successful power strokes.

The method of fluorescence resonance energy transfer (FRET) is one of the most important techniques for measuring the distance between two fluorophores and for detecting the changes in protein structure under physiological conditions. The use of green fluorescent protein is also a powerful technology that has been used to elucidate dynamic molecular events. From these we have developed a novel method to determine the three-dimensional positions of fluorophores by combining the FRET data and other structural information available. Using this method, we could determine the ATP-induced changes of three-dimensional structure of truncated Dictyostelium myosin in solution. The myosin structure with ADP in solution was found to be similar to that of the crystal structure of MgADPBeFx-bound truncated Dictyostelium myosin (type I structure), whereas myosin with ATP in solution was similar to the crystal structure of MgAdPVi-bound one (type II structure). However, the crystal structure of MgADP-bound scallop myosin (type III structure) could not be explained by any of our FRET data under various conditions. This indicates that the type III crystal structure might represent a transient intermediate conformation that could not be detected using fluorescence energy transfer.

The structure-function relationships of fast skeletal myosin isoforms remain poorly understood. To shed some light, we constructed chimeric myosins comprised of Dictyostelium myosin heavy chain backbone with carp loop sequences and analyzed their functional properties. A loop 2–10 chimeric myosin having the loop 2 sequence of the fast skeletal isoform predominantly expressed in carp acclimated to 10°C showed V max in actin-activated Mg 2+ -ATPase activity 1.4-fold higher than a loop 2–30 chimera constructed from the loop 2 sequence of the dominant isoform in carp acclimated to 30°C. These two chimera exhibited no significant differences in sliding velocity of actin filaments in in vitro motility assay. Contrastingly, both loop 1-associated chimeras, loop 1–10 and loop 1–30, did not differ in both ATPase activity and in sliding velocity of actin filaments.

A loop comprising residues 454–459 ofDictyostelium myosin II is structurally and functionally equivalent to the switch II loop of the G-protein family. The consensus sequence of the “switch II loop” of the myosin family is DIXGFE. In order to determine the functions of each of the conserved residues, alanine scanning mutagenesis was carried out on theDictyostelium myosin II heavy chain gene. Examination ofin vivo and in vitro motor functions of the mutant myosins revealed that the I455A and S456A mutants retained those functions, whereas the D454A, G457A, F458A and E459A mutants lost them. Biochemical analysis of the latter myosins showed that the G457A and E459A mutants lost the basal ATPase activity by blocking of the isomerization and hydrolysis steps of the ATPase cycle, respectively. The F458A mutant, however, lost the actin-activated ATPase activity without loss of the basal ATPase activity. These results are discussed in terms of the crystal structure of the Dictyosteliummyosin motor domain.

Three loop structures called the P-loop, switch I loop and switch II loop of myosin are major components of its ATPase site, and share structural and functional homology with the loop structures in other ATPases and GTPases such as kinesin and G-protein. Using the alanine scanning mutagenesis, structure-function relationship of the switch I and switch II loops in Dictyostelium myosin II was examined. Based on crystal structures of Dictyostelium myosin motor domain, functions of each residue in those loops are discussed.

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by ≈20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M⋅ADP⋅P i ) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP → M⋅ADP⋅P i . This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.

