The structure and function of unconventional myosins: a review

Hammer, John A.

doi:10.1007/bf00123827

Cited by 63 publications

(40 citation statements)

References 75 publications

(49 reference statements)

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“…Viral stock amplification was carried out according to the manufacturer's manual, and the titers were determined by Paragon Biotechnology. 7 Sf9 insect cells each were inoculated with both light-and heavy-chain viruses at a combined multiplicity of infection of Ϸ10. The infected cells were grown in insect medium (Paragon Biotech) for 72 hr at 27°C, released with a rubber policeman, and harvested by centrifugation at 5,000 ϫ g for 10 min.…”

Section: Full-length Acanthamoeba Myosin Ic Heavy-chain Cdnamentioning

confidence: 99%

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Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

Wang

Sellers

et al. 1998

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

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The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15-to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actinactivated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its K m for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same K m for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.In recent years, 111 members of the myosin superfamily, grouped into 15 classes, have been identified at the DNA sequence level (1), the atomic structures of actin (2) and the myosin motor domain (3) have been determined, and the actin-myosin interface has been simulated by fitting the atomic structures to reconstructions of cryoelectron microscopic images of the rigor complex (4). However, very little is known of how F-actin activates the myosin ATPase, in part because relatively few of the myosins have been purified and studied biochemically, and those that have are mostly representatives of the two largest classes: conventional, class II myosins, with 46 known members, and class I myosins, with 32 known members (the next largest, class V, contains only 7 known members).Acanthamoeba myosins IA, IB, and IC were the first unconventional (i.e., non-class II) myosins to be discovered and are the most extensively studied class I myosins. These Acanthamoeba myosins are single-headed, nonfilamentous, actinbased mechanoenzymes that appear to play important roles in several aspects of cellular and intracellular motility (refs. 5 and 6; for review, see ref.

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Section: Full-length Acanthamoeba Myosin Ic Heavy-chain Cdnamentioning

confidence: 99%

“…5 and 6; for review, see ref. 7). Their single heavy chains are composed of an Ϸ80-kDa N-terminal head domain and a Ϸ50-kDa C-terminal tail (8,9).…”

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Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

Wang

Sellers

et al. 1998

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

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“…The isoforms of myosin-I that contain an SH3 domain include myosin-Is from Acanthamoeba (4), Dictyostelium (5), Saccharomyces (6), rat (7), and human (8). In the known myosin-I sequences, the SH3 domain invariably resides in tandem with one or two proline-rich domains (5,9); a proline-rich sequence of 3BP1 has been identified as a motif that binds to the SH3 domain of Abl (10). An interaction has not been demonstrated between SH3 and proline-rich domains of myosin-I, but proline-rich domains of myosin-I have been shown to interact with actin (11)(12)(13).…”

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Identification of Acan125 as a Myosin-I-binding Protein Present with Myosin-I on Cellular Organelles of Acanthamoeba

