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UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II.

Lord M, Pollard TD - J. Cell Biol. (2004)

Bottom Line: Thus, Rng3p contributes directly to the motility activity of native Myo2.Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring.In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

ABSTRACT
We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

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Effects of mutations in Myo2p light chains and Rng3p on motility activity. (A) Amino acid sequence alignment of the NH2-terminal regions of Rlc1p, Rlc1p-N1Δ, Rlc1p-N2Δ, D. melanogaster RLC, and Homo sapien RLC homologues. Alignment was generated with MacVector 7.1.1 and Boxshade software. Single (above) and double (below) lines on the alignment denote potential Rlc1p Ser/Thr phosphorylation sites and the phospho-regulatory Thr-Ser characteristic of higher eukaryotic RLCs, respectively. The box in Rlc1p-N2Δ indicates the additional amino acid substitutions. Black, amino acid identities; gray, amino acid similarities. (B) Viability of an rlc1Δ strain (MLP 7) carrying empty vector (negative control), pGFP-rlc1 (positive control), pGFP-rlc1-N1Δ, or pGFP-rlc1-N2Δ. Transformants were streaked on an EMM Ura- plate containing 1M KCl. (C) Phenotypic quantitation of MLP 7 carrying empty vector (negative control), pGFP-rlc1 (positive control), or pGFP-rlc1-N1Δ. Transformants were grown in liquid EMM Ura− media and their nuclei stained. Nuclei/cell were visualized and scored by fluorescence microscopy. (D) Localization of GFP-Rlc1p in MLP 7 containing either pGFP-rlc1 (left panels) or pGFP-rlc1-N1Δ (right panels). Cells were grown in liquid EMM Ura− media. Fluorescence and DIC micrographs are shown. Bar, 5 μm. (E–I) Actin filament gliding assays using crude Myo2 (0.25 mg/ml impure protein) isolated in one step on glutathione-Sepharose from strains with mutations in Myo2p light chains and Rng3p. (E–G) Fluorescence micrographs of filaments labeled with rhodamine-phalloidin. Bars, 5 μm. (H and I) Quantitation of gliding rates. Conditions: samples were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Myo2 from a strain lacking Rlc1p (MLP 534 rlc1Δ 41nmt1 promoter-myo2 plus pGST-cdc4). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Arrowheads mark the 6-s time point in filaments that moved. (F) Temperature dependence of actin filament attachment to crude, wild-type Myo2 (0.25 mg/ml) purified from MLP 509 (41nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). (G) Temperature dependence of actin filament attachment to crude Myo2 (0.25 mg/ml) purified from a strain with a temperature-sensitive mutation, rng3-65 (MLP 586 rng3-65 41nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). (H) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and rng3-65 (MLP 586) temperature-sensitive backgrounds. (I) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and cdc4 temperature-sensitive backgrounds. Strains: MLP 539 cdc4-8 41nmt1 promoter-myo2, plus pGST-cdc4-8 and pGST-rlc1; MLP 641 cdc4-31 41nmt1 promoter-myo2 plus pGST-cdc4-31 and pGST-rlc1; MLP 647 cdc4-C2 41nmt1 promoter-myo2 plus pGST-cdc4-C2 and pGST-rlc1; MLP 648 cdc4-A2 41nmt1 promoter-myo2 plus pGST-cdc4-A2 and pGST-rlc1.
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fig6: Effects of mutations in Myo2p light chains and Rng3p on motility activity. (A) Amino acid sequence alignment of the NH2-terminal regions of Rlc1p, Rlc1p-N1Δ, Rlc1p-N2Δ, D. melanogaster RLC, and Homo sapien RLC homologues. Alignment was generated with MacVector 7.1.1 and Boxshade software. Single (above) and double (below) lines on the alignment denote potential Rlc1p Ser/Thr phosphorylation sites and the phospho-regulatory Thr-Ser characteristic of higher eukaryotic RLCs, respectively. The box in Rlc1p-N2Δ indicates the additional amino acid substitutions. Black, amino acid identities; gray, amino acid similarities. (B) Viability of an rlc1Δ strain (MLP 7) carrying empty vector (negative control), pGFP-rlc1 (positive control), pGFP-rlc1-N1Δ, or pGFP-rlc1-N2Δ. Transformants were streaked on an EMM Ura- plate containing 1M KCl. (C) Phenotypic quantitation of MLP 7 carrying empty vector (negative control), pGFP-rlc1 (positive control), or pGFP-rlc1-N1Δ. Transformants were grown in liquid EMM Ura− media and their nuclei stained. Nuclei/cell were visualized and scored by fluorescence microscopy. (D) Localization of GFP-Rlc1p in MLP 7 containing either pGFP-rlc1 (left panels) or pGFP-rlc1-N1Δ (right panels). Cells were grown in liquid EMM Ura− media. Fluorescence and DIC micrographs are shown. Bar, 5 μm. (E–I) Actin filament gliding assays using crude Myo2 (0.25 mg/ml impure protein) isolated in one step on glutathione-Sepharose from strains with mutations in Myo2p light chains and Rng3p. (E–G) Fluorescence micrographs of filaments labeled with rhodamine-phalloidin. Bars, 5 μm. (H and I) Quantitation of gliding rates. Conditions: samples were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Myo2 from a strain lacking Rlc1p (MLP 534 rlc1Δ 41nmt1 promoter-myo2 plus pGST-cdc4). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Arrowheads mark the 6-s time point in filaments that moved. (F) Temperature dependence of actin filament attachment to crude, wild-type Myo2 (0.25 mg/ml) purified from MLP 509 (41nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). (G) Temperature dependence of actin filament attachment to crude Myo2 (0.25 mg/ml) purified from a strain with a temperature-sensitive mutation, rng3-65 (MLP 586 rng3-65 41nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). (H) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and rng3-65 (MLP 586) temperature-sensitive backgrounds. (I) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and cdc4 temperature-sensitive backgrounds. Strains: MLP 539 cdc4-8 41nmt1 promoter-myo2, plus pGST-cdc4-8 and pGST-rlc1; MLP 641 cdc4-31 41nmt1 promoter-myo2 plus pGST-cdc4-31 and pGST-rlc1; MLP 647 cdc4-C2 41nmt1 promoter-myo2 plus pGST-cdc4-C2 and pGST-rlc1; MLP 648 cdc4-A2 41nmt1 promoter-myo2 plus pGST-cdc4-A2 and pGST-rlc1.