Nucleotide and actin binding properties of the truncated myosin head (S1dC) from Dictyostelium myosin II were studied in solution using rabbit skeletal myosin subfragment 1 as a reference material. S1dC and subfragment 1 had similar affinities for ADP analogues, ɛADP and TNP-ADP. The complexes of ɛADP and BeFx or AlF4 - were less stable with S1dC than with subfragment 1. Stern-Volmer constants for acrylamide quenching of S1dC complexes with ɛADP, ɛADP·AlF 4 - and ɛADP.BeFx were 2.6, 2.9 and 2.2 M-1, respectively. The corresponding values for subfragment 1 were 2.6, 1.5 and 1.1 M-1. The environment of the nucleotide binding site was probed by using a hydrophobic fluorescent probe, PPBA. PPBA was a competitive inhibitor of S1dC Ca2+-ATPase (Ki = 1.6 μm). The binding of nucleotides to subfragment 1 enhanced PPBA fluorescence and caused blue shifts in the wavelength of its maximum emission in the order: ATP ≈ ADP·AlF4- ≈ ADP·BeFx > ATPSγS > ADP > PPi. In the case of S1dC, the effects of different nucleotides were smaller and indistinguishable from each other. S1dC bound actin tighter than S1 (Kd = 7 nm and 60 nm, respectively). The actin activated MgATPase activity of S1dC varied between preparations, and the Vmax and Km values ranged between 3 and 7 s-1 and 60 and 190 μm, respectively. S1dC showed lower structural stability than S1 as revealed by their thermal inactivations at 35° C. These results show that the nucleotide and actin binding of S1dC and subfragment 1 are similar but there are some differences in nucleotide and phosphate analogue-induced changes and the communication between the nucleotide and actin binding sites in these proteins

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-MgADP complex belong to space group P2(1)2(1)2 with unit cell parameters of a = 105.3 A, b = 182.6 A, and c = 54.7 A, whereas the crystals of the aluminum fluoride complex belong to the orthorhombic space group C222(1) with unit cell dimensions of a = 87.9 A, b = 149.0 A, and c = 153.8 A. Chemical modification was not necessary to obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADP.BeFx is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADP.AlF4- exhibits significant domain movements. The structures suggest that the MgADP.BeFx complex mimics the ATP bound state and the MgADP.AlF4- complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADP.AlF4- complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain.

The ATPase site of myosin was located by three-dimensional electron microscopy using the avidin-biotin system. The site is about 5 nm from the tip of the myosin head, about 4 nm apart from the actin-binding site of myosin. Other functional sites on the myosin head were located by electron microscopy with the avidin-biotin system, monoclonal antibodies and site-directed antibodies. These findings enable us to estimate the domain structure of the head. The shape of the myosin heads was examined by electron microscopy using rotary-shadowing and uni-directional shadowing technique, and two types were seen: a straight one and a bent one. Bending occurs at 12 +/- 2 nm from the head-rod junction. This location corresponds with the images by three-dimensional electron microscopy and electron micrography of the crystal. The bending region locates at the boundary of domains. Bent heads increase in the presence of ADP-Vi. The location and angle of bending were almost the same under all the conditions examined. These findings suggest that the heads of straight and bent shapes are in equilibrium, and that ADP-Vi shifts the equilibrium to the bent shape. The bending of the myosin head may play an important role in the molecular mechanism of muscle contraction.

All class 2 myosins contain an N-terminal extension of approximately 80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo. Actin affinity was more than 80-fold, and the rate of ADP release approximately 40-fold decreased in this mutant. In contrast, a myosin construct that lacks only the SH3-like subdomain, corresponding to residues 33-79, displayed much smaller functional defects. In complementation experiments with myosin-2 null cells, this construct rescued myosin-2-dependent processes such as cytokinesis, fruiting body formation, and sporogenesis. An 8-fold reduction in motile activity and changes of similar extent in the affinity for ADP and filamentous actin indicate the importance of the SH3-like subdomain for correct communication between the functional regions within the myosin motor domain and suggest that local perturbations in this region can play a role in modulating myosin-2 motor activity.

