Unique sequences and predicted functions of ...

Gene 480 (2011) 10–20

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Unique sequences and predicted functions of myosins in Tetrahymena thermophila Maki Sugita, Yoshinori Iwataki, Kentaro Nakano, Osamu Numata ⁎ Structural Biosciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennohdai, Tsukuba, Ibaraki 305-8572, Japan

a r t i c l e

i n f o

Article history: Accepted 13 February 2011 Available online 19 February 2011 Keywords: Actin Ciliate Molecular evolution Unconventional myosin

a b s t r a c t Myosins are eukaryotic actin-dependent molecular motors that play important roles in many cellular events. The function of each myosin is determined by a variety of functional domains in its tail region. In some major model organisms, the functions and properties of myosins have been investigated based on their amino acid sequences. However, in protists, myosins have been little studied beyond the level of genome sequences. We therefore investigated the mRNA expression levels and amino acid sequences of 13 myosin genes in the ciliate Tetrahymena thermophila. This study is an overview of myosins in T. thermophila, which has no typical myosins, such as class I, II, or V myosins. We showed that all 13 myosins were expressed in vegetative cells. Furthermore, these myosins could be divided into 3 subclasses based on four functional domains in their tail regions. Subclass 1 comprised of 8 myosins has both MyTH4 and FERM domains, and has a potential to function in vesicle transport or anchoring between membrane and actin filaments. Subclass 2 comprised of 4 myosins has RCC1 (regulator of chromosome condensation 1) domains, which are found only in some protists, and may have unconventional features. Subclass 3 is comprised of one myosin, which has a long coiled-coil domain like class II myosin. In addition, phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters: one consists of subclasses 1 and 2, and the other consists of subclass 3. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Myosin is an actin-dependent molecular motor and is universally present in the eukaryotic kingdom. Myosins are engaged in many cellular events, such as muscle contraction, cytokinesis, vesicle transport, cell migration, and cell adhesion (reviewed by Mermall et al., 1998). The myosin heavy chain is composed of three regions: 1) a head region containing the motor domain, which interacts with actin filaments (F-actin) in an adenosine 5′-triphosphate (ATP)-dependent manner; 2) a neck region containing IQ motifs, which are binding sites of a specific myosin light chain or calmodulin; and 3) a tail region, which endows each member with a functional specificity (reviewed by Krendel and Mooseker, 2005). Myosin members are classified on the basis of phylogenetic analyses of the conserved motor domains. Small differences in the amino acid sequence in the head region lead to altered motor activity, and are strongly associated with the cellular functions of Abbreviations: ATP, adenosine 5′-triphosphate; GAP, guanosine 5′-triphosphataseactivating protein; GDP, guanosine 5′-diphosphate; GTP, guanosine 5′-triphosphate; F-actin, filamentous actin; FERM, band 4.1/ezrin/radixin/moesin; GEF, guanine nucleotide exchange factor; HMM, hidden Markov model; MyTH4, myosin tail homology 4; RACE, rapid amplification of cDNA ends; Ran, Ras-related nuclear protein; RCC1, regulator of chromosome condensation 1; SH3, Src homology 3; TH1, basic C-terminal tail homology 1; qRT-PCR, quantitative real-time polymerase chain reaction. ⁎ Corresponding author. Fax: +81 29 853 6648. E-mail address: [email protected] (O. Numata). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.02.006

each myosin class. To date, around 30 myosin classes have been identified (Foth et al., 2006; Odronitz and Kollmar, 2007). Odronitz and Kollmar (2007) have proposed that the prototype of all myosin classes is class I myosin, which shows the widest taxonomic distribution and has a simple structure comprised of only one head and a short tail. Class I, II, and V myosins are widely conserved in animals and fungi, and their functions in vitro and in vivo have been well researched, as described below. Class I myosin associates with membrane lipids by a basic C-terminal tail homology 1 (TH1) domain in the tail region, and contributes to the maintenance of membrane shape during phagocytosis, endocytosis, exocytosis, intercellular transport and amoeboid movement (reviewed by Kim and Flavell, 2008). Class II myosin forms a bipolar thick filament using a long coiled-coil domain in the tail, and generates the driving force for motor apparatus such as skeletal muscle or the contractile ring in cytokinesis (reviewed by Sellers, 2000). Class V myosin, which is composed of two heavy chains and several light chains, can use its two heads to walk along F-actin step-by-step, and can associate with vesicles and organelles via a globular domain in the each tail (reviewed by Trybus, 2008). Thus class V myosin has important roles in vesicle traffic and organelle transport. In addition, several classes of myosin contain common functional domains in their tails, such as band 4.1/ezrin/radixin/moesin (FERM) and myosin tail homology 4 (MyTH4). The FERM domain binds to membrane proteins such as integrin (Küssel-Andermann et al., 2000; Zhang et al., 2004; Etournay et al., 2007). Class VII myosin, which is

