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Characterization of the Motor Activity of Mammalian Myosin VIIA

2003; Elsevier BV; Volume: 278; Issue: 7 Linguagem: Inglês

10.1074/jbc.m210489200

1083-351X

Akira Inoue, Mitsuo Ikebe,

Neurogenetic and Muscular Disorders Research

Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg2+-ATPase activity that was markedly activated by actin with K actin of 33 μm andV max of 0.53 s−1head−1. Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K actin of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor. Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg2+-ATPase activity that was markedly activated by actin with K actin of 33 μm andV max of 0.53 s−1head−1. Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K actin of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor. Myosins are mechanochemical proteins with a motor domain containing an ATP and actin-binding region, a neck domain that interacts with light chains or calmodulin, and a tail domain that serves to anchor and position the motor domain so that it can properly interact with actin filaments at a specific intracellular location. Phylogenetic analysis revealed that myosin consists of at least 18 classes (1Hodge T. Cope M.J. J. Cell Sci. 2000; 113: 3353-3354Crossref PubMed Google Scholar, 2Yamashita R.A. Sellers J.R. Anderson J.B. J. Muscle Res. Cell Motil. 2000; 21: 491-505Crossref PubMed Scopus (47) Google Scholar). Myosin VIIs are found in human (3Hasson T. Skowron J.F. Gilbert D.J. Avraham K.B. Perry W.L. Bement W.M. Anderson B.L. Sherr E.H. Chen Z.Y. Greene L.A. Ward D.C. Corey D.P. Mooseker M.S. Copeland N.G. Jenkins N.A. Genomics. 1996; 36: 431-439Crossref PubMed Scopus (71) Google Scholar, 4Weil D. Levy G. Sahly I. Levi-Acobas F. Blanchard S. El-Amraoui A. Crozet F. Philippe H. Abitbol M. Petit C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3232-3237Crossref PubMed Scopus (150) Google Scholar), mouse (5Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (544) Google Scholar), porcine (6Bement W.M. Hasson T. Wirth J.A. Cheney R.E. Mooseker M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6549-6553Crossref PubMed Scopus (108) Google Scholar), zebrafish (7Ernest S. Rauch G.J. Haffter P. Geisler R. Petit C. Nicolson T. Hum. Mol. Genet. 2000; 9: 2189-2196Crossref PubMed Scopus (169) Google Scholar), bullfrog (8Solc C.K. Derfler B.H. Duyk G.M. Corey D.P. Auditory Neurosci. 1994; 1: 63-75Google Scholar), Caenorhabditis elegans (9Baker J.P. Titus M.A. J. Mol. Biol. 1997; 272: 523-535Crossref PubMed Scopus (47) Google Scholar), Dictyostelium discoidium (10Titus M.A. Curr. Biol. 1999; 9: 1297-1303Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), andDrosophila (11Chen T.L. Edwards K.A. Lin R.C. Coats L.W. Kiehart D.P. J. Cell Biol. 1991; 115 (abstr.): 330Google Scholar). In vertebrates, two types of genes for type VII myosin, myosin VIIA and VIIB, are identified. Among them, the entire coding region has been determined for myosin VIIA. In mammals, myosin VIIA is expressed in a variety of organs and tissues including eye, inner ear, olfactory epithelium, brain, choroid plexus, intestine, liver, kidney, adrenal gland, testis, and lymphocytes (12Chen Z.Y. Hasson T. Kelley P.M. Schwender B.J. Schwartz M.F. Ramakrishnan M. Kimberling W.J. Mooseker M.S. Corey D.P. Genomics. 1996; 36: 440-448Crossref PubMed Scopus (117) Google Scholar, 13Sahly I. El-Amraoui A. Abitbol M. Petit C. Dufier J.L. Anat. Embryol. (Berl.). 1997; 196: 159-170Crossref PubMed Scopus (86) Google Scholar, 14Wolfrum U. Liu X. Schmitt A. Udovichenko I.P. Williams D.S. Cell Motil. Cytoskeleton. 1998; 40: 261-271Crossref PubMed Scopus (111) Google Scholar). In the retina, myosin VIIA is found in the pigmented epithelial cells and is postulated to play a role in phagocytosis of cell debris that accumulates as a result of sloughing off of photoreceptor outer segments. Of interest is that defects of myosin VIIA cause the mouseshaker-1 phenotype and human Usher syndrome 1B, which are characterized by deafness, lack of vestibular function, and (in humans) progressive retinal degeneration (5Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (544) Google Scholar, 15Weil D. Blanchard S. Kaplan J. Guilford P. Gibson F. Walsh J. Mburu P. Varela A. Levilliers J. Weston M.D. Kelley P.M. Kimberlig W.J. Wagenaar M. Levi-Acobas F. Larget-Piet D. Munnich A. Steel K.P. Brown S.D.M. Petit C. Nature. 1995; 374: 60-61Crossref PubMed Scopus (866) Google Scholar). In humans, two forms of dominant and recessive nonsyndromic deafness, DFNB2 and DFNA11, are also caused by myosin VIIA mutations (16Liu X.Z. Walsh J. Mburu P. Kendrick-Jones J. Cope M.J. Steel K.P. Brown S.D. Nat. Genet. 1997; 16: 188-190Crossref PubMed Scopus (376) Google Scholar, 17Weil D. Kussel P. Blanchard S. Levy G. Levi-Acobas F. Drira M. Ayadi H. Petit C. Nat. Genet. 1997; 16: 191-193Crossref PubMed Scopus (336) Google Scholar, 18Liu X.Z. Walsh J. Tamagawa Y. Kitamura K. Nishizawa M. Steel K.P. Brown S.D. Nat. Genet. 