Starved Dictyostelium amoebae continuously change their shape and they are elongated along the front-rear axis during locomotion. In contrast, we found that disruption of the amiB gene, which had been identified as a gene required for the aggregation process during development, caused these cells to move in a manner similar to fish keratocytes. Starved amiB- cells were elongated laterally and had one large lamellipodium along the front side arc of the cell. These cells moved unidirectionally for long distances maintaining the half-moon shape, and this movement followed the predictions of the graded radial extension model, which was originally developed to describe the keratocyte movements. Furthermore, the distributions of actin, Arp2, and myosin II in amiB- cells were similar to those in keratocytes. Therefore, locomotion by keratocytes and amiB- cells appears to be driven by similar mechanisms of cytoskeletal regulation. Double knockout cells lacking both AmiB and myosin II were still able to move unidirectionally in a keratocyte-like manner, although the frequency of those movements was lower. Thus, myosin II is dispensable for the unidirectional movement, though it likely functions in the maintenance of the characteristic half-moon shape. This mutant cell can be a useful tool for further molecular genetic analysis of the mechanism of cell locomotion.

Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis.

One of the putative actin-binding sites of Dictyostelium myosin II is the beta-strand-turn-beta-strand structure (Ile(398)-Leu-Ala-Gly-Arg-Asp(403)-Leu-Val(405)), the "myopathy loop, " which is located at the distal end of the upper 50-kDa subdomain and next to the conserved arginine (Arg(397)), whose mutation in human cardiac myosin results in familial hypertrophic cardiomyopathy. The myopathy loop contains the TEDS residue (Asp(403)), which is a target of the heavy-chain kinase in myosin I. Moreover, the loop contains a cluster of hydrophobic residues (Ile(398), Leu(399), Leu(404), and Val(405)), whose side chains are fully exposed to the solvent. In our study, the myopathy loop was deleted from Dictyostelium myosin II to investigate its functional roles. The mutation abolished hydrophobic interactions of actin and myosin in the strong binding state during the ATPase cycle. Association of the mutant myosin and actin was maintained only through ionic interactions under these conditions. Without strong hydrophobic interactions, the mutant myosin still exhibited motor functions, although at low levels. It is likely that the observed defects resulted mainly from a loss of the cluster of hydrophobic residues, since replacement of Asp(403) or Arg(402) with alanine generated a mutant with less severe or no defects compared with those of the deletion mutant.

We have identified and sequenced a novel unconventional myosin termed MyoK in Dictyostelium. Like class XIV myosins, MyoK has a very short and basic tail and lacks light chain-binding IQ motifs. In contrast, a phylogenetic analysis of the motor domain (head) clearly indicated that MyoK belongs to class I myosins. Surprisingly, at the loop-1 site of the head, an insert of 142 amino acid residues was found, the longest in all myosins so far sequenced. The insert was rich in Gly and Pro and could serve as a secondary actin-binding site, as is the case with those present in the tail of some class I myosins. The expression of the MyoK transcript was stimulated at very early stages of Dictyostelium development. The growth and terminal developmental phenotype of the Dictyostelium cell were not affected by the myoK − mutation, suggesting the existence of myosin(s) with functions overlapping those of MyoK.

In muscle, the myosin head (‘crossbridge’) performs the ‘working stroke’, in which ATP is hydrolysed to generate the sliding of actin and myosin filaments. The myosin head consists of a globular motor domain and a long lever-arm domain. The ‘lever-arm hypothesis’1,2,3,4,5 predicts that during the working stroke, the lever-arm domain tilts against the motor domain, which is bound to actin in a fixed orientation. To detect this working stroke in operation, we constructed fusion proteins by connecting Aequorea victoria green fluorescent protein and blue fluorescent protein6,7,8 to the amino and carboxyl termini of the motor domain of myosin II of Dictyostelium discoideum, a soil amoeba, and measured the fluorescence resonance energy transfer between the two fluorescent proteins. We show here that the carboxy-terminal fluorophore swings at the isomerization step of the ATP hydrolysis cycle, and then swings back at the subsequent step in which inorganic phosphate is released, thereby mimicking the swing of the lever arm. The swing at the phosphate-release step may correspond to the working stroke, and the swing at the isomerization step to the recovery stroke.