Zot

1995

Journal of Biological Chemistry

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We have discovered the first protein to bind to a nonfilamentous myosin, aside from actin. This protein, Acan125, is a 125-kDa protein from Acanthamoeba that associates with the SH3 domain of Acanthamoeba myosin-IC and not the SH3 domain of human fodrin. Antibodies raised against Acan125 recognize a single protein of 125 kDa from a whole cell lysate of Acanthamoeba; antibodies to myosin-I (M1.7 and M1.8) do not recognize Acan125 on the same blot. Double labeling of Acanthamoeba show Acan125 and myosin-I to be present on the same intracellular organelle, most likely amoebastomes. Immunoprecipitation with either anti-myosin-I or anti-Acan125 antibodies coprecipitates both Acan125 and myosin-I from a lysate of Acanthamoeba, demonstrating that Acan125 interacts with native myosin-I.Binding through the Src homology domain, SH3, 1 is a recognized means of linking signal transduction proteins (1-3), but a function has not been ascribed to the SH3 domain of the cytoskeletal protein myosin-I. The isoforms of myosin-I that contain an SH3 domain include myosin-Is from Acanthamoeba (4), Dictyostelium (5), Saccharomyces (6), rat (7), and human (8). In the known myosin-I sequences, the SH3 domain invariably resides in tandem with one or two proline-rich domains (5, 9); a proline-rich sequence of 3BP1 has been identified as a motif that binds to the SH3 domain of Abl (10). An interaction has not been demonstrated between SH3 and proline-rich domains of myosin-I, but proline-rich domains of myosin-I have been shown to interact with actin (11-13). These results suggested to us that the SH3 domain of myosin-I might be available for interaction with another protein.The proposal of proteins that interact with myosin-I is rooted in efforts to reconcile reconstitution results with cellular localization studies. In vitro binding (11,14,15) and motility (16) assays are consistent with myosin-I acting mechanically on the surface of any membrane containing the ubiquitous phospholipid phosphatidylserine. But immunostaining shows myosin-I to be excluded from most cell membranes and to be concentrated at the leading edges of migrating cells (17, 18) and on selected organelles (19). The contractile vacuole of Acanthamoeba has been demonstrated to selectively bind the myosin-IC isoform (20). Myosin-IA and myosin-IB were found, using immunogold, to be associated along one side of fractionated membranes (21), as though bound to proteins.Myosin-I could associate with other proteins on membrane surfaces via interactions with SH3. SH3 domains have been shown to mediate specific associations between SH3-containing proteins and various binding partners, including phosphatidylinositol 3-kinase (22-25), p22 phox , and p47phox (26 -28), and dynamin (29 -31). In each of these studies, bacterially expressed fusion proteins of SH3 domains were used as affinity ligands to selectively extract the binding partner from a cell lysate. Selectivity may be dictated by the structures of both the SH3 domain and its binding partner (32,33). Thus, binding partners fo...

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“…Similar myosins I occur in a wide variety of species, but there are also myosins I that lack the GPA and SH3 regions, and there are others with multiple, sequential, IQ domains (1)(2)(3)8).…”

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Structural invariance of constitutively active and inactive mutants of Acanthamoeba myosin IC bound to F-actin in the rigor and ADP-bound states

Carragher

Cheng

Wang

et al. 1998

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

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The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (AMIs)-AMIA, AMIB, and AMIC-are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin's actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is ref lected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-Å resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (timeaveraged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.The myosin superfamily comprises 15 classes of protein distinguished by the sequences of their catalytic domains but also differing in the domain organization of their tails (for reviews, see refs. 1-3). The two largest and best-studied classes are conventional (double-headed, filament-forming) class II myosins and unconventional (single-headed, monomeric) class I myosins. Class I was the first of the 14 classes of unconventional myosins to be discovered. The first known class I myosins, and arguably still the best biochemically described unconventional myosins of any class, are the myosins I of Acanthamoeba castellanii (4, 5).Each of the three AMI isozymes consists of a single, relatively short (for myosins) heavy chain and a single light chain (6). The heavy chain has an Ϸ72-kDa N-terminal catalytic domain that, as with all myosins, has an ATPase site and an ATP-sensitive actin-binding site. Importantly, a single Ser or Thr residue in their catalytic domains at a position occupied by a Glu or Asp in most other myosins (1) must be phosphorylated for full expression of their actin-activated ATPase activities (6, 7). Distal to the catalytic domain is a single 6-kDa IQ domain (the presumptive light chain-binding site) followed by a Ϸ50-kDa tail. The tail contains three distinctive regions: a basic, phospholipid-binding region; a Gly-Pro-Ala-rich (GPA) region that contains a second (ATPinsensitive) actin-binding site; and a src-homology 3 domain (SH3). The three regions are sequential in AMIB, but the SH3 domain splits the GPA region in AMIC (cf. Fig. 1).Similar myosins I occur in a wide variety of species, but there are also myosins I that lack the GPA and...

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The structure and function of unconventional myosins: a review

Cited by 63 publications

References 75 publications

Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

Identification of Acan125 as a Myosin-I-binding Protein Present with Myosin-I on Cellular Organelles of Acanthamoeba

Structural invariance of constitutively active and inactive mutants of Acanthamoeba myosin IC bound to F-actin in the rigor and ADP-bound states

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