1997; 17: 268-269Crossref PubMed Scopus (270) Google Scholar).Amino acid sequence analysis of myosin VIIA has indicated that this myosin has a motor domain containing actin-binding and ATP-binding sites, and five IQ motifs at the neck domain. In the tail domain, a very short predicted coiled-coil region was found; therefore, it has been assumed that myosin VIIA forms a two-headed structure. Two "band 4.1 protein, ezrin, radixin, moesin" (FERM) 1The abbreviations used are: FERM, band 4.1 protein, ezrin, radixin, moesin; M7full, full-length myosin VIIA; DTT, dithiothreitol; NTA, nitrilotriacetic acid 1The abbreviations used are: FERM, band 4.1 protein, ezrin, radixin, moesin; M7full, full-length myosin VIIA; DTT, dithiothreitol; NTA, nitrilotriacetic acid domains that have been implicated in cytoskeletal protein interactions have been found in the tail region. There are two myosin tail homology 4 domains found in the tail region. Although similar domains are found in myosin IV and XV, the function of this domain is unclear. The tail domain of unconventional myosins has been implicated to serve as the anchoring site for the cellular target proteins. For myosin VIIa, several tail binding proteins have been identified. The type I regulatory subunit of protein kinase A binds to the FERM domain at the C terminus of myosin VIIA (19Kussel-Andermann P. El-Amraoui A. Safieddine S. Hardelin J.P. Nouaille S. Camonis J. Petit C. J. Biol. Chem. 2000; 275: 29654-29659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), although the role of protein kinase A in the function and regulation of myosin VIIA is unknown. It was shown that the FERM domain is involved in anchoring with adherens junction via a cadherin-catenins complex (20Kussel-Andermann P. El-Amraoui A. Safieddine S. Nouaille S. Perfettini I. Lecuit M. Cossart P. Wolfrum U. Petit C. EMBO J. 2000; 19: 6020-6029Crossref PubMed Scopus (183) Google Scholar). Todorov et al. reported (21Todorov P.T. Hardisty R.E. Brown S.D. Biochem. J. 2001; 354: 267-274Crossref PubMed Scopus (24) Google Scholar) that the myosin VIIA tail has an affinity for microtubule-associated protein 2B, suggesting the interaction of myosin VIIA with the microtubule-based motility system. Quite recently, El-Amraoui et al. (22El-Amraoui A. Schonn J.S. Kussel-Andermann P. Blanchard S. Desnos C. Henry J.P. Wolfrum U. Darchen F. Petit C. EMBO Rep. 2002; 3: 463-470Crossref PubMed Scopus (150) Google Scholar) reported that myosin VIIa binds to myosin VIIa- and Rab27-interacting protein, associates with melanosomes via Rab27A, and plays a role in melanosome trafficking. On the other hand, little is known about the motor function of myosin VIIa at a molecular level.Unlike the case for the well characterized conventional myosins, it becomes evident that the motor function among various members of the unconventional myosin subfamily varies uniquely from one to another, which is thought to be critical for specific physiological roles in diverse cellular motile processes. For example, recent studies have revealed that myosin V is a processive motor that can move in large steps approximating the 36-nm pseudorepeat of the actin filament (23Walker M.L. Burgess S.A. Sellers J.R. Wang F. Hammer III, J.A. Trinick J. Knight P.J. Nature. 2000; 405: 804-807Crossref PubMed Scopus (282) Google Scholar, 24Sakamoto T. Amitani I. Yokota E. Ando T. Biochem. Biophys. Res. Commun. 2000; 272: 586-590Crossref PubMed Scopus (172) Google Scholar, 25Mehta A.D. Rock R.S. Rief M. Spudich J.A. Mooseker M.S. Cheney R.E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (670) Google Scholar, 26Tanaka H. Homma K. Iwane A.H. Katayama E. Ikebe R. Saito J. Yanagida T. Ikebe M. Nature. 2002; 415: 192-195Crossref PubMed Scopus (114) Google Scholar). These characteristics are quite important to understand the physiological function of myosin V, since the processive nature of myosin V with large step size is suitable for the motors involved in cargo movement in cells.Recently, myosin VI was also demonstrated to be a processive motor with a large step size (27Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (319) Google Scholar, 28Nishikawa S. Homma K. Komori Y. Iwaki M. Wazawa T. Iwane A.H. Saito J. Ikebe R. Katayama E. Yanagida T. Ikebe M. Biochem. Biophys. Res. Commun. 2002; 290: 311-317Crossref PubMed Scopus (149) Google Scholar). A unique feature of myosin VI is that it moves backward on the actin filament (29Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Crossref PubMed Scopus (548) Google Scholar). It was originally proposed that the myosin VI unique large insertion between the neck and converter domains is responsible for the reverse directionality of myosin VI (29Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Crossref PubMed Scopus (548) Google Scholar). This view was based upon the hypothesis that the orientation of the motor domain of myosin VI against actin filament is the same direction as other myosins but the attachment of the position of the "lever arm" (a long α-helical region following the motor domain) (30Block S.M. Cell. 1996; 87: 151-157Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) is different so that the same movement of the catalytic core would rotate the lever arm in the opposite direction on actin. Since the insertion is located between the converter domain (a compact subdomain thought to amplify the conformational change of the motor domain) (31Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 32Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar) and light chain binding helix, it was hypothesized that the 53-amino acid unique insertion of myosin VI is critical in determining the reverse directionality of myosin VI. However, a recent study revealed that the myosin VI unique insertion is not important to the reverse in directionality (33Homma K. Yoshimura M. Saito J. Ikebe R. Ikebe M. Nature. 2001; 412: 831-834Crossref PubMed Scopus (62) Google Scholar). Instead the motor core domain is responsible for the change in the directionality of myosin movement (33Homma K. Yoshimura M. Saito J. Ikebe R. Ikebe M. Nature. 