Mentions: Rlc1p is not required for viability (Le Goff et al., 2000; Naqvi et al., 2000), but cells lacking Rlc1p die under stressful conditions (Fig. 6 B). Myo2 from rlc1Δ cells moved actin filaments at only 20% the rate of wild-type Myo2 (Fig. 6 E, Table I, and Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). Pre-incubation of one step purified Myo2 from rlc1Δ cells with purified GST-Rlc1p restored in vitro motility to rates close to those of wild type (Table I).


UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II.

Lord M, Pollard TD - J. Cell Biol. (2004)

Effects of mutations in Myo2p light chains and Rng3p on motility activity. (A) Amino acid sequence alignment of the NH2-terminal regions of Rlc1p, Rlc1p-N1Δ, Rlc1p-N2Δ, D. melanogaster RLC, and Homo sapien RLC homologues. Alignment was generated with MacVector 7.1.1 and Boxshade software. Single (above) and double (below) lines on the alignment denote potential Rlc1p Ser/Thr phosphorylation sites and the phospho-regulatory Thr-Ser characteristic of higher eukaryotic RLCs, respectively. The box in Rlc1p-N2Δ indicates the additional amino acid substitutions. Black, amino acid identities; gray, amino acid similarities. (B) Viability of an rlc1Δ strain (MLP 7) carrying empty vector (negative control), pGFP-rlc1 (positive control), pGFP-rlc1-N1Δ, or pGFP-rlc1-N2Δ. Transformants were streaked on an EMM Ura- plate containing 1M KCl. (C) Phenotypic quantitation of MLP 7 carrying empty vector (negative control), pGFP-rlc1 (positive control), or pGFP-rlc1-N1Δ. Transformants were grown in liquid EMM Ura− media and their nuclei stained. Nuclei/cell were visualized and scored by fluorescence microscopy. (D) Localization of GFP-Rlc1p in MLP 7 containing either pGFP-rlc1 (left panels) or pGFP-rlc1-N1Δ (right panels). Cells were grown in liquid EMM Ura− media. Fluorescence and DIC micrographs are shown. Bar, 5 μm. (E–I) Actin filament gliding assays using crude Myo2 (0.25 mg/ml impure protein) isolated in one step on glutathione-Sepharose from strains with mutations in Myo2p light chains and Rng3p. (E–G) Fluorescence micrographs of filaments labeled with rhodamine-phalloidin. Bars, 5 μm. (H and I) Quantitation of gliding rates. Conditions: samples were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Myo2 from a strain lacking Rlc1p (MLP 534 rlc1Δ 41nmt1 promoter-myo2 plus pGST-cdc4). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Arrowheads mark the 6-s time point in filaments that moved. (F) Temperature dependence of actin filament attachment to crude, wild-type Myo2 (0.25 mg/ml) purified from MLP 509 (41nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). (G) Temperature dependence of actin filament attachment to crude Myo2 (0.25 mg/ml) purified from a strain with a temperature-sensitive mutation, rng3-65 (MLP 586 rng3-65 41nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). (H) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and rng3-65 (MLP 586) temperature-sensitive backgrounds. (I) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and cdc4 temperature-sensitive backgrounds. Strains: MLP 539 cdc4-8 41nmt1 promoter-myo2, plus pGST-cdc4-8 and pGST-rlc1; MLP 641 cdc4-31 41nmt1 promoter-myo2 plus pGST-cdc4-31 and pGST-rlc1; MLP 647 cdc4-C2 41nmt1 promoter-myo2 plus pGST-cdc4-C2 and pGST-rlc1; MLP 648 cdc4-A2 41nmt1 promoter-myo2 plus pGST-cdc4-A2 and pGST-rlc1.