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one of the representative myosins containing a FERM domain, acts as an anchor between the membrane and F-actin to maintain the structural integrity of filopodia and the strong adhesion of mammalian cultured cells to the extracellular matrix (reviewed by Krendel and Mooseker, 2005). The MyTH4 domain is usually located upstream of the FERM domain, and can bind to microtubules (Weber et al., 2004). The MyTH4 and FERM domains form a pair in several myosin classes. In addition to class I, II, V and VII myosins, in this paper, we refer to other myosin classes, such as class VI, IX and XIV myosins. Class VI is the only myosin class to move towards the minus end of F-actin; all other myosin classes are plus-end motors (Ménétrey et al., 2005). Class VI myosin has a cargo binding domain in its tail and works as both a processive transporter in endocytosis or cytokinesis and a loaddependent anchor in cell adhesion (reviewed by Sweeney and Houdusse, 2010). Class IX myosin is a single-headed motor that exhibits Rho guanosine 5′-triphosphatase (GTPase)-activating protein (RhoGAP) activity in its tail (reviewed by Bähler, 2000). Activation of Rho triggers the formation of actin filament bundles (stress fibres), the formation of focal adhesions attaching cells to the extracellular matrix, and several other processes (reviewed by Hall, 1998). Therefore, Class IX myosin is thought to act like a molecular switch for signal transduction. Class XIV myosin is an Apicomplexa-specific myosin class, and includes myosin A. Apicomplexa, a member of Alveolata, is an important pathogen in humans and other animals. Myosin A, which has virtually no tail, drives gliding motility for hostcell invasion (reviewed by Soldati and Meissner, 2004). We have previously isolated actin and actin-binding proteins from the ciliates Tetrahymena pyriformis and T. thermophila, and have studied how the proteins work in many cellular events such as cytokinesis, phagocytosis and other membrane dynamics (Hirono et al., 1989; Edamatsu et al., 1992; Numata et al., 2000; Shirayama and Numata, 2003; Kuribara et al., 2006; Sugita et al., 2009; Shiozaki et al., 2009). The complete sequence of the macronuclear genome of T. thermophila has been decoded, and there are 13 myosin genes, MYO1– 13 (Eisen et al., 2006). The cellular function of Myo1 has only been analyzed by one research group: that is, Gavin and colleagues at Brooklyn College of the City, University of New York. MYO1 knockout cells fail to form and transport phagosomes, to elongate the macronucleus and segregate macronuclear DNA during cell division, and to condense chromatin and acidify the old macronucleus during the destruction of the old macronucleus during mating (Williams et al., 2000; Hosein et al., 2005; Hosein and Gavin, 2007; Garcés et al, 2009). A myosin that forms a dimer and contains a long fibrous tail was purified from T. pyriformis (Kanzawa et al., 1996). Other Tetrahymena myosins have been little investigated, except at the level of the DNA sequence, and it is unclear whether these myosins are involved in actin-related cellular events. With regard to the classification of T. thermophila myosins, Williams and Gavin (2005) postulated that Myo1–12, but not Myo13, should be classified into a new class XX. In contrast, Foth et al. (2006) proposed that myosins from T. thermophila and the closely related Apicomplexa should belong to class XIV. Subsequently, on the base of an exhaustive analysis of even more genes, Odronitz and Kollmar (2007) concluded that myosins from T. thermophila and ciliate Paramecium tetraurelia could not be classified by current genome information. We supported their assertion, and considered that the characteristics of 13 myosins in T. thermophila should be explored further using full length cDNA sequences, which provide more precise information about the characteristics of myosins than that provided by genomic sequences alone. Here, we comprehensively determined the full cDNA sequences of 13 myosins in T. thermophila. We then elucidated detailed domain structures of all myosins, inferred the function of each myosin from the predicted amino acid sequences, and compared their mRNA expression levels in vegetative cells using quantitative real-time

