Limits...
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.

Show MeSH

Related in: MedlinePlus

Purification and characterization of Myo2. (A) SDS-PAGE of proteins stained with Coomassie blue. The leftmost lane shows proteins affinity purified from a wild-type strain (MLP 479) overexpressing GST-tagged light chains from the plasmids pGST-cdc4 and pGST-rlc1. The right five lanes show samples from steps in the purification of Myo2 from a strain (MLP 374) overexpressing Myo2p from the 3nmt1 promoter and GST-tagged light chains from pGST-cdc4 and pGST-rlc1. The steps are the total cell extract after centrifuging lysed cells at 100,000 g, proteins eluted from glutathione-Sepharose, the products of thrombin cleavage, the pooled peak from gel filtration, and the pooled peak of purified Myo2 from hydroxyapatite chromatography. Polypeptides are named on the right. (B) ATP-sensitive binding of purified Myo2 to actin filaments evaluated by SDS-PAGE and staining with Coomassie blue. Samples containing 0.75 μM Myo2 in 0.5 M NaCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 2 mM MgCl2 with 0 or 10 μM actin filaments, and 0 or 2 mM ATP were centrifuged at 120,000 g for 45 min. (top) Myo2 remains in the supernatant in absence of actin filaments. (bottom) Myo2 pellets with actin filaments in the absence but not the presence of 2 mM ATP. S, supernatant; P, pellet. (C) Dependence of the solubility of Myo2 on KCl concentration. Samples of 0.5 μM Myo2 in 10 mM imidazole, 1 mM DTT, and 0 to 500 mM KCl were centrifuged at 120,000 g for 10 min. Soluble Myo2 in the supernatant was determined by both the Bradford protein assay and densitometry of samples stained on protein gels. Two independent experiments are shown based on densitometry data.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172548&req=5

fig2: Purification and characterization of Myo2. (A) SDS-PAGE of proteins stained with Coomassie blue. The leftmost lane shows proteins affinity purified from a wild-type strain (MLP 479) overexpressing GST-tagged light chains from the plasmids pGST-cdc4 and pGST-rlc1. The right five lanes show samples from steps in the purification of Myo2 from a strain (MLP 374) overexpressing Myo2p from the 3nmt1 promoter and GST-tagged light chains from pGST-cdc4 and pGST-rlc1. The steps are the total cell extract after centrifuging lysed cells at 100,000 g, proteins eluted from glutathione-Sepharose, the products of thrombin cleavage, the pooled peak from gel filtration, and the pooled peak of purified Myo2 from hydroxyapatite chromatography. Polypeptides are named on the right. (B) ATP-sensitive binding of purified Myo2 to actin filaments evaluated by SDS-PAGE and staining with Coomassie blue. Samples containing 0.75 μM Myo2 in 0.5 M NaCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 2 mM MgCl2 with 0 or 10 μM actin filaments, and 0 or 2 mM ATP were centrifuged at 120,000 g for 45 min. (top) Myo2 remains in the supernatant in absence of actin filaments. (bottom) Myo2 pellets with actin filaments in the absence but not the presence of 2 mM ATP. S, supernatant; P, pellet. (C) Dependence of the solubility of Myo2 on KCl concentration. Samples of 0.5 μM Myo2 in 10 mM imidazole, 1 mM DTT, and 0 to 500 mM KCl were centrifuged at 120,000 g for 10 min. Soluble Myo2 in the supernatant was determined by both the Bradford protein assay and densitometry of samples stained on protein gels. Two independent experiments are shown based on densitometry data.

Mentions: To improve the yield of pure Myo2, we replaced the myo2 promoter with a thiamine repressible nmt1 promoter. Three-step purification from cells overexpressing Myo2p, Cdc4p, and Rlc1p in the absence of thiamine yielded sufficient quantities (25 μg per gram of cells) of pure Myo2 (Fig. 2 A) for further characterization. GST was removed from the light chains by thrombin-cleavage before gel filtration and hydroxylapatite chromatography.


