Correspondence to: Thomas D. Pollard, The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA 92037. Tel:(858) 453-4100, ext
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Abstract |
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Fission yeast myo1+ encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1+ is not lethal, myo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1+ is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all
myo1 defects. However, it fails to rescue the
myo1
wsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.
Key Words: fission yeast, myosin-I, WASp, Arp2/3 complex, actin assembly
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Introduction |
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Myosin-I is an actin-dependent membrane-based molecular motor. Many organisms express multiple myosin-Is with similar catalytic domains but different tails containing binding sites for membrane lipids and a variety of proteins. These tail-ligand interactions are postulated to target myosin-I isoforms to various intracellular locations and/or to adapt them to different actin-dependent processes such as motility, endocytosis, phagocytosis and polarized cell growth (
The catalytic domain of all myosin-Is is followed by a region with one to five light-chain binding (IQ)1 motifs, and a basic tail domain called tail homology 1 (TH1) that binds acidic phospholipids (
The COOH terminus of WASp/Scar proteins, containing an acidic A-domain, stimulates actin assembly by interacting with Arp2/3 complex. In animal cells, WASp/Scar proteins provide a link to signaling molecules affecting the actin cytoskeleton (reviewed by
Before any knowledge of this work on budding yeast myosin-Is, we identified a WASp-like acidic A-domain at the COOH terminus of Myo1p, a myosin-I from Schizosaccharomyces pombe. Here we show with quantitative assays that Myo1p tail binds to and stimulates nucleation activity of Arp2/3 complex. Consistent with a role in regulating the actin cytoskeleton, disruption of myo1+ causes abnormal morphology due to depolarization of actin patches. Unlike budding yeast and other known long-tailed myosin-Is, all defects associated with loss of Myo1p were rescued by a construct containing only the head and TH1 domain. This construct was also sufficient for localization to cortical patches. Although deletion of myo1+ is not lethal, additional disruption of the COOH terminus of Wsp1p, a fission yeast WASp homologue, results in synthetic lethality. Myo1p, Wsp1p, or Myo1p lacking the A-domain rescues this lethality. This differs from budding yeast, where deletion of A-domains from WASp and myosin-I resulted in severe growth defects. Interestingly, Myo1p lacking the TH3 and A-domains, a construct that fully complements loss of Myo1p, fails to rescue the absence of both A-domains from Wsp1p and Myo1p, implicating the TH3 domain in myosin-Imediated Arp2/3 complex activation in fission yeast. Our results suggest that in cooperation with Wsp1p, Myo1p directly regulates actin assembly.
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Materials and Methods |
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Strains, Media, and Transformation
Table 1 lists the S. pombe strains used in this study. Fission yeast culture and genetic manipulations were carried out by standard methods. Transformation of S. pombe was achieved by electroporation (
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Identification and Cloning of myo1+ and wsp1+
Three uncharacterized myosin heavy chain genes in the Sanger database were identified by blasting (
We cloned a 5-kb EcoRI fragment containing the myo1+ locus from SPBC146 into pBluescript. This construct is pBSmyo1. Total RNA from mid-log wild-type cells was amplified by 5'-RACE PCR (Life Technologies) and the products were subcloned and sequenced.
We identified a WASp-like gene in cosmid SPAC4F10. This wsp1+ gene was previously submitted to Genbank (GenBank/EMBL/DDBJ accession number AAB92587). We identified exons in wsp1+ by comparing its sequence with other WASp sequences and searching for the usually conserved 5' (GTAC) and 3' (CCAG) splice sites. We amplified a 3-kb fragment containing the entire wsp1+ locus from genomic DNA, cloned it into pBluescript, and sequenced it. This construct is pBSwsp1.
Construction of myo1+ or wsp1+ Disruption Strains
Integration of linear myo1+ and wsp1+ disruption constructs (see Fig 1 A and 6 A) into his3-D1 or leu1-32 wild-type diploid cells was screened by PCR. Stable transformed diploids were sporulated on malt extract and individual spores were dissected from tetrads and germinated on Yeast Extract Supplemented (YES). In either disruption, four viable spores were obtained, of which two were His+ or Leu+ containing the disrupted allele. We verified disruptions in viable haploids by PCR and Southern blot. Disruption of wsp1+ allows expression of only the NH2-terminal 346 residues, since an in-frame stop codon was created at the 5' ligation site of the leu+ insert.