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polymerase chain reaction (qRT-PCR). Finally, we classified the 13 T. thermophila myosins (Myo1–13) into 3 subclasses based on functional domains in the tail region. 2. Materials and methods 2.1. Preparation of cDNA from vegetative growing cells T. thermophila CH1 strain was cultured in Neff medium (1% protease peptone No. 3, 0.5% yeast extract, 0.87% D-glucose, and 3.3 mM FeCl2) in a shaking incubator at 30 °C. Cells (5 × 105 cells/ml) were collected by centrifugation for 5 min at 1100g. Total RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan), and then treated with RNase-free DNase (Stratagene, CA, USA) for 30 min at room temperature. First-strand cDNA was synthesized from the total RNA template using the Super Script™ first-strand synthesis system for RT-PCR (Invitrogen, Tokyo, Japan). PCR reactions were performed in a 50 μl solution containing 3 mM dNTP mix, 5.5 mM MgCl2, 1.5 μg random hexamers, 150 U Super Script™ II reverse transcriptase, and 200 μg total RNA. After synthesis, the cDNA was treated with RNase H for 20 min at 37 °C. Myosin cDNAs were amplified by PCR using primers designed from the predicted exon sequences in the Tetrahymena genome project database (http://www.lifesci.ucsb. edu/~genome/Tetrahymena/). Rapid amplification of cDNA ends (RACE) was performed using the SMART RACE cDNA Amplification kit (Clontech Laboratories, CA, USA). The sizes of the PCR products were confirmed by agarose gel electrophoresis, and the corresponding products were sequenced by Sanger's method using Terminator reaction ready mix (Applied Biosystems, Tokyo, Japan). 2.2. qRT-PCR Primers for qRT-PCR were designed using PRIMER3 (http://frodo. wi.mit.edu/cgi-bin/primer3/primer3_www.cg; Table S1). The specificity of primer sequences was confirmed by T. thermophila BLASTN searches (http://seq.ciliate.org/cgi-bin/blast-tgd.pl). qRT-PCR was performed using the Thermal Cycler Dice™ Real-Time System and a SYBR® Premix Ex Taq™ II kit (Takara Bio, Shiga, Japan). A 1 μl aliquot of the cDNA prepared as described above was used as the PCR template. The thermocycling conditions for the PCR reaction were as follows: initial denaturation at 95 °C for 10 s, followed by 40 cycles of denaturation (95 °C for 5 s) and annealing and extension (55 °C for 30 s). For each gene transcript, the relative transcript level compared to the Myo1 transcript was calculated by the ΔΔCt method. The specificity of the PCR was verified by denaturing curve analysis. 2.3. Domain analyses Amino acid sequences of myosins were compared using ClustalW sequence alignment program version 1.83 at DDBJ (http://clustalw. ddbj.nig.ac.jp/top-j.html). Functional domains were investigated using the Pfam protein families database version 24.0 at the Sanger institute (http://pfam.sanger.ac.uk/). The IQ motifs in the neck and tail region of myosins were identified using Pfam and the Calmodulin target database (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home. html) with default settings. Prediction of coiled-coil formation was calculated by using the MTIDK matrix in COILS server at EMBnet (http://www.ch.embnet.org/software/COILS_form.html). We judged that sequences with scores N0.7 in a matrix using a 28-residues scanning window were coiled-coil domains. 2.4. Phylogenetic tree A phylogenetic unrooted tree was developed using a neighborjoining algorithm and bootstrap analysis (1000 replicates) as implemented in ClustalW with default settings. The data were

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visualized using TreeView version 1.6.6 (http://taxonomy.zoology.gla. ac.uk/rod/treeview.html). 3. Results and Discussion 3.1. cDNA sequences and mRNA expression levels of T. thermophila myosin genes Williams and Gavin (2005) predicted the structures of T. thermophila myosins on the basis of genomic DNA sequences (Table 1). Because the study of cDNA sequences provides important information about protein structure, we prepared and sequenced cDNAs encoding Myo1–13 from total RNA in vegetative cells (Table 1; Fig. S1). Our ability to isolate cDNAs for each myosin suggests that all 13 myosin genes are not pseudogenes and that all 13 myosin genes are expressed together in vegetative cells. The molar ratios of Myo1– 13 mRNA transcripts were then estimated by qRT-PCR. Myo4, Myo8 and Myo10 were expressed at higher levels than Myo1 (Fig. 1). Myo2 showed the lowest expression level among the T. thermophila myosins (Fig. 1). The expression ratios of the T. thermophila myosins revealed in this study are supported by microarray data, derived from cells at the mid-log phase stage (3.5 × 105 cells/ml), in the Tetrahymena gene expression database (http://tged.ihb.ac.cn/Default.aspx), which became available in 2009. Several functional domains, such as the MyTH4, FERM and regulator of chromosome condensation 1 (RCC1) domains, were predicted from the primary structure based on the cDNA sequences of T. thermophila myosins (Table 1; Fig. 2) and their position in the head or neck and tail regions was determined. The head region is defined as the region from the N-terminus of the protein to the C-terminal of the motor domain. Because it is difficult to define the boundary between the neck and tail regions, we defined a combined neck and tail region as the region downstream of the head region through to the C-terminal of the protein (Fig. 2). When we made a comparison between our analysis of cDNA sequences and the previous report of genomic sequences (reviewed by Williams and Gavin, 2005), we found that our analysis had identified several new features in T. thermophila myosins, and provided a more accurate determination of predicted protein structure in several ways. Firstly, our data suggests that the previously reported predicted amino acid sequences of Myo3–13 are incorrect because of inaccuracies in the prediction of exon/intron boundaries in the Tetrahymena genome project database (Table 1; Fig. S1); our data differs from the previous report in the numbers of predicted residues in Myo3–13. Secondly, we located