2001; 412: 831-834Crossref PubMed Scopus (62) Google Scholar). This finding has raised the possibility that other classes of myosin may be capable of moving to the minus-end of actin filaments. Actually, it was found quite recently that myosin IX is another myosin that shows reverse directionality (34Inoue A. Saito J. Ikebe R. Ikebe M. Nat. Cell Biol. 2002; 4: 302-306Crossref PubMed Scopus (78) Google Scholar, 35Inoue A. Ikebe M. Mol. Biol. Cell. 2000; 11 (abstr.): 375Google Scholar).There is no doubt that myosin VIIA plays a role in various cellular motile processes. How myosin VIIA functions in cellular motile events is unknown. This is at least partly due to a lack of biochemical information on myosin VIIA. Furthermore, little is known about the motor characteristic of myosin VIIA at the molecule level. The present study was initiated to clarify the motor function of myosin VIIA at a molecular level.DISCUSSIONA number of unconventional myosins have been found during the last decade, and it has been anticipated that these newly found myosins play a key role in diverse cellular motile processes. Whereas their unique C-terminal domain would be important for targeting each myosin to a specific cellular component, it is anticipated that the uniqueness of the motor properties of each myosin is also critical for its physiological function, in particular motile processes in cells. For myosin VIIA, where gene disruption is responsible for hereditary deafness and blindness (5Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (544) Google Scholar, 15Weil D. Blanchard S. Kaplan J. Guilford P. Gibson F. Walsh J. Mburu P. Varela A. Levilliers J. Weston M.D. Kelley P.M. Kimberlig W.J. Wagenaar M. Levi-Acobas F. Larget-Piet D. Munnich A. Steel K.P. Brown S.D.M. Petit C. Nature. 1995; 374: 60-61Crossref PubMed Scopus (866) Google Scholar, 16Liu X.Z. Walsh J. Mburu P. Kendrick-Jones J. Cope M.J. Steel K.P. Brown S.D. Nat. Genet. 1997; 16: 188-190Crossref PubMed Scopus (376) Google Scholar, 18Liu X.Z. Walsh J. Tamagawa Y. Kitamura K. Nishizawa M. Steel K.P. Brown S.D. Nat. Genet. 1997; 17: 268-269Crossref PubMed Scopus (270) Google Scholar), little is known regarding its motor function at a molecular level. The present study has determined the motor properties of myosin VIIA. In order to prepare mammalian myosin VIIA, we decided to express recombinant myosin VIIA rather than to purify it from tissues for several reasons. First, it is known that various types of myosins are present in the same tissue and it would be difficult to completely eliminate the contamination of other myosins. This problem can be overcome by overexpressing myosin VIIA in Sf9 cells and purifying with a histidine tag. Also, large quantities of the protein can be made and prepared in a short period of time. This is critical for preventing the denaturation and degradation of the protein during preparation.We produced a M7IQ5 construct that contains the entire head domain plus coiled-coil domain. It has been thought that myosin VIIA is a double-headed myosin because of the presence of the coiled-coil domain (12Chen Z.Y. Hasson T. Kelley P.M. Schwender B.J. Schwartz M.F. Ramakrishnan M. Kimberling W.J. Mooseker M.S. Corey D.P. Genomics. 1996; 36: 440-448Crossref PubMed Scopus (117) Google Scholar). However, the length of the coiled-coil domain is relatively short, and there is no conclusive evidence for the two-headed structure of myosin VIIA. Our results clearly demonstrated that M7IQ5, having an entire coiled-coil domain of myosin VIIA, is single-headed. However, quite interestingly, the full-length myosin VIIA formed a two-headed structure. The result suggests that the relatively short coiled-coil domain of myosin VIIA is not sufficient to stabilize the two-headed structure, and the tail domains of myosin VIIA in the two heavy chains interact with each other, which contributes to form a stable two-headed structure of myosin VIIA.Myosin VIIA showed maximum actin-translocating velocity of 0.16 ± 0.02 μm/s at 25 °C. This value agrees with that of mouse myosin VIIA (0.19 μm/s) as recently reported by Udovichenko et al. (53Udovichenko I.P. Gibbs D. Williams D.S. J. Cell Sci. 2002; 115: 445-450Crossref PubMed Google Scholar). However, to our surprise, myosin VIIA requires an extremely high ATP concentration for its motility activity. Consistently, a high ATP concentration was needed for the saturation of the actin-activated ATPase activity of myosin VIIA and the ATP-induced dissociation of myosin VIIA from actin (Table I). These results suggest either an extremely slow ATP binding or a weak affinity of myosin VIIA for ATP. A very slow rate of ATP binding has been reported formyr1 (54Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). On the other hand, ADP significantly inhibited the actin-activated ATPase activity of myosin VIIA. Consistently, the actin-translocating velocity of myosin VIIA was also inhibited by ADP. These results suggest the relatively strong binding of myosin VIIA to ADP. Of particular interest is the observation that ADP markedly reduced K actin, thus increasing the apparent affinity for actin during the ATPase cycle (Table I). Consistently, the addition of ADP markedly increased the binding of myosin VIIA to actin filaments in the presence of ATP. Even in the presence of 1 mm ATP, the addition of ADP enhanced the binding of myosin VIIA, with nearly 100% of myosin VIIA binding to actin filaments. These results suggest that a significant fraction of myosin VIIA during the ATPase cycle is in a strong binding state in the presence of ADP, presumably in a myosin VIIA/ADP form.If this is true, then it is plausible that myosin VIIA plays a role in connecting actin cytoskeleton and cellular components to maintain stress. Myosin VIIA is associated with cross-links between adjacent stereocilia (55Hasson T. Gillespie P.G. Garcia J.A. MacDonald R.B. Zhao Y. Yee A.G. Mooseker M.S. Corey D.P. J. Cell Biol. 1997; 137: 1287-1307Crossref PubMed Scopus (441) Google Scholar), suggesting a role in maintaining their structural integrity. The present results are consistent with this earlier finding and provide a molecular basis for this notion.The weak affinity for actin in the presence of ATP suggests the low duty ratio of myosin VIIA. However, the tight binding of ADP usually suggests slow ADP dissociation. If so, the myosin would tend to give high duty ratio. In the presence of ADP, myosin movement is inhibited, but the inhibition required a relatively high ADP concentration. A likely scenario would be that the ADP affinity is not very high, but because ATP binding is weak, relatively low ADP concentration decreases the ATPase activity. In the presence of sufficiently high ADP concentration, myosin movement is inhibited by the presence of myosin-ADP complex. If this is the case, myosin VII may function to maintain tension in cells where a significant fraction binds ADP. Tension maintenance in cells has also been proposed for myr1 (54Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).Directionality of myosin movement is another important issue to understand the physiological relevance of each myosin motor. The present results clearly indicate that myosin VIIA is a (+)-ended motor, unlike myosin VI. Quite recently, it was assumed thatDictyostelium myosin VII would be a plus end-directed motor, because it may function in the assembly and disassembly of adhesion proteins at the plasma membrane, and the actin at areas of membrane extension is arranged with the plus ends outermost (55Hasson T. Gillespie P.G. Garcia J.A. MacDonald R.B. Zhao Y. Yee A.G. Mooseker M.S. Corey D.P. J. Cell Biol. 1997; 137: 1287-1307Crossref PubMed Scopus (441) Google Scholar, 56Tuxworth R.I. Weber I. Wessels D. Addicks G.C. Soll D.R. Gerisch G. Titus M.A. Curr. Biol. 2001; 11: 318-329Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The present results agree with this assumption. Whereas myosin VI and VIIA are present in the sensory hair cells in the inner ear and the mutation of these two classes of myosins causes auditory dysfunction, the present results strongly suggest that the function of these two types of myosins in the sensory hair cells must be distinct from each other. Myosins are mechanochemical proteins with a motor domain containing an ATP and actin-binding region, a neck domain that interacts with light chains or calmodulin, and a tail domain that serves to anchor and position the motor domain so that it can properly interact with actin filaments at a specific intracellular location. Phylogenetic analysis revealed that myosin consists of at least 18 classes (1Hodge T. Cope M.J. J. Cell Sci. 2000; 113: 3353-3354Crossref PubMed Google Scholar, 2Yamashita R.A. Sellers J.R. Anderson J.B. J. Muscle Res. Cell Motil. 2000; 21: 491-505Crossref PubMed Scopus (47) Google Scholar). Myosin VIIs are found in human (3Hasson T. Skowron J.F. Gilbert D.J. Avraham K.B. Perry W.L. Bement W.M. Anderson B.L. Sherr E.H. Chen Z.Y. Greene L.A. Ward D.C. Corey D.P. Mooseker M.S. Copeland N.G. Jenkins N.A. Genomics. 1996; 36: 431-439Crossref PubMed Scopus (71) Google Scholar, 4Weil D. Levy G. Sahly I. Levi-Acobas F. Blanchard S. El-Amraoui A. Crozet F. Philippe H. Abitbol M. Petit C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3232-3237Crossref PubMed Scopus (150) Google Scholar), mouse (5Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (544) Google Scholar), porcine (6Bement W.M. Hasson T. Wirth J.A. Cheney R.E. Mooseker M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6549-6553Crossref PubMed Scopus (108) Google Scholar), zebrafish (7Ernest S. Rauch G.J. Haffter P. Geisler R. Petit C. Nicolson T. Hum. Mol. Genet. 2000; 9: 2189-2196Crossref PubMed Scopus (169) Google Scholar), bullfrog (8Solc C.K. Derfler B.H. Duyk G.M. Corey D.P. Auditory Neurosci. 1994; 1: 63-75Google Scholar), Caenorhabditis elegans (9Baker J.P. Titus M.A. J. Mol. Biol. 1997; 272: 523-535Crossref PubMed Scopus (47) Google Scholar), Dictyostelium discoidium (10Titus M.A. Curr. Biol. 1999; 9: 1297-1303Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), andDrosophila (11Chen T.L. Edwards K.A. Lin R.C. Coats L.W. Kiehart D.P. J. Cell Biol. 1991; 115 (abstr.): 330Google Scholar). In vertebrates, two types of genes for type VII myosin, myosin VIIA and VIIB, are identified. Among them, the entire coding region has been determined for myosin VIIA. In mammals, myosin VIIA is expressed in a variety of organs and tissues including eye, inner ear, olfactory epithelium, brain, choroid plexus, intestine, liver, kidney, adrenal gland, testis, and lymphocytes (12Chen Z.Y. Hasson T. Kelley P.M. Schwender B.J. Schwartz M.F. Ramakrishnan M. Kimberling W.J. Mooseker M.S. Corey D.P. Genomics. 1996; 36: 440-448Crossref PubMed Scopus (117) Google Scholar, 13Sahly I. El-Amraoui A. Abitbol M. Petit C. Dufier J.L. Anat. Embryol. (Berl.). 1997; 196: 159-170Crossref PubMed Scopus (86) Google Scholar, 14Wolfrum U. Liu X. Schmitt A. Udovichenko I.P. Williams D.S. Cell Motil. Cytoskeleton. 1998; 40: 261-271Crossref PubMed Scopus (111) Google Scholar). In the retina, myosin VIIA is found in the pigmented epithelial cells and is postulated to play a role in phagocytosis of cell debris that accumulates as a result of sloughing off of photoreceptor outer segments. Of interest is that defects of myosin VIIA cause the mouseshaker-1 phenotype and human Usher syndrome 1B, which are characterized by deafness, lack of vestibular function, and (in humans) progressive retinal degeneration (5Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (544) Google Scholar, 15Weil D. Blanchard S. Kaplan J. Guilford P. Gibson F. Walsh J. Mburu P. Varela A. Levilliers J. Weston M.D. Kelley P.M. Kimberlig W.J. Wagenaar M. Levi-Acobas F. Larget-Piet D. Munnich A. Steel K.P. Brown S.D.M. Petit C. Nature. 