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The myo1+ locus is flanked 5' by a coatomer beta subunit gene and 3' closely by a ubiquinone biosynthesis monooxygenase gene. The coatomer beta subunit gene has the same transcriptional orientation as myo1+, but the ubiquinone monooxygenase gene is in opposite orientation. Disruption of myo1+ by removing an NdeI-SalI fragment was lethal and could not be rescued by transforming plasmids carrying full-length myo1+ gene (pUR-myo1 or pSGP-myo1). This NdeI-SalI fragment includes sequences coding for residues 2491217 and 179 bp of 3' untranslated region of myo1+ gene, leaving the open reading frame of the downstream ubiquinone monooxygenase gene intact. Since we were unable to rescue this myo1+ disruption with complementing plasmids, we concluded that the function of the downstream ubiquinone monooxygenase gene was most likely affected, perhaps at the level of transcript stability.
Construction of Expression and Complementation Constructs
We used sequences of other myosin-Is for which tail domains have been defined proteolytically (
We made constructs expressing mutant Myo1p with COOH-terminal deletions designed to integrate at the puc1+ locus. We cloned a NotI fragment containing S. pombe puc1+ (myo1 cells were transformed with these integrating constructs linearized by XhoI in the middle of the puc1+. Stable Ura+ transformants were selected and streaked to permissive and nonpermissive conditions.
pUR19-wsp1 was made by subcloning wsp1+ from pBSwsp1 into a unique EcoRI site in pUR19, an ars1-containing fission yeast vector that carries the ura4+ marker (
Microscopy
Cells were stained with calcofluor (
Actin Polymerization and Binding Assays
Actin was purified from rabbit skeletal muscle (
Online Supplemental Material
The online version of this article includes detailed methods used in constructing myo1+ and wsp1+ strains and two additional figures showing phylogenetic analysis of myosins, alignment of IQ motifs, 5' RACE PCR of myo1+ and latrunculin-A disruption of GFP-Myo1p patches. Available at http://www.jcb.org/cgi/content/full/151/4/789/DC1
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Results |
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Identification of the myo1+ Gene
As of August 2000, the S. pombe genome contained five genes with significant homology to Acanthamoeba myosin-IA catalytic domain. Of these five, two were previously characterized myosin-IIs called myo2+ and myp2+ (
Myo1p and other known fungal myosin-Is have two similar IQ motifs, especially the first (Figure S1). Beyond the IQ motifs, Myo1p is a typical long-tailed myosin-I with the addition of a COOH-terminal A-domain. The basic TH1 domain has a calculated pI of 10. The TH2 domain is rich in Pro (20%), Ala (18%), Ser (12%), and Thr (11%). Abundant Ser and Thr in TH2 are unusual, found only in S. cerevisiae Myo3p and Myo5p, but not in other long-tailed myosin-Is. The A-domain is found on all fungal myosin-Is reported so far, but not on animal or protozoa myosin-Is. Two independent 5'-RACE products obtained from total S. pombe RNA using different sets of antisense and nested primers (Fig 1 A, primers 79) established the presence of a 44-bp intron separating the ATG codon from the remaining coding sequence. myo1+ transcript begins 43 bp upstream of the ATG.
Targeted Disruption of myo1+
We disrupted myo1+ by replacing >50% of the catalytic domain and the first IQ motif with the his3+ gene (Fig 1 A). This disruption allowed expression of only the NH2-terminal 248 residues. Amplification with primers inside the his3+ gene and primers outside the disruption construct verified that myo1+ was disrupted in a His+ diploid (Fig 1 A, primers 1+3, 2+3, 4+6, and 4+5). All four progeny of this diploid were viable in YES at 25°C. Of these four colonies, two were His+ (myo1::his3+) and always smaller in size. In liquid Edinburgh minimal media (EMM) at 25°C,
myo1 cells grew with a doubling time of 12.5 h (n = 2) compared with 6.1 h (n = 4) for wild-type. These mid-log
myo1 cells had aberrant morphology: 16% were round, slightly swollen, or irregularly shaped (Table II; Fig 1 C); 13.7% had abnormal septal material (Fig 1 B); and 37% had a septum, which was generally abnormally thick. Actin patches were delocalized in aberrantly shaped
myo1 cells (Fig 1 C, arrow), but those with rod shapes usually had polarized actin patches at growing ends or an actin ring in the middle of dividing cells.