Familial hypertrophic cardiomyopathy(FHC) is caused by missence mutations in β-myosin heavy chain or other various sarcomeric proteins. To elucidate the functional impact of FHC mutations in myosin heavy chain, we generated Dictyostelium discoideum myosin II mutants equivalent to human FHC mutations by site-directed mutagenesis, and characterized their molecular-basis motor function. The current mutants, i.e. R397Q, F506C, G575R, A699R, K703Q and K703W are equivalent to R403Q, F513C, G584R, G716R, R719Q and R719W FHC mutants respectively. We measured the molecular-basis force and the sliding velocity generated by these myosin mutants. The measurement revealed that the A699R, K703Q and K703W myosins exhibited the lowest level of force with their preserved actin-activated MgATPase activity. F506C mutant showed the least impairment of the motile and enzymatic activities. The motor function of R397Q and G575R myosins were classified as intermediate. These results suggest that ELC binding domain might be important for force production.

In order to determine the functional roles of the conserved sequence (NXNSSRFG) of the "switch I" loop (residues 233-240 in Dictyostelium myosin II), alanine scanning mutagenesis was performed on Dictyostelium myosin II. N233A and S237A mutant myosins did not bind a fluorescent analog of ADP, mant-deoxyADP, at the low concentration range (micromolar and had low level of ATPase activities. They were nonmotile when examined by the in vitro motility assay. Dictyostelium cells expressing these myosins showed worse phenotypes than that of myosin-null cells. In contrast to these mutant myosins, R238A myosin tightly bound mant-deoxyADP. However, the mutant had a defect in the ATP hydrolysis step and exhibited the lowest ATPase activities among the mutants examined here. The R238A myosin was nonmotile. R238C or R238H mutations, which mimic the Usher syndrome mutations, generated myosins with similar functional defects to those of the R238A mutation. Cells expressing the R238A myosin exhibited the phenotype similar to that of the myosin-null cells. N235A, S236A, F239A, and G240A myosins retained moderate levels of ATPase activities and could drive sliding of actin filaments at various speeds. Phenotypes of cells expressing them were very similar to that of the wild-type cells. Taken together, these results suggest that side chains of N233 and S237 may play essential roles in holding a nucleotide in the ATPase pocket and that R238 may play crucial roles in the ATP hydrolysis step, while those of the other residues in the switch I loop are not essential for the process.

The cysteine residue called SH2 in the skeletal myosin heavy chain is conserved among various species. Cys 678 in Dictyostelium myosin II is equivalent to SH2 in skeletal myosin. Using the Dictyostelium myosin II heavy chain gene, SH2 was mutated to Gly, Ala, Ser, or Thr. These mutant myosins were expressed in Dictyostelium myosin-null cells. To investigate how these mutations affect the motor functions of myosin, we examined the phenotypes of the transformed cells. We also purified the mutant myosins, and characterized them by measuring the actin-activated MgATPase activity, sliding velocity of actin filaments and force level. All of these mutant myosins complemented the myosin-specific defects of the myosin-null cells. Consistent with these observed phenotypes, all of the purified mutant myosins retained similar actin-activated MgATPase activities and force levels to those of the wild-type myosin (WT). However, the sliding velocities of actin filaments were significantly different (WT > or = Ser > Ala >> Thr > Gly). In particular, the Gly and Thr mutants exhibited a striking decrease in velocity, while the Ser mutant exhibited velocity comparable to that of the wild-type myosin. Thus, mutations of SH2 resulted in uncoupling of ATP hydrolysis and the sliding.

Recent studies have revealed that familial hypertrophic cardiomyopathy (FHC) is caused by missence mutations in myosin heavy chain or other sarcomeric proteins. To investigate the functional impact of FHC mutations in myosin heavy chain, mutants of Dictyostelium discoideum myosin II equivalent to human FHC mutations were generated by site-directed mutagenesis, and their motor function was characterized at the molecular level. These mutants, i.e., R397Q, F506C, G575R, A699R, K703Q, and K703W are respectively equivalent to R403Q, F513C, G584R, G716R, R719Q, and R719W FHC mutants. We measured the force generated by these myosin mutants as well as the sliding velocity and the actin-activated ATPase activity. These measurements showed that the A699R, K703Q, and K703W myosins exhibited unexpectedly weak affinity with actin and the lowest level of force, though their ATPase activity remained rather high. F506C mutant which has been reported to have benign prognosis exhibited the least impairment of the motile and enzymatic activities. The motor functions of R397Q and G575R myosins were classified as intermediate. These results suggest that the force level of mutant myosin molecule may be one of the key factors for pathogenesis which affect the prognosis of human FHC.