1995; 374: 60-61Crossref PubMed Scopus (866) Google Scholar). In humans, two forms of dominant and recessive nonsyndromic deafness, DFNB2 and DFNA11, are also caused by myosin VIIA mutations (16Liu X.Z. Walsh J. Mburu P. Kendrick-Jones J. Cope M.J. Steel K.P. Brown S.D. Nat. Genet. 1997; 16: 188-190Crossref PubMed Scopus (376) Google Scholar, 17Weil D. Kussel P. Blanchard S. Levy G. Levi-Acobas F. Drira M. Ayadi H. Petit C. Nat. Genet. 1997; 16: 191-193Crossref PubMed Scopus (336) Google Scholar, 18Liu X.Z. Walsh J. Tamagawa Y. Kitamura K. Nishizawa M. Steel K.P. Brown S.D. Nat. Genet. 1997; 17: 268-269Crossref PubMed Scopus (270) Google Scholar). Amino acid sequence analysis of myosin VIIA has indicated that this myosin has a motor domain containing actin-binding and ATP-binding sites, and five IQ motifs at the neck domain. In the tail domain, a very short predicted coiled-coil region was found; therefore, it has been assumed that myosin VIIA forms a two-headed structure. Two "band 4.1 protein, ezrin, radixin, moesin" (FERM) 1The abbreviations used are: FERM, band 4.1 protein, ezrin, radixin, moesin; M7full, full-length myosin VIIA; DTT, dithiothreitol; NTA, nitrilotriacetic acid 1The abbreviations used are: FERM, band 4.1 protein, ezrin, radixin, moesin; M7full, full-length myosin VIIA; DTT, dithiothreitol; NTA, nitrilotriacetic acid domains that have been implicated in cytoskeletal protein interactions have been found in the tail region. There are two myosin tail homology 4 domains found in the tail region. Although similar domains are found in myosin IV and XV, the function of this domain is unclear. The tail domain of unconventional myosins has been implicated to serve as the anchoring site for the cellular target proteins. For myosin VIIa, several tail binding proteins have been identified. The type I regulatory subunit of protein kinase A binds to the FERM domain at the C terminus of myosin VIIA (19Kussel-Andermann P. El-Amraoui A. Safieddine S. Hardelin J.P. Nouaille S. Camonis J. Petit C. J. Biol. Chem. 2000; 275: 29654-29659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), although the role of protein kinase A in the function and regulation of myosin VIIA is unknown. It was shown that the FERM domain is involved in anchoring with adherens junction via a cadherin-catenins complex (20Kussel-Andermann P. El-Amraoui A. Safieddine S. Nouaille S. Perfettini I. Lecuit M. Cossart P. Wolfrum U. Petit C. EMBO J. 2000; 19: 6020-6029Crossref PubMed Scopus (183) Google Scholar). Todorov et al. reported (21Todorov P.T. Hardisty R.E. Brown S.D. Biochem. J. 2001; 354: 267-274Crossref PubMed Scopus (24) Google Scholar) that the myosin VIIA tail has an affinity for microtubule-associated protein 2B, suggesting the interaction of myosin VIIA with the microtubule-based motility system. Quite recently, El-Amraoui et al. (22El-Amraoui A. Schonn J.S. Kussel-Andermann P. Blanchard S. Desnos C. Henry J.P. Wolfrum U. Darchen F. Petit C. EMBO Rep. 2002; 3: 463-470Crossref PubMed Scopus (150) Google Scholar) reported that myosin VIIa binds to myosin VIIa- and Rab27-interacting protein, associates with melanosomes via Rab27A, and plays a role in melanosome trafficking. On the other hand, little is known about the motor function of myosin VIIa at a molecular level. Unlike the case for the well characterized conventional myosins, it becomes evident that the motor function among various members of the unconventional myosin subfamily varies uniquely from one to another, which is thought to be critical for specific physiological roles in diverse cellular motile processes. For example, recent studies have revealed that myosin V is a processive motor that can move in large steps approximating the 36-nm pseudorepeat of the actin filament (23Walker M.L. Burgess S.A. Sellers J.R. Wang F. Hammer III, J.A. Trinick J. Knight P.J. Nature. 2000; 405: 804-807Crossref PubMed Scopus (282) Google Scholar, 24Sakamoto T. Amitani I. Yokota E. Ando T. Biochem. Biophys. Res. Commun. 2000; 272: 586-590Crossref PubMed Scopus (172) Google Scholar, 25Mehta A.D. Rock R.S. Rief M. Spudich J.A. Mooseker M.S. Cheney R.E. Nature. 1999; 400: 590-593Crossref PubMed Scopus (670) Google Scholar, 26Tanaka H. Homma K. Iwane A.H. Katayama E. Ikebe R. Saito J. Yanagida T. Ikebe M. Nature. 2002; 415: 192-195Crossref PubMed Scopus (114) Google Scholar). These characteristics are quite important to understand the physiological function of myosin V, since the processive nature of myosin V with large step size is suitable for the motors involved in cargo movement in cells. Recently, myosin VI was also demonstrated to be a processive motor with a large step size (27Rock R.S. Rice S.E. Wells A.L. Purcell T.J. Spudich J.A. Sweeney H.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13655-13659Crossref PubMed Scopus (319) Google Scholar, 28Nishikawa S. Homma K. Komori Y. Iwaki M. Wazawa T. Iwane A.H. Saito J. Ikebe R. Katayama E. Yanagida T. Ikebe M. Biochem. Biophys. Res. Commun. 2002; 290: 311-317Crossref PubMed Scopus (149) Google Scholar). A unique feature of myosin VI is that it moves backward on the actin filament (29Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Crossref PubMed Scopus (548) Google Scholar). It was originally proposed that the myosin VI unique large insertion between the neck and converter domains is responsible for the reverse directionality of myosin VI (29Wells A.L. Lin A.W. Chen L.Q. Safer D. Cain S.M. Hasson T. Carragher B.O. Milligan R.A. Sweeney H.L. Nature. 