Fig. 1. Expression levels of T. thermophila myosin mRNAs. The molar ratios of 12 myosin transcripts to the Myo1 transcript were estimated by the ΔΔCt method in qRT-PCR. Each value represents the mean ± SD of three independent tests.

several IQ motifs in each of the myosins, whereas the previous report found no IQ motif in Myo2, Myo4–7, Myo9 or Myo13 (Table 1; Fig. 2). Thirdly, we found 6–8 RCC1 domains in Myo3 and Myo10–12, whereas no RCC1 domains were identified in the previous study (Table 1; Fig. 2). Fourthly, we identified both MyTH4 and FERM domains in Myo1, Myo2, and Myo4–9, whereas the previous study reported that only Myo4 had a MyTH4 domain, only Myo7 had a FERM domain, and Myo6 and Myo8 did not have any functional domains in their tail regions (Table 1; Fig. 2). These discrepancies are probably caused by differences in the sequence information and search algorithms. We employed a hidden Markov model (HMM) (Finn et al., 2010) for the functional domain analyses, whereas Williams and Gavin (2005) employed BLAST2. HMM specializes in the motif recognition, whereas BLAST2 prioritizes processing speed over accuracy. In addition, we consider that a careful visual inspection of the sequence is important. We next describe the features of T. thermophila myosins in detail. 3.2. Motor domain We compared the primary amino acid sequences in the head regions of T. thermophila myosins with those of the typical myosins in classes I, II, V, VI and IX. In the motor regions of T. thermophila

Table 1 Myosin genes and their products in T. thermophila. Gene name

Accession no.

Deduced gene product in this study

MYO1 MYO2 MYO3 MYO4 MYO5 MYO6 MYO7 MYO8 MYO9 MYO10 MYO11 MYO12 MYO13

U87268 AB472065 AB472066 AB222209 AB472067 AB222077 AB472068 AB222079 AB222080 AB472069 AB472070 AB222078 AB221084

1810 1796 1876 1797 1639 1640 1783 1643 1586 1919 1963 1926 1357

Residues

a

IQ

b

5 5 13 5 5 7 5 4 4 5 10 12 6

CC 1 1 3 3 1 1 1 2 1 1 1 0 3

Deduced gene product in Williams and Gavin (2005) c

Domain

d

MyTH4/FERM MyTH4/FERM RCC1 MyTH4/FERM MyTH4/FERM MyTH4/FERM MyTH4/FERM MyTH4/FERM MyTH4/FERM RCC1 RCC1 RCC1 Coiled-coil

Residuesa

IQb

CCc

Domaind

1810 1796 1823 1705 1874 1568 1696 1560 1545 1928 1942 1800 1306

1 0 2 0 0 0 0 1 0 2 5 2 0

1 1 0 2 1 1 1 0 1 0 1 0 1

MyTH4/FERM MyTH4/FERM – MyTH4 MyTH4/FERM – FERM – MyTH4/FERM – – – Coiled-coil

The comparison of the basic information of T. thermophila myosins in this study with those in the report in Williams and Gavin (2005). a Number of predicted residues. b Number of IQ motifs and IQ-like sequences. c Number of coiled-coil domains. d Name of domain in the tails.

M. Sugita et al. / Gene 480 (2011) 10–20

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Fig. 2. Schematic diagrams of the functional domains in T. thermophila myosins. We investigated functional domains from amino acid sequences predicted from cDNA of 13 myosins in T. thermophila. Color keys to the domain names are given on the right. Asterisks on the top of RCC1 domains show the location of the RCC1's active center as a RanGEF. The dotted lines show our defined boundary between head and neck regions. The scale bar at bottom right indicates length of 500 amino acids.

