No abstract

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.Myosins are motor proteins that translocate actin filaments upon hydrolysis of ATP, and thus they play a critical role in diverse forms of cell contractility and motility. During the last decade a number of myosin-like proteins have been found, and the myosins are currently organized into 15 classes based upon phylogenetic sequence comparisons of the motor domains (1-5). Class V myosin was originally identified in brain as a calmodulin-binding protein that had actin-dependent ATPase activity (6). Myosin V is a member of the myosin superfamily that is expressed in variety of cell types and is involved in a variety of membrane trafficking and organelle transport functions (1-5). Myosin V has two heads that are connected with a long coiledcoil domain; however, in contrast to conventional myosin, it contains a globular C-terminal domain and does not form thick filaments (7). The head domain is composed of a globular motor domain and an elongated neck domain that is associated with a number of light chains. The sequence at the neck region contains six IQ motifs that have been implicated as calmodulin or myosin light chain binding consensus motifs as found in a variety of calmodulin-binding proteins and myosins (7). Since light chains play a critical role in the regulation of various conventional myosins, it has been proposed that the IQ domain serves as a regulatory component of myosin V. The role of the IQ motif and bound calmodulin serving as a regulatory component of unconventional myosins is best studied for mammalian myosin Is. For both brush border myosin I (8 -10) and myosin I␤ (11-13), high Ca 2ϩ inhibits motor activity due to Ca 2ϩ binding to the calmodulin light chain. Since 1 mol of bound calmodulin dissociates from myosin I at high Ca 2ϩ , it was originally thought that this dissociation of calmodulin was responsible for the inhibition of myosin I...

Class-V myosin proceeds along actin filaments with large ( approximately 36 nm) steps. Myosin-V has two heads, each of which consists of a motor domain and a long (23 nm) neck domain. In accordance with the widely accepted lever-arm model, it was suggested that myosin-V steps to successive (36 nm) target zones along the actin helical repeat by tilting its long neck (lever-arm). To test this hypothesis, we measured the mechanical properties of single molecules of myosin-V truncation mutants with neck domains only one-sixth of the native length. Our results show that the processivity and step distance along actin are both similar to those of full-length myosin-V. Thus, the long neck domain is not essential for either the large steps or processivity of myosin-V. These results challenge the lever-arm model. We propose that the motor domain and/or the actomyosin interface enable myosin-V to produce large processive steps during translocation along actin.

Myosin is an actin-based molecular motor that constitutes a diverse superfamily. In contrast to conventional myosin, which binds to actin for only a short time during cross-bridge cycling, recent studies have demonstrated that class V myosin moves along actin filaments for a long distance without dissociating. This would make it suitable for supporting cargo movement in cells. Because myosin V has a two-headed structure with an expanded neck domain, it has been postulated to 'walk' along the 36-nm helical repeat of the actin filament, with one head attached to the actin and leading the other head to the neighbouring helical pitch. Here, we report that myosin IXb, a single-headed myosin, moves processively on actin filaments. Furthermore, we found that myosin IXb is a minus-end-directed motor. In addition to class VI myosin, this is the first myosin superfamily member identified that moves in the reverse direction. The processive movement of the single-headed myosin IXb cannot be explained by a 'hand-over-hand' mechanism. This suggests that an alternative mechanism must be operating for the processive movement of single-headed myosin IXb.

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with Factin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.Myosin is a molecular motor that interacts with actin filaments and converts chemical energy of ATP to mechanical work. The molecular structure of conventional myosin is characterized by its globular head domain and the filament forming ␣-helical coiled-coil tail domain. Although the former characteristic is shared by a number of unconventional myosins, only conventional myosin, classified as the second class of myosin in myosin super family (1-6), carries the latter one. The amino acid sequence of the tail shows the seven-residue repeat, characteristic of coiled-coil proteins (7), and in addition to this, the myosin tail shows a 28-residue repeat in which charged residues face toward the outside of the coiled-coil structure (8 -11). Because filament formation of myosin is abolished at high ionic strength, these charged residues in the tail have been thought to be responsible for the assembly of myosin molecules. Use of proteolytic fragments (12, 13) as well as recombinant expressed fragments (14 -16) has revealed that the C-terminal region of LMM 1 is important for the assembly of the rod. For skeletal myosin, deletion of the N-terminal residues of LMM did not affect the solubility at low ionic strength, but deletion of 92 residues from the C terminus caused an increase in solubility (16). Hodge et al. (17) reported that, by expressing the rod portion of nonmuscle myosin II by Escherichia coli expression system, the 35-residue C-terminal nonhelical region influences filament formation. Recently it was reported that the deletion of 29 residues near the C terminus of skeletal myosin heavy chain disrupts the filament formation (18). On the other hand, for amoeba myosin, the C-ter...