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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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Cdc4p and Rlc1p are the light chains for Myo2p. Quantitative analysis of GST-pull down experiments with GST-tagged myosin light chains, GST-tagged Myo2p heavy chain, or GFP-tagged candidate myosin light chains. Proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE, immunoblotted for Myo2p, Myo1p, or GFP, detected by ECL, and quantitated by densitometry of the bands. (A) TP 150 cells overexpressed GST-tagged candidate light chains cdc4, rlc1, cam1, or cam2. The GST-fusion proteins in cell extracts were affinity purified on glutathione-Sepharose and copurifying Myo2p (black bars) and Myo1p (gray bars) were quantitated by densitometry. Amounts were estimated relative to the maximal signal, which was given a value of 1. (B) Glutathione-Sepharose was used to purify GST-tagged proteins from cells expressing myo2-GST alone, YFP-cdc4 alone, rlc1-GFP alone, cam2-GFP alone, myo2-GST plus YFP-cdc4, myo2-GST plus rlc1-GFP, or myo2-GST plus cam2-GFP. Relative amounts of Myo2p (black bars) and copurifying light chains (gray bars) were quantitated by densitometry of bands detected after immunoblotting.
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fig1: Cdc4p and Rlc1p are the light chains for Myo2p. Quantitative analysis of GST-pull down experiments with GST-tagged myosin light chains, GST-tagged Myo2p heavy chain, or GFP-tagged candidate myosin light chains. Proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE, immunoblotted for Myo2p, Myo1p, or GFP, detected by ECL, and quantitated by densitometry of the bands. (A) TP 150 cells overexpressed GST-tagged candidate light chains cdc4, rlc1, cam1, or cam2. The GST-fusion proteins in cell extracts were affinity purified on glutathione-Sepharose and copurifying Myo2p (black bars) and Myo1p (gray bars) were quantitated by densitometry. Amounts were estimated relative to the maximal signal, which was given a value of 1. (B) Glutathione-Sepharose was used to purify GST-tagged proteins from cells expressing myo2-GST alone, YFP-cdc4 alone, rlc1-GFP alone, cam2-GFP alone, myo2-GST plus YFP-cdc4, myo2-GST plus rlc1-GFP, or myo2-GST plus cam2-GFP. Relative amounts of Myo2p (black bars) and copurifying light chains (gray bars) were quantitated by densitometry of bands detected after immunoblotting.

Mentions: Before designing strategies to purify Myo2, we wished to confirm that Cdc4p and Rlc1p are the only light chains required for Myo2 function. The S. pombe genome encodes five proteins with sequences similar to Cdc4p: Cam1p (calmodulin; 38% identity/62% similarity to Cdc4p); SPAC29A4.05p (35%/58%); Cdc31p (27%/51%); Rlc1p (23%/49%); and a Cnb1p orthologue (21%/49%). We ruled out Cdc31p and Cnb1p as myosin light chains, because Cdc31p (a centrin) has a role in spindle pole body duplication (Paoletti et al., 2003), and Cnb1p is a regulatory subunit of calcineurin B phosphatase (Cyert and Thorner, 1992). Cam1p localizes at polarized growth sites and the contractile ring (Moser et al., 1997; Eng et al., 1998), whereas SPAC29A4.05p had not been characterized. We named SPAC29A4.05p Cam2p because its amino acid sequence is 41% identical/65% similar to Cam1p. Like GST-Cdc4p and GST-Rlc1p, overexpressed GST-Cam2p binds the Myo2 heavy chain to glutathione-Sepharose beads, whereas GST-Cam1p does not (Fig. 1 A). In contrast, only native Cdc4p and Rlc1p copurified with native Myo2p-GST (Fig. 1 B), suggesting that the Myo2p heavy chain binds these two light chains in preference to Cam2p. Cam2p localizes as patches at sites of polarized growth (unpublished data), like type-I myosin, Myo1p (Lee et al., 2000; Toya et al., 2001). In addition, like Cam1p (Toya et al., 2001), Cam2p associates with Myo1p (Fig. 1 A). Thus, both Cam1p and Cam2p are likely to be light chains for Myo1p. Given these results, we chose to purify Myo2p in combination with Cdc4p and Rlc1p.