1999; 401: 505-508Crossref PubMed Scopus (548) Google Scholar). This view was based upon the hypothesis that the orientation of the motor domain of myosin VI against actin filament is the same direction as other myosins but the attachment of the position of the "lever arm" (a long α-helical region following the motor domain) (30Block S.M. Cell. 1996; 87: 151-157Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) is different so that the same movement of the catalytic core would rotate the lever arm in the opposite direction on actin. Since the insertion is located between the converter domain (a compact subdomain thought to amplify the conformational change of the motor domain) (31Houdusse A. Kalabokis V.N. Himmel D. Szent-Gyorgyi A.G. Cohen C. Cell. 1999; 97: 459-470Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 32Dominguez R. Freyzon Y. Trybus K.M. Cohen C. Cell. 1998; 94: 559-571Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar) and light chain binding helix, it was hypothesized that the 53-amino acid unique insertion of myosin VI is critical in determining the reverse directionality of myosin VI. However, a recent study revealed that the myosin VI unique insertion is not important to the reverse in directionality (33Homma K. Yoshimura M. Saito J. Ikebe R. Ikebe M. Nature. 2001; 412: 831-834Crossref PubMed Scopus (62) Google Scholar). Instead the motor core domain is responsible for the change in the directionality of myosin movement (33Homma K. Yoshimura M. Saito J. Ikebe R. Ikebe M. Nature. 2001; 412: 831-834Crossref PubMed Scopus (62) Google Scholar). This finding has raised the possibility that other classes of myosin may be capable of moving to the minus-end of actin filaments. Actually, it was found quite recently that myosin IX is another myosin that shows reverse directionality (34Inoue A. Saito J. Ikebe R. Ikebe M. Nat. Cell Biol. 2002; 4: 302-306Crossref PubMed Scopus (78) Google Scholar, 35Inoue A. Ikebe M. Mol. Biol. Cell. 2000; 11 (abstr.): 375Google Scholar). There is no doubt that myosin VIIA plays a role in various cellular motile processes. How myosin VIIA functions in cellular motile events is unknown. This is at least partly due to a lack of biochemical information on myosin VIIA. Furthermore, little is known about the motor characteristic of myosin VIIA at the molecule level. The present study was initiated to clarify the motor function of myosin VIIA at a molecular level. DISCUSSIONA number of unconventional myosins have been found during the last decade, and it has been anticipated that these newly found myosins play a key role in diverse cellular motile processes. Whereas their unique C-terminal domain would be important for targeting each myosin to a specific cellular component, it is anticipated that the uniqueness of the motor properties of each myosin is also critical for its physiological function, in particular motile processes in cells. For myosin VIIA, where gene disruption is responsible for hereditary deafness and blindness (5Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (544) Google Scholar, 15Weil D. Blanchard S. Kaplan J. Guilford P. Gibson F. Walsh J. Mburu P. Varela A. Levilliers J. Weston M.D. Kelley P.M. Kimberlig W.J. Wagenaar M. Levi-Acobas F. Larget-Piet D. Munnich A. Steel K.P. Brown S.D.M. Petit C. Nature. 1995; 374: 60-61Crossref PubMed Scopus (866) Google Scholar, 16Liu X.Z. Walsh J. Mburu P. Kendrick-Jones J. Cope M.J. Steel K.P. Brown S.D. Nat. Genet. 1997; 16: 188-190Crossref PubMed Scopus (376) Google Scholar, 18Liu X.Z. Walsh J. Tamagawa Y. Kitamura K. Nishizawa M. Steel K.P. Brown S.D. Nat. Genet. 1997; 17: 268-269Crossref PubMed Scopus (270) Google Scholar), little is known regarding its motor function at a molecular level. The present study has determined the motor properties of myosin VIIA. In order to prepare mammalian myosin VIIA, we decided to express recombinant myosin VIIA rather than to purify it from tissues for several reasons. First, it is known that various types of myosins are present in the same tissue and it would be difficult to completely eliminate the contamination of other myosins. This problem can be overcome by overexpressing myosin VIIA in Sf9 cells and purifying with a histidine tag. Also, large quantities of the protein can be made and prepared in a short period of time. This is critical for preventing the denaturation and degradation of the protein during preparation.We produced a M7IQ5 construct that contains the entire head domain plus coiled-coil domain. It has been thought that myosin VIIA is a double-headed myosin because of the presence of the coiled-coil domain (12Chen Z.Y. Hasson T. Kelley P.M. Schwender B.J. Schwartz M.F. Ramakrishnan M. Kimberling W.J. Mooseker M.S. Corey D.P. Genomics. 1996; 36: 440-448Crossref PubMed Scopus (117) Google Scholar). However, the length of the coiled-coil domain is relatively short, and there is no conclusive evidence for the two-headed structure of myosin VIIA. Our results clearly demonstrated that M7IQ5, having an entire coiled-coil domain of myosin VIIA, is single-headed. However, quite interestingly, the full-length myosin VIIA formed a two-headed structure. The result suggests that the relatively short coiled-coil domain of myosin VIIA is not sufficient to stabilize the two-headed structure, and the tail domains of myosin VIIA in the two heavy chains interact with each other, which contributes to form a stable two-headed structure of myosin VIIA.Myosin VIIA showed maximum actin-translocating velocity of 0.16 ± 0.02 μm/s at 25 °C. This value agrees with that of mouse myosin VIIA (0.19 μm/s) as recently reported by Udovichenko et al. (53Udovichenko I.P. Gibbs D. Williams D.S. J. Cell Sci. 2002; 115: 445-450Crossref PubMed Google Scholar). However, to our surprise, myosin