To identify tropomyosin-binding site(s) on the surface of actin molecule, we examined the effect of mutagenesis introduced to subdomain 4 of actin. Because the sequence of Gln228-Ser232 of Dictyostelium actin differs from that of Tetrahymena actin that does not bind tropomyosin, the Dictyostelium/Tetrahymena chimeric actin was produced. Also, Lys238 and Glu241 were replaced with alanine (mutant 645) to study the role of charged residues which are located at both ends of a beta-sheet. As a control experiment, a negative charge was introduced near to the N-terminus (mutant 663). To facilitate the separation of mutant actins without affecting the normal function, Glu360 was replaced with histidine. As a control mutant to such mutants, the mutant 647 (E360H) was produced. Mutant actins were expressed in Dictyostelium cells. All mutant actins were functional: they (i) polymerize and (ii) activate ATPase activity of rabbit skeletal myosin subfragment-1 (S1). The mutant 663 (G2E) showed tropomyosin binding and activated myosin ATPase almost as well as rabbit skeletal actin. However, the tropomyosin binding of the mutant 645 (K238A/E241A/E360H) became magnesium dependent. The chimeric actin (mutant 646: QTAAS-to-KAYKE replacement and E360H) showed decreased tropomyosin binding even in the presence of magnesium ions. These results indicate that the tropomyosin-binding sites of "on"-state actin are on subdomain 4. Surprisingly, the chimeric actin showed more cooperative calcium regulation than rabbit skeletal actin in the presence of tropomyosin-troponin. The mutant actin 645 can hardly activate S1 ATPase irrespective of calcium concentration in the presence of tropomyosin-troponin, even though this actin by itself can activate S1 ATPase. The steric blocking or cooperative/allosteric mechanism of thin filament regulation is discussed.

To understand the underlying mechanism of force generation by myosin motor, it is crucial to know which part of the molecule is essential for the process. Recent structure determination of myosin motor domain at atomic resolution has revealed that the domain comprises two smaller domains, the "ATPase domain" consisting of only an N-terminal segment of the heavy chain and the "neck domain" consisting of a long α-helix of the heavy chain and two light chains. This atomic structure begs the question of whether both domains are required for force generation. To answer it, we genetically truncated the head to generate a recombinant fragment composed of the "ATPase domain" alone. The truncated head drove sliding movement of actin filaments and generated force in a novel in vitro assay system, which allows us to hold a specific site of the head on a glass surface. These results indicate that the compact ATPase domain functions as a force-generating machinery of the myosin motor.

Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/D363/E364 are not essential for ATP-driven sliding and force generation.

We constructed several mutant actin genes from the Dictyostelium actin 15 gene by the site-directed mutagenesis. Mutations were designed to change acidic residues in actin subdomain 1 to histidine residues. Amino acid replacements were: D1H (single replacement of Aspl to His), D4H, D1H/D4H (double replacements of Asp1 and Asp4 to histidine), D1H/E3H/D4H (triple replacements of Aspl, Glu3 and Asp4 to histidine), D24H/D25H, E99H/E100H, E360H/E361H, and D363H/E364H. Mutant genes were then expressed in Dictyostelium cells. In vitro motility assays were carried out for purified actins to see whether the mutations affect sliding motion of actin filaments driven by HMM. The assays showed that replacement of N-terminal acidic residues inhibited the sliding. Replacement of D24/D25 and E99/E100 also resulted in inhibition of the sliding motion. However, replacement of acidic residues at the C-terminal cluster E360/E361/D363/E363 did not resulted in loss of motility.