myosins, the following regions (Fig. 3A) were well conserved: the switch I, switch II, and P-loop regions, which are required for ATP hydrolysis, the relay helix, which transmits the conformational change from the ATP hydrolysis region to the converter domain, and the actin-binding site (reviewed by Houdusse and Sweeney, 2001; Holmes et al., 2004). Myo10 had a 3 amino acids insertion in the switch I region. Furthermore, the glycine residue downstream of SH1 is an important residue for the function of converter domains (Kad et al., 2007). In Myo3, Myo6 and Myo8–10, the glycine residue was replaced by another residue, such as serine, threonine or methionine (Fig. 3A). It is currently difficult to predict the effect that the above insertions and substitutions would have on the motor activities of the myosins. There is no insertion between the converter and lever arm in any of the T. thermophila myosins. An insertion at this position is detected only in class VI myosin (Fig. 3A, class VI insertion 2), which is the only class myosin to move towards the minus end of F-actin (Ménétrey et al., 2005). Therefore, the absence of an insertion suggests that T. thermophila myosins is a plus-end directed motor like most major class myosins. It has been suggested that the diversity in the length and amino acid sequence of the loop 2 region contributes to the variety of motor activities in myosin classes because loop 2 is exposed on the surface of the motor near an actin-binding site (reviewed by Spudich, 1994). For example, class V myosin is able to associate to F-actin with a relatively high affinity through a positive charge in loop 2 even in the weak binding state of the head to actin; this may prevent diffusion of the head away from the F-actin and increase the degree of processive motion of class V myosin (Yengo and Sweeney, 2004). Moreover, although class IX myosin is single-headed, it can move as a processive motor due to a large insertion with a highly positive charge in loop 2 (Nalavadi et al., 2005; Table 2; Fig. 3A). The sequences of loop 2 in T. thermophila myosins were as diverse as those in other myosins (Fig. 3A). We calculated the electric charge of loop 2 in class I, II, V, IX and T. thermophila myosins according to the method of Yengo and Sweeney (2004) (Table 2). The electric charge of loop 2 in

T. thermophila myosins was similar to that of loop 2 in class I and II myosins, and different to the more highly positively charged loop 2 in class V and IX myosins. According to the charge in loop 2, it seems likely that T. thermophila myosins have a relatively low affinity to Factin and move as non-processive motors. 3.3. N-terminal additional sequence We found N-terminal additional sequences in all T. thermophila myosins except Myo9 (Fig. 2). Additional sequences in T. thermophila myosins except Myo13 are unidentified. The N-terminal additional sequence of Myo13 possessed a Src homology 3 (SH3)-like domain, which has also been detected in several members of the myosin family: i.e., class II, V, VI, VII, X and other class myosins (Fig. 3B; Odronitz and Kollmar, 2007). The function of the SH3-like domain has been only partially clarified. The N-terminal SH3-like domain in class II myosin acts in protein–protein interactions and associates with the motor domain to maintain enzymatic activity. The SH3-like domain in class II myosin is near the essential light chain, and it is suggested that the domain functions by being in close contact with two heads of the myosin (van Duffelen et al., 2005; Fujita-Becker et al., 2006). It seems likely that the SH3-like domain in Myo13 affects the activity of the motor in a similar way to the SH3-like domain in class II myosin. 3.4. IQ motif The myosin light chains, which include calmodulin or other related Ca2+-binding EF-hand proteins, regulate the activity of the myosin heavy chain by binding to IQ motifs in the neck region. There is good correspondence among the number of IQ motifs, the lever arm length and the size of the working stroke (Rock et al., 2005; Sakamoto et al., 2005). We therefore investigated the IQ motif [(I/L/V/F/M)QXXX(R/K) GXXX(R/K)XX(I/L/V/F/W/Y), where X denotes any amino acid] in T. thermophila myosins using the Pfam protein families database and the Calmodulin target database. IQ motifs are categorized into two types

14 M. Sugita et al. / Gene 480 (2011) 10–20 Fig. 3. Sequence alignment of the head regions in major class myosins and T. thermophila myosins. (A) Alignment of motor domains in major class myosins and T. thermophila myosins. Gray boxes indicate conserved regions, and the rectangular frame shows glycine residues near the SH1 helix. (B) Alignment of the N-terminal SH3-like domains in Myo13 and class II myosins. Gray boxes show conserved residues. The meaning of the abbreviations are as follows: Hs, Homo sapiens; Dd, Dictyostelium discoideum; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; Ss, Sus scrofa; Dr, Danio rerio; Ci, Ciona intestinalis; Ce, Caenorhabditis elegans; Tt, Tetrahymena thermophila; M, muscle; SM, smooth muscle; and NM, non-muscle. The GenBank accession numbers are as follows: Hs M, NP_000248; Hs SM, NP_001035202; Hs NM, NP_005955; DdMhc2, XP_637740; SpMyo2, NP_588114; HsMyoIA, NP_005370; HsMyoIB, NP_001123630; HsMyoIC, NP_001074248; SpMyo1, NP_595402; HsMyo5A, NP_000250; DmMyoV, NP_477186; SpMyo51, NP_596233; HsMyo6, NP_004990; Ss Myo6, NP_999186; DrMyo6, NP_001004110; CiMyo6, XP_002124197; DmMyo6, NP_524478; HsMyoIX, AAI40870; and CeMyo9, NP_490755.

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Fig. 3 (continued).

M. Sugita et al. / Gene 480 (2011) 10–20

M. Sugita et al. / Gene 480 (2011) 10–20

Fig. 3 (continued).