VIIA requires an extremely high ATP concentration for its motility activity. Consistently, a high ATP concentration was needed for the saturation of the actin-activated ATPase activity of myosin VIIA and the ATP-induced dissociation of myosin VIIA from actin (Table I). These results suggest either an extremely slow ATP binding or a weak affinity of myosin VIIA for ATP. A very slow rate of ATP binding has been reported formyr1 (54Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). On the other hand, ADP significantly inhibited the actin-activated ATPase activity of myosin VIIA. Consistently, the actin-translocating velocity of myosin VIIA was also inhibited by ADP. These results suggest the relatively strong binding of myosin VIIA to ADP. Of particular interest is the observation that ADP markedly reduced K actin, thus increasing the apparent affinity for actin during the ATPase cycle (Table I). Consistently, the addition of ADP markedly increased the binding of myosin VIIA to actin filaments in the presence of ATP. Even in the presence of 1 mm ATP, the addition of ADP enhanced the binding of myosin VIIA, with nearly 100% of myosin VIIA binding to actin filaments. These results suggest that a significant fraction of myosin VIIA during the ATPase cycle is in a strong binding state in the presence of ADP, presumably in a myosin VIIA/ADP form.If this is true, then it is plausible that myosin VIIA plays a role in connecting actin cytoskeleton and cellular components to maintain stress. Myosin VIIA is associated with cross-links between adjacent stereocilia (55Hasson T. Gillespie P.G. Garcia J.A. MacDonald R.B. Zhao Y. Yee A.G. Mooseker M.S. Corey D.P. J. Cell Biol. 1997; 137: 1287-1307Crossref PubMed Scopus (441) Google Scholar), suggesting a role in maintaining their structural integrity. The present results are consistent with this earlier finding and provide a molecular basis for this notion.The weak affinity for actin in the presence of ATP suggests the low duty ratio of myosin VIIA. However, the tight binding of ADP usually suggests slow ADP dissociation. If so, the myosin would tend to give high duty ratio. In the presence of ADP, myosin movement is inhibited, but the inhibition required a relatively high ADP concentration. A likely scenario would be that the ADP affinity is not very high, but because ATP binding is weak, relatively low ADP concentration decreases the ATPase activity. In the presence of sufficiently high ADP concentration, myosin movement is inhibited by the presence of myosin-ADP complex. If this is the case, myosin VII may function to maintain tension in cells where a significant fraction binds ADP. Tension maintenance in cells has also been proposed for myr1 (54Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).Directionality of myosin movement is another important issue to understand the physiological relevance of each myosin motor. The present results clearly indicate that myosin VIIA is a (+)-ended motor, unlike myosin VI. Quite recently, it was assumed thatDictyostelium myosin VII would be a plus end-directed motor, because it may function in the assembly and disassembly of adhesion proteins at the plasma membrane, and the actin at areas of membrane extension is arranged with the plus ends outermost (55Hasson T. Gillespie P.G. Garcia J.A. MacDonald R.B. Zhao Y. Yee A.G. Mooseker M.S. Corey D.P. J. Cell Biol. 1997; 137: 1287-1307Crossref PubMed Scopus (441) Google Scholar, 56Tuxworth R.I. Weber I. Wessels D. Addicks G.C. Soll D.R. Gerisch G. Titus M.A. Curr. Biol. 2001; 11: 318-329Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The present results agree with this assumption. Whereas myosin VI and VIIA are present in the sensory hair cells in the inner ear and the mutation of these two classes of myosins causes auditory dysfunction, the present results strongly suggest that the function of these two types of myosins in the sensory hair cells must be distinct from each other. A number of unconventional myosins have been found during the last decade, and it has been anticipated that these newly found myosins play a key role in diverse cellular motile processes. Whereas their unique C-terminal domain would be important for targeting each myosin to a specific cellular component, it is anticipated that the uniqueness of the motor properties of each myosin is also critical for its physiological function, in particular motile processes in cells. For myosin VIIA, where gene disruption is responsible for hereditary deafness and blindness (5Gibson F. Walsh J. Mburu P. Varela A. Brown K.A. Antonio M. Beisel K.W. Steel K.P. Brown S.D. Nature. 1995; 374: 62-64Crossref PubMed Scopus (544) Google Scholar, 15Weil D. Blanchard S. Kaplan J. Guilford P. Gibson F. Walsh J. Mburu P. Varela A. Levilliers J. Weston M.D. Kelley P.M. Kimberlig W.J. Wagenaar M. Levi-Acobas F. Larget-Piet D. Munnich A. Steel K.P. Brown S.D.M. Petit C. Nature. 1995; 374: 60-61Crossref PubMed Scopus (866) Google Scholar, 16Liu X.Z. Walsh J. Mburu P. Kendrick-Jones J. Cope M.J. Steel K.P. Brown S.D. Nat. Genet. 1997; 16: 188-190Crossref PubMed Scopus (376) Google Scholar, 18Liu X.Z. Walsh J. Tamagawa Y. Kitamura K. Nishizawa M. Steel K.P. Brown S.D. Nat. Genet. 