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

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

Cdc4p and Rlc1p are the light chains for Myo2p. Quantitative analysis of GST-pull down experiments with GST-tagged myosin light chains, GST-tagged Myo2p heavy chain, or GFP-tagged candidate myosin light chains. Proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE, immunoblotted for Myo2p, Myo1p, or GFP, detected by ECL, and quantitated by densitometry of the bands. (A) TP 150 cells overexpressed GST-tagged candidate light chains cdc4, rlc1, cam1, or cam2. The GST-fusion proteins in cell extracts were affinity purified on glutathione-Sepharose and copurifying Myo2p (black bars) and Myo1p (gray bars) were quantitated by densitometry. Amounts were estimated relative to the maximal signal, which was given a value of 1. (B) Glutathione-Sepharose was used to purify GST-tagged proteins from cells expressing myo2-GST alone, YFP-cdc4 alone, rlc1-GFP alone, cam2-GFP alone, myo2-GST plus YFP-cdc4, myo2-GST plus rlc1-GFP, or myo2-GST plus cam2-GFP. Relative amounts of Myo2p (black bars) and copurifying light chains (gray bars) were quantitated by densitometry of bands detected after immunoblotting.
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fig1: Cdc4p and Rlc1p are the light chains for Myo2p. Quantitative analysis of GST-pull down experiments with GST-tagged myosin light chains, GST-tagged Myo2p heavy chain, or GFP-tagged candidate myosin light chains. Proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE, immunoblotted for Myo2p, Myo1p, or GFP, detected by ECL, and quantitated by densitometry of the bands. (A) TP 150 cells overexpressed GST-tagged candidate light chains cdc4, rlc1, cam1, or cam2. The GST-fusion proteins in cell extracts were affinity purified on glutathione-Sepharose and copurifying Myo2p (black bars) and Myo1p (gray bars) were quantitated by densitometry. Amounts were estimated relative to the maximal signal, which was given a value of 1. (B) Glutathione-Sepharose was used to purify GST-tagged proteins from cells expressing myo2-GST alone, YFP-cdc4 alone, rlc1-GFP alone, cam2-GFP alone, myo2-GST plus YFP-cdc4, myo2-GST plus rlc1-GFP, or myo2-GST plus cam2-GFP. Relative amounts of Myo2p (black bars) and copurifying light chains (gray bars) were quantitated by densitometry of bands detected after immunoblotting.
Mentions: Before designing strategies to purify Myo2, we wished to confirm that Cdc4p and Rlc1p are the only light chains required for Myo2 function. The S. pombe genome encodes five proteins with sequences similar to Cdc4p: Cam1p (calmodulin; 38% identity/62% similarity to Cdc4p); SPAC29A4.05p (35%/58%); Cdc31p (27%/51%); Rlc1p (23%/49%); and a Cnb1p orthologue (21%/49%). We ruled out Cdc31p and Cnb1p as myosin light chains, because Cdc31p (a centrin) has a role in spindle pole body duplication (Paoletti et al., 2003), and Cnb1p is a regulatory subunit of calcineurin B phosphatase (Cyert and Thorner, 1992). Cam1p localizes at polarized growth sites and the contractile ring (Moser et al., 1997; Eng et al., 1998), whereas SPAC29A4.05p had not been characterized. We named SPAC29A4.05p Cam2p because its amino acid sequence is 41% identical/65% similar to Cam1p. Like GST-Cdc4p and GST-Rlc1p, overexpressed GST-Cam2p binds the Myo2 heavy chain to glutathione-Sepharose beads, whereas GST-Cam1p does not (Fig. 1 A). In contrast, only native Cdc4p and Rlc1p copurified with native Myo2p-GST (Fig. 1 B), suggesting that the Myo2p heavy chain binds these two light chains in preference to Cam2p. Cam2p localizes as patches at sites of polarized growth (unpublished data), like type-I myosin, Myo1p (Lee et al., 2000; Toya et al., 2001). In addition, like Cam1p (Toya et al., 2001), Cam2p associates with Myo1p (Fig. 1 A). Thus, both Cam1p and Cam2p are likely to be light chains for Myo1p. Given these results, we chose to purify Myo2p in combination with Cdc4p and Rlc1p.

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