Three functionally important sites of myosin head, i.e. ATPase site, actin-binding site and SH1 thiol, were identified in its three-dimensional structure by electron microscopy. These sites were also mapped along primary structures of myosin heavy and light chains by chemical methods such as affinity labeling, chemical crosslinking and "end-label fingerprinting".

Subfragment 1 (S1) prepared from rabbit skeletal muscle myosin was digested with trypsin to cleave the 95K heavy chain into three pieces, i.e., the 23K, 50K, and 20K fragments. The trypsin-treated S1 was then cross-linked with p-nitrophenyl iodoacetate. The cross-linker bridged one of the reactive thiols (SH2) in the 20K fragment and a lysine residue in the 23K fragment [Hiratsuka, T. (1987) Biochemistry 26, 3168-3173]. Location of the lysine residue was mapped along the 23K fragment by "end-label fingerprinting", which employed site-directed antibodies against the N-terminus of the 23K fragment and against the C-terminus of the 24K fragment (the 23K fragment plus nine extra residues at its C-terminus). The mapping revealed that Lys-184 or Lys-189 was the residue cross-linked with SH2. Since the cross-linker used here spans only several angstroms, the result indicates that Lys-184 or Lys-189 is very close to SH2 in the three-dimensional structure of myosin head. Examination of the primary structure of the 23K fragment has revealed that these lysine residues are in and very close to the so-called "glycine-rich loop", whose sequence is highly homologous to those of nucleotide-binding sites of various nucleotide-binding proteins.

To elucidate the interaction of calmodulin with calmodulin binding proteins, we studied the location of the interaction sites on calmodulin by using a chemical cross-linking reagent. Calmodulin prepared from wheat germ was cross-linked to myosin light chain kinase and troponin-I with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked products were cleaved partially with cyanogen bromide and cross-linked sites were determined by peptide mapping analysis using SDS-urea polyacrylamide gel electrophoresis. Peptides which contain the cross-linked site were displaced from their position because of the attached fragments of myosin light chain kinase or troponin I. The peptide of calmodulin from the N-terminal to Met-73 in the cross-linked product with myosin light chain kinase had the same mobility as that of uncross-linked calmodulin on the map though the amount of the peptide was decreased in the cross-linked product. The peptide from the N-terminal to Met-110 in the cross-linked product was displaced from its position. Similar change in the mobility of the calmodulin peptides was also observed in the cross-linked products with troponin I. It was concluded, therefore, that at least one cross-linked site for myosin light chain kinase and one for troponin I were located between Met-73 and Met-110 of the wheat germ calmodulin.

When the rigor complex of actin and myosin subfragment 1 (S1) was treated with a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, covalently linked complexes of actin and S1 heavy chain with apparent molecular weights of 165,000 and 175,000 were generated. Measurements of the molar ratio of actin to S1 heavy chain in the 165K and 175K products showed that they were 1:1 complexes of actin and S1 heavy chain. Chemical cleavages of the cross-linked products followed by peptide mappings revealed that two distinct segments of S1 heavy chain spanning the 18K-20K region and the 27K-35K region from its C terminus participated in cross-linking with actin. Cross-linking of actin to the former site generated the 165K peptide while the latter site was responsible for generating the 175K peptide.

A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.

We determined the spatial location of the N terminus of the heavy chain of rabbit skeletal muscle myosin by electron microscopy, using a site-directed antibody raised against its N-terminal eight residues as an electron microscopic probe. By examining rotary-shadowed images of the heavy meromyosin-antibody complex, we measured distances between the head-rod junction and the attachment site of the antibody bound on the head. The average distance was estimated to be about 12 nm. The result indicates that the N terminus of the heavy chain is located at the middle region of the head.