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

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

Purification and characterization of Myo2. (A) SDS-PAGE of proteins stained with Coomassie blue. The leftmost lane shows proteins affinity purified from a wild-type strain (MLP 479) overexpressing GST-tagged light chains from the plasmids pGST-cdc4 and pGST-rlc1. The right five lanes show samples from steps in the purification of Myo2 from a strain (MLP 374) overexpressing Myo2p from the 3nmt1 promoter and GST-tagged light chains from pGST-cdc4 and pGST-rlc1. The steps are the total cell extract after centrifuging lysed cells at 100,000 g, proteins eluted from glutathione-Sepharose, the products of thrombin cleavage, the pooled peak from gel filtration, and the pooled peak of purified Myo2 from hydroxyapatite chromatography. Polypeptides are named on the right. (B) ATP-sensitive binding of purified Myo2 to actin filaments evaluated by SDS-PAGE and staining with Coomassie blue. Samples containing 0.75 μM Myo2 in 0.5 M NaCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 2 mM MgCl2 with 0 or 10 μM actin filaments, and 0 or 2 mM ATP were centrifuged at 120,000 g for 45 min. (top) Myo2 remains in the supernatant in absence of actin filaments. (bottom) Myo2 pellets with actin filaments in the absence but not the presence of 2 mM ATP. S, supernatant; P, pellet. (C) Dependence of the solubility of Myo2 on KCl concentration. Samples of 0.5 μM Myo2 in 10 mM imidazole, 1 mM DTT, and 0 to 500 mM KCl were centrifuged at 120,000 g for 10 min. Soluble Myo2 in the supernatant was determined by both the Bradford protein assay and densitometry of samples stained on protein gels. Two independent experiments are shown based on densitometry data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172548&req=5

fig2: Purification and characterization of Myo2. (A) SDS-PAGE of proteins stained with Coomassie blue. The leftmost lane shows proteins affinity purified from a wild-type strain (MLP 479) overexpressing GST-tagged light chains from the plasmids pGST-cdc4 and pGST-rlc1. The right five lanes show samples from steps in the purification of Myo2 from a strain (MLP 374) overexpressing Myo2p from the 3nmt1 promoter and GST-tagged light chains from pGST-cdc4 and pGST-rlc1. The steps are the total cell extract after centrifuging lysed cells at 100,000 g, proteins eluted from glutathione-Sepharose, the products of thrombin cleavage, the pooled peak from gel filtration, and the pooled peak of purified Myo2 from hydroxyapatite chromatography. Polypeptides are named on the right. (B) ATP-sensitive binding of purified Myo2 to actin filaments evaluated by SDS-PAGE and staining with Coomassie blue. Samples containing 0.75 μM Myo2 in 0.5 M NaCl, 10 mM imidazole, pH 7.0, 1 mM EGTA, 2 mM MgCl2 with 0 or 10 μM actin filaments, and 0 or 2 mM ATP were centrifuged at 120,000 g for 45 min. (top) Myo2 remains in the supernatant in absence of actin filaments. (bottom) Myo2 pellets with actin filaments in the absence but not the presence of 2 mM ATP. S, supernatant; P, pellet. (C) Dependence of the solubility of Myo2 on KCl concentration. Samples of 0.5 μM Myo2 in 10 mM imidazole, 1 mM DTT, and 0 to 500 mM KCl were centrifuged at 120,000 g for 10 min. Soluble Myo2 in the supernatant was determined by both the Bradford protein assay and densitometry of samples stained on protein gels. Two independent experiments are shown based on densitometry data.
Mentions: To improve the yield of pure Myo2, we replaced the myo2 promoter with a thiamine repressible nmt1 promoter. Three-step purification from cells overexpressing Myo2p, Cdc4p, and Rlc1p in the absence of thiamine yielded sufficient quantities (25 μg per gram of cells) of pure Myo2 (Fig. 2 A) for further characterization. GST was removed from the light chains by thrombin-cleavage before gel filtration and hydroxylapatite chromatography.

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.

Show MeSH
Related in: MedlinePlus