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M. Sugita et al. / Gene 480 (2011) 10–20

17

Fig. 3 (continued).

according to their degree of conservation: a complete IQ motif conserves GXXX(R/K)XX(I/L/V/F/W/Y), whereas an IQ-like sequence does not fully conserve this motif. The complete IQ motif can bind to calmodulin without Ca2+, whereas the IQ-like sequence binds to calmodulin in the calcium-dependent manner (Houdusse and Cohen, 1995; Munshi et al., 1996). We identified one complete IQ motif in Myo3 and three complete IQ motifs in Myo12; all were in the neck and tail regions of their respective myosins (Fig. 2; Fig. S1). In contrast, IQlike sequences were present not only in the neck and tail region but also in a head region of most T. thermophila myosins (Fig. 2; Fig. S1). It is unlikely that the IQ-like sequences in head regions function properly as light chain-binding sites because these regions are not exposed on the surface. In the neck and tail region, we found 2 IQ-like sequences in Myo1, Myo4, and Myo7, 3 sequences in Myo2, Myo8,

Myo9, and Myo13, 4 sequences in Myo10, 5 sequences in Myo5 and Myo6, 9 sequences in Myo3 and Myo11, and 10 sequences in Myo12. Class V myosin has light chains comprised of calmodulin. In class V myosin, calmodulin binds to IQ motifs depending on Ca2+ concentration, because each IQ motif has a slightly different sequence allowing for a variety of calcium-dependent properties. The activity of class V myosin is regulated by the number of calmodulin binding to IQ motifs (reviewed by Taylor, 2007). It is possible that Ca2+-calmodulin binds to the IQ motifs of T. thermophila myosins and regulates the motor activities, because T. thermophila myosins have different numbers of complete IQ motifs and IQ-like sequences. Gonda et al. (1999) reported that T. thermophila calmodulin is localized to a cleavage furrow during cytokinesis, and Ca2+-calmodulin regulates the formation of the contractile ring. Thus, it is possible that calmodulin acts as a myosin light chain and interacts with myosin in a contractile ring in T. thermophila.

Table 2 Electric charges in loop 2.

3.5. Three subclasses of T. thermophila myosins

Class

Name of myosin

Positivea

Negativeb

Scorec

I

Hs MyoIA Hs MyoIB Hs MyoIC Sp Myo1 Hs Muscle Hs Smooth Muscle Hs Non Muscle Dd Mhc2 Sp Myo2 Hs Myo5A Dm MyoV Sp Myo51 Hs MyoIXA Ce Myo9 Tt Myo1 Tt Myo2 Tt Myo4 Tt Myo5 Tt Myo6 Tt Myo7 Tt Myo8 Tt Myo9 Tt Myo3 Tt Myo10 Tt Myo11 Tt Myo12 Tt Myo13

3 3 4 3 5 7 6 3 6 9 7 5 36 24 3 7 6 6 2 4 4 2 5 4 2 2 6

1 1 4 2 2 4 4 1 3 4 4 1 17 10 2 5 3 3 2 3 3 3 3 5 2 2 3

+2 +2 0 +1 +3 +3 +2 +2 +3 +5 +3 +4 + 19 + 14 +1 +2 +3 +3 0 +1 +1 -1 +2 -1 0 0 +3

II

V

IX Subclass 1 (MyTH4/FERM)

Subclass 2 (RCC1)

Subclass 3 (Coiled-coil)

The charges in loop 2 in each class were estimated by the method of Yengo and Sweeney (2004). The meanings of the abbreviations are as follows: Hs, Homo sapiens; Sp, Schizosaccharomyces pombe; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; and Tt, Tetrahymena thermophila. The GenBank accession numbers are as follows: HsMyoIA, NP_005370; HsMyoIB, NP_001123630; HsMyoIC, NP_001074248; SpMyo1, NP_595402; Hs M, NP_000248; Hs SM, NP_001035202; Hs NM, NP_005955; DdMhc2, XP_637740; SpMyo2, NP_588114; HsMyo5A, NP_000250; DmMyoV, NP_477186; SpMyo51, NP_596233; HsMyoIX, AAI40870; and CeMyo9, NP_490755. a Number of residues with positive charge (arginine and lysine). b Number of residues with negative charge (asparaginic acid and glutamic acid). c The score of a minus b.