1997; 17: 268-269Crossref PubMed Scopus (270) Google Scholar), little is known regarding its motor function at a molecular level. The present study has determined the motor properties of myosin VIIA. In order to prepare mammalian myosin VIIA, we decided to express recombinant myosin VIIA rather than to purify it from tissues for several reasons. First, it is known that various types of myosins are present in the same tissue and it would be difficult to completely eliminate the contamination of other myosins. This problem can be overcome by overexpressing myosin VIIA in Sf9 cells and purifying with a histidine tag. Also, large quantities of the protein can be made and prepared in a short period of time. This is critical for preventing the denaturation and degradation of the protein during preparation. We produced a M7IQ5 construct that contains the entire head domain plus coiled-coil domain. It has been thought that myosin VIIA is a double-headed myosin because of the presence of the coiled-coil domain (12Chen Z.Y. Hasson T. Kelley P.M. Schwender B.J. Schwartz M.F. Ramakrishnan M. Kimberling W.J. Mooseker M.S. Corey D.P. Genomics. 1996; 36: 440-448Crossref PubMed Scopus (117) Google Scholar). However, the length of the coiled-coil domain is relatively short, and there is no conclusive evidence for the two-headed structure of myosin VIIA. Our results clearly demonstrated that M7IQ5, having an entire coiled-coil domain of myosin VIIA, is single-headed. However, quite interestingly, the full-length myosin VIIA formed a two-headed structure. The result suggests that the relatively short coiled-coil domain of myosin VIIA is not sufficient to stabilize the two-headed structure, and the tail domains of myosin VIIA in the two heavy chains interact with each other, which contributes to form a stable two-headed structure of myosin VIIA. Myosin VIIA showed maximum actin-translocating velocity of 0.16 ± 0.02 μm/s at 25 °C. This value agrees with that of mouse myosin VIIA (0.19 μm/s) as recently reported by Udovichenko et al. (53Udovichenko I.P. Gibbs D. Williams D.S. J. Cell Sci. 2002; 115: 445-450Crossref PubMed Google Scholar). However, to our surprise, myosin VIIA requires an extremely high ATP concentration for its motility activity. Consistently, a high ATP concentration was needed for the saturation of the actin-activated ATPase activity of myosin VIIA and the ATP-induced dissociation of myosin VIIA from actin (Table I). These results suggest either an extremely slow ATP binding or a weak affinity of myosin VIIA for ATP. A very slow rate of ATP binding has been reported formyr1 (54Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). On the other hand, ADP significantly inhibited the actin-activated ATPase activity of myosin VIIA. Consistently, the actin-translocating velocity of myosin VIIA was also inhibited by ADP. These results suggest the relatively strong binding of myosin VIIA to ADP. Of particular interest is the observation that ADP markedly reduced K actin, thus increasing the apparent affinity for actin during the ATPase cycle (Table I). Consistently, the addition of ADP markedly increased the binding of myosin VIIA to actin filaments in the presence of ATP. Even in the presence of 1 mm ATP, the addition of ADP enhanced the binding of myosin VIIA, with nearly 100% of myosin VIIA binding to actin filaments. These results suggest that a significant fraction of myosin VIIA during the ATPase cycle is in a strong binding state in the presence of ADP, presumably in a myosin VIIA/ADP form. If this is true, then it is plausible that myosin VIIA plays a role in connecting actin cytoskeleton and cellular components to maintain stress. Myosin VIIA is associated with cross-links between adjacent stereocilia (55Hasson T. Gillespie P.G. Garcia J.A. MacDonald R.B. Zhao Y. Yee A.G. Mooseker M.S. Corey D.P. J. Cell Biol. 1997; 137: 1287-1307Crossref PubMed Scopus (441) Google Scholar), suggesting a role in maintaining their structural integrity. The present results are consistent with this earlier finding and provide a molecular basis for this notion. The weak affinity for actin in the presence of ATP suggests the low duty ratio of myosin VIIA. However, the tight binding of ADP usually suggests slow ADP dissociation. If so, the myosin would tend to give high duty ratio. In the presence of ADP, myosin movement is inhibited, but the inhibition required a relatively high ADP concentration. A likely scenario would be that the ADP affinity is not very high, but because ATP binding is weak, relatively low ADP concentration decreases the ATPase activity. In the presence of sufficiently high ADP concentration, myosin movement is inhibited by the presence of myosin-ADP complex. If this is the case, myosin VII may function to maintain tension in cells where a significant fraction binds ADP. Tension maintenance in cells has also been proposed for myr1 (54Coluccio L.M. Geeves M.A. J. Biol. Chem. 1999; 274: 21575-21580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Directionality of myosin movement is another important issue to understand the physiological relevance of each myosin motor. The present results clearly indicate that myosin VIIA is a (+)-ended motor, unlike myosin VI. Quite recently, it was assumed thatDictyostelium myosin VII would be a plus end-directed motor, because it may function in the assembly and disassembly of adhesion proteins at the plasma membrane, and the actin at areas of membrane extension is arranged with the plus ends outermost (55Hasson T. Gillespie P.G. Garcia J.A. MacDonald R.B. Zhao Y. Yee A.G. Mooseker M.S. Corey D.P. J. Cell Biol. 1997; 137: 1287-1307Crossref PubMed Scopus (441) Google Scholar, 56Tuxworth R.I. Weber I. Wessels D. Addicks G.C. Soll D.R. Gerisch G. Titus M.A. Curr. Biol. 2001; 11: 318-329Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The present results agree with this assumption. Whereas myosin VI and VIIA are present in the sensory hair cells in the inner ear and the mutation of these two classes of myosins causes auditory dysfunction, the present results strongly suggest that the function of these two types of myosins in the sensory hair cells must be distinct from each other.