Both ATP hydrolysis by myosin and the accompanying cyclic association-dissociation of actin and myosin are essential for muscle contraction. It is important for understanding the molecular mechanism of contraction to know the three-dimensional locations of the two major functional sites of myosin: the ATPase site and the actin-binding site. We have determined the position of the ATPase site of myosin using three-dimensional image reconstruction from electron micrographs and site-specific labelling with the avidin-biotin system. The ATPase site is about 5 nm from the tip of the myosin head and is about 4 nm away from the actin-binding site of myosin. This is the first report of the three-dimensional location of an enzyme active site by electron microscopy.

One of the reactive thiols in the myosin head, SH 1 , was covalently labeled with a biotin derivative, N -iodoacetyl- N′ -biotinylhexylenediamine. When 50% of the SH 1 thiol was modified with the biotin reagent as judged from measurements of ATPase activities, the biotinylated myosin bound one mole of avidin per mole of myosin at the saturating level. The avidin-myosin complex was readily formed in the presence of MgADP or MgATP. Peptide maps of the biotinylated myosin revealed that SH 1 is actually the site of biotinylation with N -iodoacetyl- N′ -biotinylhexylenediamine. Electron microscopic examination of the avidin-myosin complex showed that the attachment site of avidin on the myosin head is 130 A from the head-rod junction, indicating that the SH 1 thiol is located there.

We have cloned a novel unconventional myosin gene myoM in Dictyostelium. Phylogenetic analysis of the motor domain indicated that MyoM does not belong to any known subclass of the myosin superfamily. Following the motor domain, two calmodulin-binding IQ motifs, a putative coiled-coil region, and a Pro, Ser and Thr-rich domain, lies a combination of dbl homology and pleckstrin homology domains. These are conserved in Rho GDP/GTP exchange factors (RhoGEFs). We have identified for the first time the RhoGEF domain in the myosin sequences. The growth and terminal developmental phenotype of Dictyostelium cells were not affected by the myoM− mutation. Green fluorescent protein-tagged MyoM, however, accumulated at crown-shaped projections and membranes of phase lucent vesicles in growing cells, suggesting its possible roles in macropinocytosis.

To examine the spatial relationship between SH1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1-avidin complex bound F-actin and its Mg2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH1 is separated from the actin binding site on subfragment-1 surface by at least 17–20 Å.

Site-directed antibodies were raised against three synthetic peptides whose sequences correspond to a region around the reactive lysine residue and two protease-sensitive regions of subfragment 1 (S1) of skeletal muscle myosin (one at the junction of the 23,000 Mr and 50,000 Mr segments, the J1 junction; and the other at the junction of the 50,000 Mr and 20,000 Mr segments of the heavy chain, the J2 junction). The antisera cross-reacted with intact myosin with titres of 5 × 104 (anti-J1 antiserum) and 104 (anti-J2 and anti-reactive lysine residue antisera). Site-specific antibodies purified by S1-Sepharose readily bound to myosin. Electron microscopic examinations of antibody-myosin complexes revealed that the J1 and J2 junctions are located 15 nm and 16 nm from the head-rod junction, respectively, while the reactive lysine residue region is 13 nm from the junction.

The ATPase site of rabbit skeletal myosin was covalently labeled by an ADP analogue that carried a biotin moiety on its adenine ring and a photoreactive phenyl azide group on its ribose ring [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 212-216]. The ADP analogue was tightly trapped into the ATPase site in the presence of vanadate ions and then covalently cross-linked to the site by UV irradiation. The N-terminal 23,000-dalton tryptic fragment of the heavy chain was selectively labeled with the analogue. Further mapping of the labeled segment along the 23-kDa fragment was carried out by "end-label fingerprinting" which employed site-directed antibodies against both ends of the N-terminal heavy chain fragment. The mapping revealed that a hydrophobic segment of approximately 10 residues next to Trp-130, which was reported to be in proximity to the adenine ring of ADP bound to the ATPase site [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579], was the site of covalent labeling with the ADP analogue. The result indicates that the hydrophobic segment is close to the ribose ring of ADP bound to the ATPase site.