myo1 cells failed to form colonies at 17°C, 36°C, or, in the presence of 1 M KCl, at 25°C, where they died swollen, branched, abnormally shaped, and lysed. This terminal phenotype developed within 5 h after shifting from 25° to 36°C (Fig 1B and Fig C) or to 1 M KCl at 25°C (not shown) in liquid EMM-His. Less than 1% of wild-type cells had abnormal morphology under these conditions (Table 2). Calcofluor staining revealed the most striking effects of myo1+ deletion in cells shifted to 36°C. More than half of
myo1 cells (Table II; Fig 1 B) had severely thickened septa, and abnormal septal material at one end, on one side, or all around the cell. Even those
myo1 cells with normal rod morphology often had abnormally placed septal material. At 36°C,
myo1 cells with abnormal morphology had no actin patches (Fig 1 C). Since DAPI stained the nuclei of these fixed cells (not shown), the absence of actin patches was not likely due to failure of rhodamine-phalloidin to penetrate the cell wall or plasma membrane. Wild-type cells maintained normal septal deposition and polarized actin patches at 36°C.
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Mating was defective in myo1 cells. When crossed with wild-type cells of opposite mating type on malt extract agar, zygotes were rare. Iodine vapor also revealed same coloring as nonmated controls, indicating that
myo1 mated very inefficiently.
Complementation of myo1 Phenotypes and Localization of GFP-Myo1p
To evaluate the function of the tail domains of Myo1p, we integrated constructs expressing full-length Myo1p (H/1/2/3/A) or Myo1p with COOH-terminal deletions (H/1/2/3, H/1/2, H/1, and H) under control of the native myo1+ promoter into myo1 cells. All constructs, except H, restored growth at 17° (not shown), 36°, and 25°C with 1 M KCl (Fig 2 A).
myo1 cells transformed with construct H were indistinguishable from cells transformed with empty vector. Similarly, all constructs, except H, rescued
myo1 mating defects assessed by iodine vapor staining. Thus, the head plus TH1 are the minimum domains needed to complement the absence of functional myo1+.
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GFP-Myo1p and GFP-H/1 restored wild-type growth to myo1 cells at 36° or 25°C with 1 M KCl (Fig 2B and Fig C), showing that the GFP tag did not interfere with function. In the presence of thiamine, where they were expressed at a low level, as verified by microscopy, transformants had near wild-type morphology and colony size. In contrast, GFP fusions of head or tail alone (GFP-H, GFP-1, GFP-1/2/3/A, and GFP-2/3/A) failed to complement
myo1 defects.
Full-length GFP-Myo1p localized to patches in wild-type as well as myo1 cells. Three-dimensional reconstructions made by deconvolution microscopy showed that all GFP-Myo1p patches were located at the periphery of living cells (Fig 3B and Fig C), so they appeared in different focal planes by conventional fluorescence microscopy (Fig 3 A). Like actin patches, these Myo1p patches usually concentrated at both growing ends or in the middle of dividing cells, and were dynamic, since we observed them moving along the cell cortex. We found that GFP-Myo1p patches partially colocalized with actin patches (Fig 3D and Fig E). Staining of GFP-Myo1pexpressing cells with rhodamine-phalloidin revealed that
25% of patches contained only GFP-Myo1p (green patches) and
15% contained only actin (red patches). Approximately 60% of patches contained a variable ratio of actin and GFP-Myo1p, since these patches ranged in color from yellow to orange (350 patches counted). Latrunculin-A reversibly dispersed GFP-Myo1p from patches to a diffuse cytoplasmic fluorescence, indicating that cortical localization of Myo1p depended on intact actin filaments. Expression levels from the nmt1+ promoter varied from cell to cell, producing patches of different intensities but otherwise indistinguishable. Overexpression of GFP-Myo1p, while not toxic, produced uniform fluorescence throughout the cell periphery and large fluorescent aggregates in the cytoplasm. We conclude that expression of GFP-Myo1p at low levels mimics endogenous Myo1p localization.
