Cloning and Characterization of a Myosin from Characean Alga, the Fastest Motor Protein in the World

Kashiyama, Taku; Kimura, Naohiro; Mimura, Tetsuro; Yamamoto, Keiichi

doi:10.1093/oxfordjournals.jbchem.a022699

Cited by 58 publications

(39 citation statements)

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“…Based on several reports describing the inhibition of kinesin-based movements by microtubule-associated proteins (Lopez and Sheetz, 1993;Ebneth et al, 1998;Ashby et al, 2006;Dixit et al, 2008;Vershinin et al, 2008), it seems probable that excess binding of ABD2:GFP to the actin cytoskeleton interferes with the translocation of myosin motor proteins along the filaments. The observed restriction of Golgi and TMV trafficking likely involves the inhibition of class XI myosins, which move along actin filaments (Tominaga et al, 2003) and are responsible for the dynamic movements of Golgi complexes and other organelles (Avisar et al, 2008b;Peremyslov et al, 2008;Prokhnevsky et al, 2008;Sparkes et al, 2008) as well as for those of the ER (Kashiyama et al, 2000;Morimatsu et al, 2000;Kimura et al, 2003;Yokota et al, 2009). The myosin-driven Golgi movements occur along actin filaments that align the ER (Boevink et al, 1998) and have been proposed to reflect myosin-supported movements of ER membrane proteins (Runions et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

et al. 2009

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The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments.Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosinassociated protein trafficking along the ER membrane.The mechanism by which plant viruses move from cell to cell and systemically to cause systemic infection has been the subject of intense studies (for review, see

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Section: Discussionmentioning

confidence: 99%

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

et al. 2009

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“…Z34294) but now contains at least 13 members ( Figure 5). Class XI myosins are also present in Characean algae (Kashiyama et al, 2000), where they are thought to underlie the rapid cytoplasmic streaming of organelles, and in the biflagellate green alga Chlamydomonas (acc. no.…”

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A Millennial Myosin Census

Berg

Powell

Cheney

2001

MBoC

715

621

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The past decade has seen a remarkable explosion in our knowledge of the size and diversity of the myosin superfamily. Since these actin-based motors are candidates to provide the molecular basis for many cellular movements, it is essential that motility researchers be aware of the complete set of myosins in a given organism. The availability of cDNA and/or draft genomic sequences from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium discoideum has allowed us to tentatively define and compare the sets of myosin genes in these organisms. This analysis has also led to the identification of several putative myosin genes that may be of general interest. In humans, for example, we find a total of 40 known or predicted myosin genes including two new myosins-I, three new class II (conventional) myosins, a second member of the class III/ninaC myosins, a gene similar to the class XV deafness myosin, and a novel myosin sharing at most 33% identity with other members of the superfamily. These myosins are in addition to the recently discovered class XVI myosin with N-terminal ankyrin repeats and two human genes with similarity to the class XVIII PDZ-myosin from mouse. We briefly describe these newly recognized myosins and extend our previous phylogenetic analysis of the myosin superfamily to include a comparison of the complete or nearly complete inventories of myosin genes from several experimentally important organisms. INTRODUCTIONMyosins are actin-based motors known or hypothesized to play fundamental roles in many forms of eukaryotic motility such as cell crawling, cytokinesis, phagocytosis, growth cone extension, maintenance of cell shape, and organelle/particle trafficking. Although actin polymerization alone can drive some forms of motility, myosins appear to power an assortment of movements and are important in processes such as signal transduction (Bahler, 2000) and establishment of polarity (Yin et al., 2000). Recent evidence even implicates myosins in the polymerization of actin (Evangelista et al., 2000;Lechler et al., 2000;Lee et al., 2000). To understand the molecular basis of actin-based motility, it is thus critical to identify the pool of candidate motor proteins.Members of the myosin superfamily are defined by the presence of a heavy chain with a conserved ϳ80 kDa catalytic domain. In most myosins, the catalytic domain is followed by an ␣-helical light chain-binding region consisting of one or more IQ motifs. Most myosins also have a Cterminal tail and/or an N-terminal extension thought to endow class-specific properties such as membrane binding or kinase activity. Class II myosins are familiar from studies of muscle contraction, but the myosin superfamily also contains a large number of other myosins with quite different tail domains. Although the conventional-unconventional dichotomy is clearly artificial in terms of structure and evolution, it is operationally useful because of the historical emphasis on...

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“…This velocity is about 10 times faster than that of the fast skeletal muscle myosin and the Chara myosin is the fastest motor protein known so far. We have cloned cDNA of the Chara myosin heavy chain (4) and succeeded in expressing functional motor domain (5,6). The velocity of the expressed motor domain measured by in vitro motility assay was comparable to that of the native Chara myosin if we consider the difference in the lever arm length.…”

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Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

Ito

Yamaguchi

Yanase

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected Vmax and Kapp of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected Kapp and the velocity, although it did not affect Vmax. Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.actin ͉ ATPase ͉ motility ͉ cytoplasmic streaming ͉ molecular engineering C ytoplasmic streaming in characean algal cells is extremely fast, and this streaming is brought about by the movement of myosin-coated organelles along actin filament bundles fixed inside the cell (1). Myosin purified from Chara corallina could translocate actin filaments in the in vitro motility assay at a velocity comparable to that of the cytoplasmic streaming (approximately 50 m s Ϫ1 ) (2, 3). This velocity is about 10 times faster than that of the fast skeletal muscle myosin and the Chara myosin is the fastest motor protein known so far. We have cloned cDNA of the Chara myosin heavy chain (4) and succeeded in expressing functional motor domain (5, 6). The velocity of the expressed motor domain measured by in vitro motility assay was comparable to that of the native Chara myosin if we consider the difference in the lever arm length. Relevant steps in actomyosin ATPase cycle to the sliding velocity are ADP release from actomyosin and ATP reassociation resulting in actin-myosin dissociation. Kinetic analyses of Chara myosin motor domain revealed that both the ADP release and the ATP-induced dissociation from actin were very fast (6). Time spent in the strongly bound state with actin, which was estimated from these rates, was less than 1 ms. This very short strongly bound state, together with a large step size (7), are probably the reason for the very high velocity of Chara myosin. Actin-activated ATPase activity of expressed Chara motor domain was approximately 500 s Ϫ1 head Ϫ1

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Cloning and Characterization of a Myosin from Characean Alga, the Fastest Motor Protein in the World

Cited by 58 publications

References 0 publications

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

A Millennial Myosin Census

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

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