Cell Motility and the Cytoskeleton 39:195–200 (1998) Views and Reviews Yeast Myosin II: A New Subclass of Unconventional Conventional Myosins? Karen M. May, Thein Z. Win, and Jeremy S. Hyams* Department of Biology, University College, London, UK Myosin II is the founder member of a large and structurally diverse clan of actin-based motor proteins. The native myosin II molecule is a hexamer consisting of two heavy chains, two essential light chains (ELC), and two regulatory light chains (RLC). For convenience, the myosin IIs are often subdivided into four subclasses: vertebrate skeletal and cardiac muscle myosin II form one subclass, vertebrate smooth muscle and nonmuscle myosin II a second, invertebrate muscle a third, and protozoan myosin II a fourth [Sellers and Goodson, 1995]. Different mechanisms of regulation may exist between myosins within a single subclass yet all myosin IIs share a common three-domain structure; the N-terminus of the heavy chain forms two globular heads that contain the ATP- and actin-binding sites and the a-helical neck region that is stabilised by the binding of the two classes of light chains, whilst the C-terminus forms an extended coiled-coil tail that can consist of anywhere between 700 and 1,200 amino acids. In nonmuscle cells, myosin II has at least two well-defined functions, cell locomotion and cytokinesis. Yeast cells do not locomote, and their mechanism of cytokinesis involves the deposition of a cross-wall or septum. However, in the fission yeast, Schizosaccharomyces pombe, deposition of the septum is anticipated by the appearance of a contractile actomyosin ring [Marks and Hyams, 1985; May et al., 1997; Kitayama et al., 1997] and actin is also present at the bud neck during cytokinesis in the budding yeast, Saccharomyces cerevisiae [Kilmartin and Adams, 1984]. Here we report a phylogenetic analysis of the N-terminal head domains of the myosin IIs from both yeasts, a structural analysis of the tail domains of these proteins and we speculate as to the nature of the light chains that regulate their function. On the basis of these findings, we propose that the yeast myosin IIs constitute a divergent fifth class of ‘‘unconventional’’ conventional myosins. Cell Motil. Cytoskeleton 39:195–200, 1998. r 1998 Wiley-Liss, Inc. INTRODUCTION Recent years have seen a rapid expansion of the myosin superfamily. Based on sequence homology within the head or motor domain, at least 13 myosin classes are now recognised [Mooseker and Cheney, 1995; Cope et al., 1996]. Since members of individual classes are also related by tail structure, it has been proposed that heads and tails coevolved to perform a specific function [Cope et al., 1996]. Most cells express at least two myosin classes but only in the budding yeast, Saccharomyces r 1998 Wiley-Liss, Inc. cerevisiae, which expresses three, do we have a complete catalogue of all the myosins expressed within a single organism [Brown, 1997]. Whilst the cellular role of the budding yeast myosin Is in the polarisation of the actin cytoskeleton and in endocytosis [Goodson et al., 1996; *Correspondence to: Jeremy S. Hyams, Department of Biology, University College, Gower Street, London WC1E 6BT, UK Received 4 December 1997; accepted 9 December 1997 May et al. Figure 1. 196 Yeast Myosin II Goodson and Spudich, 1995; Geli and Riezman, 1996] and the myosin Vs [Johnston et al., 1991; Haarer et al., 1994; Govindan et al., 1995] in a post-Golgi step in the secretory pathway are relatively well established, myosin II, encoded by the gene MYO1, has received surprisingly little attention. Cells deleted for MYO1 are viable but have defective cell wall organization at the mother-bud neck and are consequently defective for cell division, or at least cell separation [Watts et al., 1987; Rodriguez and Paterson, 1990]. Analysis of the head domain of MYO1 places budding yeast on an early branch of the evolutionary history of myosin II with other simple eukaryotes such as Acanthamoeba and Dictyostelium as its closest relatives [Cheney et al., 1993; Mooseker and Cheney, 1995; Cope et al., 1996]. However, unusually, and in contrast to the protozoan myosin IIs, the MYO1 tail contains nine proline residues that might be expected to affect its ability to form the typical extended coiled-coil structure [Watts et al., 1987]. THEREBY HANGS A TAIL Recently, we [May et al., 1997] and Kitayama et al. , independently isolated a myosin II from the fission yeast, Schizosaccharomyces pombe, which we designated myo21 [May et al., 1996]. myo21 is an essential gene encoding a protein of 1526 amino acids. Phylogenetic analysis identifies the closest relatives of myo21 as MYO1 [1851 amino acids] and a second fission yeast myosin II gene identified by the Sanger Centre sequencing project, which we have designated myo221 (2104 amino acids; Fig. 1). Like MYO1, the tails of both myo21 and myo221 contain numerous proline residues, nine in the case of myo21 and 27 in myo221. Analysis using the PairCoil program of Berger et al.  predicts that the tails of all three yeast myosin IIs consist of numerous short segments of coiled-coil [May et al., 1997] (Fig. 2a–c), rather than a single extended structure, as seen in both the protozoan (Fig. 2d) and vertebrate (Fig. 2e) myosin IIs. Thus, both the amino Fig. 1. Phylogenetic analysis of the myosin superfamily. An unrooted phylogenetic tree showing the major myosin classes. The four previously defined myosin II subclasses are within the large shaded area (MII). myo21, myo221, and MYO1 (small shaded area) clearly fall within the myosin IIs but are most similar to each other. The 42 myosin head sequences were aligned using the CLUSTALW software. The tree was constructed using the NEIGHBOR joining method from the PHYLIP suite of programs (Felsenstein, 1989). For accession numbers, see Cope et al. ; GenBank accession numbers for myo21 and myo221 are U75357 and Z98762 respectively. To estimate the degree of confidence in branching, the tree was bootstrapped 100 times. 