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GFP-H/1, a construct that rescued myo1 defects, localized to discrete patches similar to GFP-Myo1p in
myo1 (Fig 4) and wild-type cells. However, the level of expression was more variable from cell to cell and cytoplasmic fluorescence was greater for GFP-H/1 than for GFP-Myo1p. GFP fusions that failed to complement
myo1 were either mislocalized to the nucleus (Fig 4, GFP-1 and GFP-1/2/3/A), aggregated (GFP-H), or diffuse in the cytoplasm (GFP-2/3/A). Nuclear localization of GFP-1 and GFP-1/2/3/A were confirmed by staining with DAPI.
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Myo1p Tail Binds and Activates Arp2/3 Complex
The sequence of Myo1p A-domain is similar to A-domains of WASp/Scar proteins that bind Arp2/3 complex (Fig 5 A). Among known myosin tails, only fungal myosin-Is have A-domains. Two assays established that Myo1p tail interacts with Arp2/3 complex. In a supernatant depletion assay, purified amoeba Arp2/3 complex bound a fusion protein GST-2/3/A (GST fused to the NH2 terminus of TH2/3/A-domains of Myo1p) immobilized on beads with a Kd of 5 µM (Fig 5 B). Control glutathione beads did not deplete Arp2/3 complex from the supernatant.
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Purified GST-2/3/A stimulated actin filament nucleation by amoeba (Fig 5 C) and bovine Arp2/3 complex (not shown). GST alone did not promote actin polymerization by Arp2/3 complex. Separately, neither GST-2/3/A nor the Arp2/3 complex had an appreciable effect on the time course of spontaneous polymerization, but together they reduced the lag at the outset of polymerization up to threefold (not shown) and generated ninefold more filament ends (Fig 5 D). These effects plateaued at concentrations of GST-2/3/A > 1 µM. GST-2/3/A had lower affinity and activity than the GST-WA-domain of WASp (
GST-2/3/A did not pellet with muscle actin filaments in actin polymerization buffer (not shown). Based on the concentrations used, the minimum value of the Kd for GST-2/3/A binding muscle actin filaments is 20 µM.
Targeted Disruption of wsp1+ and Its Genetic Interaction with myo1+
We identified a WASp-like gene, wsp1+, in the Sanger database as a potential activator of Arp2/3 complex. The genomic sequence of wsp1+ has four exons encoding domains similar to WASp proteins: WH1 domain, a polyproline region, WH2 domain, and an acidic A-domain, but no GBD/CRIB sequence that might bind Cdc42p.
We made a wsp1+ disruption strain (wsp1), which would produce a COOH-terminally truncated Wsp1p protein (Fig 6 A). Tetrad dissection of individual asci and random spore analysis of a
wsp1/wsp1+ diploid revealed that COOH-terminal truncation of Wsp1p is not lethal. Amplification with specific primers from the genomic locus verified that wsp1+ was disrupted in Leu+ haploids (Fig 6 A).
wsp1 cells formed smaller colonies than wild-type in selective EMM, were sensitive to 1 M KCl (Fig 6 B) and mated inefficiently. A genomic clone of wsp1+ (pUR19-wsp1) fully complemented the salt phenotype and mating defects. Like
myo1 cells,
wsp1 cells had depolarized actin patches and morphological defects (Fig 6 C). Disruption of wsp1+ did not cause faulty targeting of septal material, but mid-log
wsp1 cells grown at 32°C did have more uniseptated cells than wild-type (Table II; Fig 6 C). The lack of aberrant septal targeting suggests that, unlike Myo1p, Wsp1p is not involved in proper septal deposition.
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To test for genetic interactions between wsp1+ and myo1+, we crossed wsp1 with
myo1 cells, by transforming each strain first with a complementing plasmid before mating (pUR-wsp1 for
wsp1, pSGP573-myo1 for
myo1). The progeny from the cross were examined by random spore analysis after germination at 25°C. Marker analysis of 657 progeny indicated that wild-type (250, His-, and Leu-),
myo1 (192 His+), and
wsp1 (215 Leu+) spores could be recovered. No spores carrying disruption of myo1+ and wsp1+ (0 His+ and Leu+) were recovered, so at the permissive temperature for both disruptions,
wsp1 is synthetically lethal with
myo1.