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fig6: Effects of mutations in Myo2p light chains and Rng3p on motility activity. (A) Amino acid sequence alignment of the NH2-terminal regions of Rlc1p, Rlc1p-N1Δ, Rlc1p-N2Δ, D. melanogaster RLC, and Homo sapien RLC homologues. Alignment was generated with MacVector 7.1.1 and Boxshade software. Single (above) and double (below) lines on the alignment denote potential Rlc1p Ser/Thr phosphorylation sites and the phospho-regulatory Thr-Ser characteristic of higher eukaryotic RLCs, respectively. The box in Rlc1p-N2Δ indicates the additional amino acid substitutions. Black, amino acid identities; gray, amino acid similarities. (B) Viability of an rlc1Δ strain (MLP 7) carrying empty vector (negative control), pGFP-rlc1 (positive control), pGFP-rlc1-N1Δ, or pGFP-rlc1-N2Δ. Transformants were streaked on an EMM Ura- plate containing 1M KCl. (C) Phenotypic quantitation of MLP 7 carrying empty vector (negative control), pGFP-rlc1 (positive control), or pGFP-rlc1-N1Δ. Transformants were grown in liquid EMM Ura− media and their nuclei stained. Nuclei/cell were visualized and scored by fluorescence microscopy. (D) Localization of GFP-Rlc1p in MLP 7 containing either pGFP-rlc1 (left panels) or pGFP-rlc1-N1Δ (right panels). Cells were grown in liquid EMM Ura− media. Fluorescence and DIC micrographs are shown. Bar, 5 μm. (E–I) Actin filament gliding assays using crude Myo2 (0.25 mg/ml impure protein) isolated in one step on glutathione-Sepharose from strains with mutations in Myo2p light chains and Rng3p. (E–G) Fluorescence micrographs of filaments labeled with rhodamine-phalloidin. Bars, 5 μm. (H and I) Quantitation of gliding rates. Conditions: samples were applied to flow cells in 25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM ATP, 100 mM DTT, and 10 nM labeled actin filaments. (E) Myo2 from a strain lacking Rlc1p (MLP 534 rlc1Δ 41nmt1 promoter-myo2 plus pGST-cdc4). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Arrowheads mark the 6-s time point in filaments that moved. (F) Temperature dependence of actin filament attachment to crude, wild-type Myo2 (0.25 mg/ml) purified from MLP 509 (41nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). (G) Temperature dependence of actin filament attachment to crude Myo2 (0.25 mg/ml) purified from a strain with a temperature-sensitive mutation, rng3-65 (MLP 586 rng3-65 41nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). (H) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and rng3-65 (MLP 586) temperature-sensitive backgrounds. (I) Temperature dependence of in vitro motility rates of crude Myo2 purified from wild-type (MLP 509) and cdc4 temperature-sensitive backgrounds. Strains: MLP 539 cdc4-8 41nmt1 promoter-myo2, plus pGST-cdc4-8 and pGST-rlc1; MLP 641 cdc4-31 41nmt1 promoter-myo2 plus pGST-cdc4-31 and pGST-rlc1; MLP 647 cdc4-C2 41nmt1 promoter-myo2 plus pGST-cdc4-C2 and pGST-rlc1; MLP 648 cdc4-A2 41nmt1 promoter-myo2 plus pGST-cdc4-A2 and pGST-rlc1.
Mentions: Rlc1p is not required for viability (Le Goff et al., 2000; Naqvi et al., 2000), but cells lacking Rlc1p die under stressful conditions (Fig. 6 B). Myo2 from rlc1Δ cells moved actin filaments at only 20% the rate of wild-type Myo2 (Fig. 6 E, Table I, and Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). Pre-incubation of one step purified Myo2 from rlc1Δ cells with purified GST-Rlc1p restored in vitro motility to rates close to those of wild type (Table I).

Bottom Line: Thus, Rng3p contributes directly to the motility activity of native Myo2.Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring.In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

ABSTRACT
We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

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Related in: MedlinePlus