197 acid sequence of the head domains and the unique and unusual structure of their tails appear to place these yeast proteins in a distinct subclass of myosin II. Despite this, the fission yeast protein at least appears to function as a bona fide actin-based motor, most notably as a component of the contractile cytokinetic actomyosin ring [May et al., 1997; Kitayama et al., 1997]. Cells deleted for myo221 are defective for cytokinesis (T.Z. Win and J.S. Hyams, unpublished results), although the precise function of this protein remains to be determined. A LITTLE LIGHT ON LIGHT CHAINS What do we know about the regulation of the yeast myosin IIs? Both the MYO1 and myo21 sequences contain IQ motifs that define the binding sites for both essential and regulatory light chains [Houdusse and Cohen, 1995; Watts et al., 1987; May et al., 1997]. In both Drosophila [Karess et al., 1991] and Dictyostelium [Pollenz et al., 1992], mutations in the genes encoding either light chain result in a defect in cytokinesis. Exhaustive genetic screens for cytokinesis mutants in fission yeast [Nurse et al., 1976; Chang et al., 1996] have identified only a single light chain candidate. cdc41 encodes an EF hand protein that shares features of both ELCs and RLCs, but cannot be assigned unambiguously to either class [McCollum et al., 1995]. As with the Dictyostelium and Drosophila light chain mutants [see above], cdc42 mutants are defective for cytokinesis [Nurse et al., 1976] due to a failure to assemble a functional actin ring [Marks et al., 1987; McCollum et al., 1995]. The Cdc4 protein colocalises with myosin II in the contractile ring [McCollum et al., 1995]. A search of the budding yeast genome also failed to identify conventional ELC or RLC genes (K.M. May and J.S. Hyams, unpublished results) [Wang et al., 1997], although it did reveal an open reading frame sharing 42% identity with cdc41 (Fig. 3), designated MLC1 (R.C. Stephens and T.N. Davis, 1997, unpublished observations, cited in the Saccharomyces Genetic Database). Like cdc41, MLC1 encodes an EF-hand protein with four putative Ca21-binding loops and, also like cdc41, it is an essential gene. Phylogenetic analysis places both proteins together, with the protozoan ELCs as the closest relative. However, the bootstrap values for the branching of the yeast light chains are too low to ascribe these proteins to any of the three classes of EF hand protein with any confidence (Fig. 4). A detailed characterisation of MLC1 is now essential to clarify whether the yeast myosin IIs are not only structurally distinct from other members of this myosin class but are also regulated by a unique class of light chains. The recent demonstra- 198 May et al. Fig. 2. Coiled-coil predictions for myosin II sequences. The ability of S. pombe myo21 (A), myo221 (B), and S cerevisiae MYO1 (C) to form coiled-coils was predicted using the PairCoil program of Berger et al. (1995). The horizontal axis shows amino acid number from the N-terminus. Unlike Acanthamoeba (D) and human embryonic skeletal (ESk) muscle (E) myosin IIs, the tails of which (roughly from amino acid 800 to the C-terminus) consist of an almost unbroken stretch of coiled-coil, the yeast myosin IIs form only short segments of coiledcoil. The breaks coincide with the positions of the proline residues in the yeast and Acanthamoeba sequences. Yeast Myosin II 199 UNITED BY DIVISION Fig. 3. Alignment of the putative yeast myosin light chains. Both Cdc41 and Mlc1 have four potential Ca21-binding loops (doubleheaded arrow), although, unlike calmodulin, three of these (I, II, and IV) can be inferred not to bind Ca21. tion that calmodulin colocalises with myosin II to the cleavage plane during cytokinesis in fission yeast may also prove to be relevant to these speculations [Moser et al., 1997]. The sequencing of myosin genes has already revealed considerable diversity amongst the myosin superfamily [Mooseker and Cheney, 1995; Cope et al., 1996]. Although still relatively superficially characterised, the yeast myosin IIs appear to offer a new twist in the tale. For the present, the biological significance of the unique structure of the yeast myosin II tails and their unusual light chains remains unknown. In fission yeast, an ‘‘unconventional’’ conventional myosin can participate in a cytokinetic mechanism that, superficially at least, resembles the equivalent process in animal cells. 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