To test for functional redundancy between the tail of Myo1p and Wsp1p, we crossed myo1 cells containing integrated Myo1p mutants (H/1/2/3/A, H/1/2/3, H/1/2, H/1, and H) with
wsp1 cells carrying a modified pUR-wsp1. We added the kanamycin resistance gene to pUR-wsp1 (= pUR-wsp1/Kanr) to identify progeny carrying ura4+-marked wsp1+ in the presence of ura4+-marked Myo1p mutants. The progeny from these crosses were examined by random spore analysis after germination at 25°C in YES. We analyzed >400 progeny for each cross. Progeny carrying disruption of myo1+ and wsp1+ in the presence of integrated H/1/2/3/A or H/1/2/3 were recovered with expected mendelian segregation and independent of pUR-wsp1/Kanr. H/1/2, H/1, or H progeny carrying both disruptions were always kanamycin resistant, indicating the presence of wsp1+, and were recovered with significantly lower than expected frequency. We conclude that H/1/2/3/A and H/1/2/3, but not H/1/2, H/1 or H, rescue the lethality of
myo1
wsp1.
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Discussion |
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Judging from the available sequence information (95%) at the Sanger Genome Center, fission yeast potentially contains only one type-I myosin gene, making it attractive for detailed analysis of myosin-I functions. Multiple myosin-I genes in other organisms are interesting in terms of their specialized functions, but have been a burden experimentally in studies of basic functions.
Functions of Myo1p
Deletion of myo1+ causes defects in cell morphology and actin organization. Nonpermissive conditions greatly enhance these defects, leading to branched and rounded cells and eventually to cell death. Fission yeast cells grow in a polarized fashion, using the actin cytoskeleton to deliver essential materials to the growing ends of cells. The morphology of myo1 cells suggests that Myo1p has a role in regulating the sites of polarized growth, like the S. pombe tea and orb gene products (
Consistent with a role in regulating polarized growth, Myo1p and Pak1p/Shk1p localize to growing ends. Localization of Myo1p depends on an intact actin cytoskeleton. However, not all Myo1p-containing patches are actin patches and not all actin patches contain Myo1p (Fig 3D and Fig E). This intriguing observation indicates that there are distinct populations of patches that vary in molecular composition. If Myo1p is a target of Pak1p/Shk1p, it would be interesting to investigate whether these Myo1p-containing actin-deficient patches also contain Pak1p/Shk1p. Genetic interactions between pak1+/shk1+ and myo1+, phosphorylation of Myo1p by Pak1p/Shk1p, and localization of both proteins will be the subject of future work. We expect that Myo1p is part of a complicated and redundant system of proteins establishing polarity, since mutation in a variety of genes (for example, tea, orb, and now wsp1+) clearly exhibit similar phenotypes.
Like Pak1p/Shk1p (-factorinduced mating projections (
Cells lacking Myo1p accumulate septal components abnormally, especially at high temperature (Fig 1 B) and in high salt. Even under permissive conditions, most myo1 cells have a thick septum, and twice the number of
myo1 cells have septa compared with wild-type. This function of Myo1p may be distinct from its role in maintaining cell shape, as many
myo1 cells with normal rod morphology deposit cell wall abnormally. Thus, Myo1p may contribute to proper septation, perhaps transporting vesicles containing septal material to the division site.
Contribution of Myo1p Domains to Function
All known defects of myo1 cells are corrected by a construct with just the head and TH1 domains, a protein similar to a short-tailed myosin-Ilike brush border myosin-I. This is surprising since important functions have been attributed to conserved parts of myosin-I tails that are missing in this construct: TH2 binds actin filaments (
myo1 cells. The rest of the tail is not only unnecessary for function, but the tail does not even localize properly without the head.
