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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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Domain organization and sequence comparisons of UCS proteins. Amino acid sequence comparisons (identities/similarities) were generated from sequence alignments (MacVector 7.1.1). Conservation amongst NH2-terminal tetratricopeptide repeat (TPR) domains (light gray), central regions (gray), and COOH-terminal UCS domains (black) are compared individually. Amino acid sequence length and total conservation are shown on the right for each protein. (A) Comparison of animal and fungal UCS proteins with H. sapiens general cell form (Hs GC; Price et al., 2002), H. sapiens striated muscle form (Hs SM; Price et al., 2002), Danio rerio (Dr Unc45r; zebrafish; Etheridge et al., 2002), D. melanogaster (Dm; GenBank/EMBL/DDBJ accession no. AAK93568), C. elegans (Ce Unc-45; Venolia et al., 1999); P. anserina (Pa Cro1p; Berteaux-Lecellier et al., 1998); S. pombe (Sp Rng3p; Wong et al., 2000); and Saccharomyces cerevisiae (Sc She4p; Jansen et al., 1996). (B) Comparison of fungal UCS proteins with Rng3p.
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fig7: Domain organization and sequence comparisons of UCS proteins. Amino acid sequence comparisons (identities/similarities) were generated from sequence alignments (MacVector 7.1.1). Conservation amongst NH2-terminal tetratricopeptide repeat (TPR) domains (light gray), central regions (gray), and COOH-terminal UCS domains (black) are compared individually. Amino acid sequence length and total conservation are shown on the right for each protein. (A) Comparison of animal and fungal UCS proteins with H. sapiens general cell form (Hs GC; Price et al., 2002), H. sapiens striated muscle form (Hs SM; Price et al., 2002), Danio rerio (Dr Unc45r; zebrafish; Etheridge et al., 2002), D. melanogaster (Dm; GenBank/EMBL/DDBJ accession no. AAK93568), C. elegans (Ce Unc-45; Venolia et al., 1999); P. anserina (Pa Cro1p; Berteaux-Lecellier et al., 1998); S. pombe (Sp Rng3p; Wong et al., 2000); and Saccharomyces cerevisiae (Sc She4p; Jansen et al., 1996). (B) Comparison of fungal UCS proteins with Rng3p.

Mentions: Like Unc45, most UCS-domain proteins consist of three domains: an NH2-terminal tetratricopeptide repeat (TPR); a central region; and a COOH-terminal UCS domain (Hutagalung et al., 2002). TPR domains can bind either Hsp70 or Hsp90 chaperones (Blatch and Lassle, 1999). The TPR domain of Unc45 is specific for Hsp90 in vitro (Barral et al., 2002). The central domain is well conserved only in animals, whereas the UCS domain is well conserved in all UCS proteins (Hutagalung et al., 2002; Fig. 7 A). When overexpressed in Sf9 cells, a truncated form of Unc45 lacking its TPR domain copurified with endogenous insect Hsp70, but not Hsp90. (Barral et al., 2002). This truncated form of Unc45 also bound C. elegans myosin-II in vitro. Because Unc45 inhibited thermal aggregation of scallop myosin subfragment 1, Barral et al. (2002) concluded that Unc45 acts both as a molecular chaperone and a Hsp90 cochaperone supporting proper folding of myosin heads and subsequent assembly of muscle thick filaments.


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

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

Domain organization and sequence comparisons of UCS proteins. Amino acid sequence comparisons (identities/similarities) were generated from sequence alignments (MacVector 7.1.1). Conservation amongst NH2-terminal tetratricopeptide repeat (TPR) domains (light gray), central regions (gray), and COOH-terminal UCS domains (black) are compared individually. Amino acid sequence length and total conservation are shown on the right for each protein. (A) Comparison of animal and fungal UCS proteins with H. sapiens general cell form (Hs GC; Price et al., 2002), H. sapiens striated muscle form (Hs SM; Price et al., 2002), Danio rerio (Dr Unc45r; zebrafish; Etheridge et al., 2002), D. melanogaster (Dm; GenBank/EMBL/DDBJ accession no. AAK93568), C. elegans (Ce Unc-45; Venolia et al., 1999); P. anserina (Pa Cro1p; Berteaux-Lecellier et al., 1998); S. pombe (Sp Rng3p; Wong et al., 2000); and Saccharomyces cerevisiae (Sc She4p; Jansen et al., 1996). (B) Comparison of fungal UCS proteins with Rng3p.
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fig7: Domain organization and sequence comparisons of UCS proteins. Amino acid sequence comparisons (identities/similarities) were generated from sequence alignments (MacVector 7.1.1). Conservation amongst NH2-terminal tetratricopeptide repeat (TPR) domains (light gray), central regions (gray), and COOH-terminal UCS domains (black) are compared individually. Amino acid sequence length and total conservation are shown on the right for each protein. (A) Comparison of animal and fungal UCS proteins with H. sapiens general cell form (Hs GC; Price et al., 2002), H. sapiens striated muscle form (Hs SM; Price et al., 2002), Danio rerio (Dr Unc45r; zebrafish; Etheridge et al., 2002), D. melanogaster (Dm; GenBank/EMBL/DDBJ accession no. AAK93568), C. elegans (Ce Unc-45; Venolia et al., 1999); P. anserina (Pa Cro1p; Berteaux-Lecellier et al., 1998); S. pombe (Sp Rng3p; Wong et al., 2000); and Saccharomyces cerevisiae (Sc She4p; Jansen et al., 1996). (B) Comparison of fungal UCS proteins with Rng3p.
Mentions: Like Unc45, most UCS-domain proteins consist of three domains: an NH2-terminal tetratricopeptide repeat (TPR); a central region; and a COOH-terminal UCS domain (Hutagalung et al., 2002). TPR domains can bind either Hsp70 or Hsp90 chaperones (Blatch and Lassle, 1999). The TPR domain of Unc45 is specific for Hsp90 in vitro (Barral et al., 2002). The central domain is well conserved only in animals, whereas the UCS domain is well conserved in all UCS proteins (Hutagalung et al., 2002; Fig. 7 A). When overexpressed in Sf9 cells, a truncated form of Unc45 lacking its TPR domain copurified with endogenous insect Hsp70, but not Hsp90. (Barral et al., 2002). This truncated form of Unc45 also bound C. elegans myosin-II in vitro. Because Unc45 inhibited thermal aggregation of scallop myosin subfragment 1, Barral et al. (2002) concluded that Unc45 acts both as a molecular chaperone and a Hsp90 cochaperone supporting proper folding of myosin heads and subsequent assembly of muscle thick filaments.

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