Domain analyses using Pfam and the COILS database of coiled-coil proteins indicated that T. thermophila myosins could be classified into the following three subclasses on the basis of functional domains in the tail region: subclass 1 contained both MyTH4 and FERM domains and was comprised of Myo1, Myo2 and Myo4–9; subclass 2 contained the RCC1 domain and was comprised of Myo3 and Myo10–12; and subclass 3 contained the long coiled-coil domain and was comprised of Myo13 alone (Table 1; Fig. 2). 3.5.1. Myosins containing MyTH4 and FERM domains (subclass 1) MyTH4 and FERM domains have been identified in class VII, X, XII, XV and other myosins in animals and some protists, but not in yeast and higher plants (Foth et al., 2006). In the protist Dictyostelium discoideum, class VII myosin binds to the plasma membrane through the FERM domain and functions in phagocytosis by forming an anchor between the actin cytoskeleton and the membrane (Titus, 1999; Tuxworth et al., 2001). In T. thermophila, it has been suggested that Myo1, which contains MyTH4 and FERM domains, is involved in the intercellular transport of phagosomes, and it has been predicted that the FERM domain binds to the phagosome membrane (Hosein et al., 2005; Hosein and Gavin, 2007). Because Myo1 gene disruption does not completely suppress the formation and transport of phagosomes (Williams et al., 2000), we suggest that the function of Myo1 may overlap with that of other subclass 1 myosins (i.e., Myo2 and Myo4–9). Thus, the subclass 1 myosins may have the potential to regulate membrane dynamics. The fact that T. thermophila possesses 8 myosins in subclass 1 may be closely related to the frequent vesicular transport and membrane-anchoring processes that occur in T. thermophila. In addition, all subclass 1 myosins were expressed in vegetative cells. Therefore, the redundancy of subclass 1 myosins suggests that some of the subclass 1 myosins can substitute for each other functionally, and that each subclass 1 myosin may divide up the role of membrane dynamics. We speculate that Myo8, in particular, has an important role in the process of membrane

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dynamics, because Myo8 shows the highest mRNA expression level among subclass 1 myosins (Fig. 1). 3.5.2. Myosins containing RCC1 domains (subclass 2) Myosins containing RCC1 domains have been found only in Alveolata, such as T. thermophila, P. tetraurelia (Odronitz and Kollmar, 2007), and 6 species of Apicomplexa (Foth et al., 2006). The RCC1 domain was first identified in the RCC1 protein (Nishimoto et al., 1978; Ohtsubo et al., 1987). The RCC1 protein, which contains RCC1 domains and a nuclear localization signal, can bind to a small GTPbinding protein such as Ras-related nuclear protein (Ran), and can function as a guanine nucleotide exchange factor (GEF) (Renault et al., 1998). The RCC1 domain forms a blade and 5–7 blades form a β-propeller structure. The RCC1 protein has 7 RCC1 domains, which together form the 7 blades and one of the 7 blades has an active center for RanGEF activity (Renault et al., 1998; reviewed by Hadjebi et al., 2008). In subclass 2 myosins (i.e., Myo3 and Myo10–12), the tandem repeat of 6–8 RCC1 domains suggests that the domains may form a β-propeller structure consisting of 6–8 blades, and the second or third RCC1 domain in the tandem repeat contains an active center for RanGEF activity (Fig. 2; Fig. S1). It is likely that the RCC1 domains of subclass 2 myosins can work as RanGEFs because the domains have a sufficient number of blades to form a β-propeller and an active center for RanGEF activity. Ran regulates mitotic spindle assembly and nuclear transport of RNAs and proteins through nuclear pore complexes (reviewed by Clarke and Zhang, 2008). If the RCC1 domains of subclass 2 myosins function as RanGEFs, these myosins may play roles in mitosis and nuclear transport as novel nuclear

myosins. In addition, subclass 2 myosins do not have a clear coiledcoil domain for dimerization (Table 1; Fig. 2). Therefore, these myosins probably work as a monomer like class I or class IX myosins. Myo10 may be of particular importance because it shows the highest mRNA expression level among subclass 2 myosins. Experiments involving Myo10 gene disruption should help to elucidate the functions of subclass 2 myosin. 3.5.3. Myosin containing long coiled-coil domain (subclass 3) Myo13 contains three coiled-coil domains in the tail, the longest of which is 185 amino acids in length. Myo13 has a coiled-coil domain in the region immediately downstream of its neck region, suggesting that Myo13 forms a dimer. If Myo13 binds in an antiparallel fashion through C-terminal coiled-coil domains and forms a bipolar tetramer, Myo13 may generate a contractile force by hauling actin filaments. Relatively long coiled-coil domains are found in several myosins such as MyoA and MyoB in the ciliate P. tetraurelia (Odronitz and Kollmar, 2007). Further studies using gene knockout techniques to investigate the role of Myo13 in cell motility may shed light on the functions of this myosin. The mRNA expression level of Myo13 was one-thirds that of Myo1 (Fig. 1). Because subclass 3 containing a long coiled-coil domain consists only of Myo13, we speculate that Myo13 might have an important role in T. thermophila. 3.6. Genetic relationship between subclasses Differences in the features in the tail region of myosins are commonly associated with differences among their motors (Richards