Head and TH1 sequences are required and sufficient for proper localization of Myo1p. GFP-H/1 localizes like full-length Myo1p to cortical patches, but when expressed alone as a GFP-fusion protein, TH1 mislocalizes to the nucleus. TH1 contains many potential nuclear localization sequences, clusters of arginines and lysines, such as KKQRRR in the first 10 residues of this domain. Artifactual nuclear localization shows that associations of TH1 with acidic phospholipids and actin filaments are insufficient to prevent transport of a GFP-1 construct into the nucleus. The head, but not TH2/3/A, overcomes this nuclear targeting. This intriguing observation indicates that the motor domain of a myosin-I participates in targeting the protein in the cell. Similar work on mammalian myosin-Is (
Role of Myo1p in Actin Assembly
The COOH-terminal A-domain of Myo1p is similar to the COOH-terminal A-domains of WASp/Scar proteins. Similar motifs on budding yeast myosin-Is and Bee1p/Las17p as well as other WASp proteins mediate physical interactions with Arp2/3 complex. In budding and fission yeasts, ablation of Arp2/3 complex subunits is lethal or severely debilitating (myo1 and is synthetically lethal with
myo1, indicating that these genes may share functions in regulating actin dynamics.
Interestingly, the minimal Myo1p construct for myosin-I function and localization, H/1, fails to rescue the myo1
wsp1 double-mutant lethality. Additional TH2 and TH3 domains are required to rescue the double-mutant, and rescued cells grow normally at 25°C in YES (data not shown). This differs from budding yeast, where deletion of A-domains from WASp and myosin-I result in severe growth defects. Our findings implicate TH3 domain in myosin-Imediated activation of Arp2/3 complex in fission yeast, perhaps by recruiting an additional A-domain containing protein.
Function and Evolution of Myosin-I Tails
Work on other organisms has shown that tail domains are important for myosin-I function and localization, but the domains required appear to differ from organism to organism. In S. cerevisiae, a point mutation in TH3 or deletion of TH2 and TH3 domains disperses myosin-Icontaining patches into diffuse cytoplasmic staining or alters the asymmetric distribution of patches to buds. These myosin-I mutants fail to complement defects of null cells (
These functional differences in myosin-I tails may reflect a loss or gain of particular tail-ligand interactions subsequent to the divergence of these organisms during evolution. Phylogenetic analysis revealed that all myosin-I genes had a common ancestor in an early eukaryote more than one billion years ago. The sequences of their head domains distinguish them from other classes of myosin. On the other hand, analysis of their tail sequences suggested that myosin-I genes acquired their TH3 domains relatively late, in more than one independent event after the separation of contemporary organisms from their common ancestors (
Addition of a COOH-terminal A-domain on fungal myosin-Is appears to have occurred once, soon after fungi diverged from animals and plants, 800 million years ago. No known animal, plant, or protozoan myosin-Is has an A-domain. The A-domain on fungal myosin-Is was in place 550 million years ago when S. pombe diverged from the lineage giving rise to S. cerevisiae and A. nidulans. In fact, the four fungal myosin-Is are closely related throughout: the head sequences form a tight phylogenetically related cluster, the IQ motifs are similar, TH2 is abundant in serine and threonine (except MYOA), and all have an A-domain at their COOH termini (Fig 5 A and S1, see supplemental material).
Since acquisition of the A-domain by fungal myosin-Is was a recent event, we hypothesize that other proteins may have also acquired A-domains late in their evolution. Similar acidic A-domain sequences are observed in various proteins not related to the WASp/Scar family or myosin-I, such as S. cerevisiae Abp1p and the intracellular domain of Toxoplasma gondii thrombospondin-related anonymous protiens (TRAP-related proteins). It would be interesting to investigate whether any of these A-domaincontaining proteins may link nonfungal myosin-Is to Arp2/3 complex.
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Footnotes |
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The online version of this article contains supplemental material.
1 Abbreviations used in this paper: EMM, Edinburgh minimal media; GFP, green fluorescent protein; IQ motif, light-chain binding motif; TH, tail homology.
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Acknowledgements |
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The authors thank Dr. Susan L. Forsburg for extensive advice and guidance on S. pombe. We thank Dr. David Ow for sharing his unpublished results on wsp1+. We thank Harry Higgs and Don Kaiser for Arp2/3 complex, Laurent Blanchoin for input on actin filament disruption experiments, and Debbie T. Liang and other members of S.L. Forsburg laboratory for reagents and helpful suggestions.
This work was supported by National Institutes of Health research grant GM-26132 to T.D. Pollard.
Submitted: 19 June 2000
Revised: 12 September 2000
Accepted: 13 September 2000
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References |
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