Fig. 4. Phylogenetic tree produced from the amino acid sequences of the motor domains in T. thermophila myosins and members of the major classes of myosins in various species. The values in the tree show the number of bootstrap operations. The meanings of the abbreviations are as follows: Hs, Homo sapiens; Dd, Dictyostelium discoideum; Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; Ss, Sus scrofa; Ci, Ciona intestinalis; Ce, Caenorhabditis elegans; Tt, Tetrahymena thermophila; Pt, Paramecium tetraurelia; M, muscle; SM, smooth muscle; and NM, non-muscle. The GenBank accession numbers are as follows: Hs M, NP_000248; Hs SM, NP_001035202; Hs NM, NP_005955; DdMhc2, XP_637740; SpMyo2, NP_588114; HsMyoIA, NP_005370; HsMyoIB, NP_001123630; HsMyoIC, NP_001074248; SpMyo1, NP_595402; HsMyo5A, NP_000250; DmMyoV, NP_477186; SpMyo51, NP_596233; HsMyo6, NP_004990; SsMyo6, NP_999186; DrMyo6, NP_001004110; CiMyo6, XP_002124197; DmMyo6, NP_524478; HsMyoIX, AAI40870; and CeMyo9, NP_490755. The dataset of P. tetraurelia myosins (MyoA, MyoB, MyoC, MyoD, MyoE and MyoF) was obtained from Odronitz and Kollmar (2007).

M. Sugita et al. / Gene 480 (2011) 10–20

and Cavalier-Smith, 2005). Here, T. thermophila myosins were divided into three subclasses by the features of the tail region such as MyTH4 and FERM, RCC1 and coiled-coil domains. As a result of the phylogenetic analysis by sequence alignment of motor domains, T. thermophila myosins were separated from the clusters of other typical myosin classes. In addition, T. thermophila myosins were further separated into two clusters (Fig. 4): one consists of T. thermophila subclass 1 and subclass 2 myosins, and the other consists of Myo13, the T. thermophila subclass 3 myosin. Myo13 belongs to same cluster as P. tetraurelia MyoA and MyoB (Fig. 4). Even though these three myosins are phylogenetically distinct from class II myosins, they may share a similar function to class II myosin because of their long coiledcoil domains. We speculate that the taxonomic position of T. thermophila myosins should be explored by further studies of full length cDNA sequences in a wide range of organisms. We anticipate that these phylogenetic relationships will become even more defined as the cDNA sequences of more myosins from a variety of protists such as Stramenopile and Excavata become available. 4. Conclusion We investigated the mRNA expression levels and primary structural features of 13 myosins in the ciliate T. thermophila. Based on cDNA sequences of T. thermophila myosins, we identified several new features in these myosins, and provided more accurate predicted myosin structures than those in a previous study which relied on genome information. The results indicate that T. thermophila myosins can be classified into the following three groups: the MyTH4/FERM subclass (subclass 1), which may function in membrane dynamics; the RCC1 subclass (subclass 2), which is detected only in Alveolata and may have a unique function; and the coiledcoil subclass (subclass 3), which may form a bipolar tetramer and may generate a contractile force with actin filaments. The phylogenetic analysis on the basis of motor domains showed that T. thermophila myosins are separated into two clusters. Subclasses 1 and 2 belong to the same cluster, and subclass 3 belongs to the other cluster. Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2011.02.006. Acknowledgments We are very grateful to Dr. H. Kuwayama and Ms. M. Kunitani (University of Tsukuba) for technical support in the qRT-PCR experiments, and Dr. N. Kanzawa (Sophia University) for useful discussions. We also thank Mr. T. Nakahara (University of Tsukuba) for support in the phylogenetic analyses, and other members of our laboratory for useful discussions. This research was supported by a grant from the Sumitomo Foundation to K.N. and by the Japan Society for the Promotion of Science (JSPS) Research Fellowship for Young Scientists granted to M.S. References Bähler, M., 2000. Are class III and class IX myosins motorized signalling molecules? Biochim. Biophys. Acta 1496, 52–59. Clarke, P.R., Zhang, C., 2008. Spatial and temporal coordination of mitosis by Ran GTPase. Nat. Rev. Mol. Cell Biol. 9, 464–477. Edamatsu, M., Hirono, M., Watanabe, Y., 1992. Tetrahymena profilin is localized in the division furrow. J. Biochem. 112, 637–642. Eisen, J.A., et al., 2006. Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol. 4, 1620–1642. Etournay, R., Zwaenepoel, I., Perfettini, I., Legrain, P., Petit, C., El-Amraoui, A., 2007. Shroom2, a myosin-VIIa- and actin-binding protein, directly interacts with ZO-1 at tight junctions. J. Cell Sci. 120, 2838–2850. Finn, R.D., et al., 2010. The Pfam protein families database. Nucleic Acids Res. 38, 211–222. Foth, B.J., Goedecke, M.C., Soldati, D., 2006. New insights into myosin evolution and classification. Proc. Natl. Acad. Sci. USA 103, 3681–3686.

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