UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II.
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.
Affiliation: Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
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.
- Cytoskeletal Proteins/physiology*
- Myosin Type II/chemistry*
- Schizosaccharomyces pombe Proteins/metabolism/physiology*
- Adenosine Triphosphatases/metabolism
- Amino Acid Sequence
- Dose-Response Relationship, Drug
- Glutathione Transferase/metabolism
- Image Processing, Computer-Assisted
- Microscopy, Fluorescence
- Microscopy, Interference
- Molecular Sequence Data
- Protein Structure, Tertiary
- Sequence Homology, Amino Acid
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fig3: Rng3p stimulates Myo2 activity. (A) Lanes 1–5 show samples from steps in the purification of Myo2 from a strain (MLP 676) containing a chromosomal rng3-GFP3 fusion overexpressing Myo2p from the 41nmt1 promoter and GST-tagged light chains from pGST-cdc4 and pGST-rlc1. Samples were run on an SDS-PAGE gel, immunoblotted and probed with Myo2 heavy chain antibodies (top) and GFP antibodies (bottom). (B) Bead binding assay for interaction of chromosomal Rng3p-GST with Myo2. Strains: FY 435 carrying pGST-rlc1, MLP 694 (rng3-GST), MLP 693 (rng3-GST, myo2-E1), and MLP 695 (wild-type). GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain (top) and Rng3p-GST (α-GST; bottom). pGST-rlc1, positive control with GST-Rlc1p. wild type, negative control lacking a GST fusion. (C) Bead binding assay for interaction of overexpressed GST-Rng3p (pGST-rng3-FL) and GST-Rng3-UCS domain (pGST-rng3-UCS) with Myo2. Strains: FY 435 (wild-type) and TP 73 (myo2-E1) carrying plasmids pGST-rng3, pGST-rng3-UCS, pGST-cam1, and pGST-rlc1. GST proteins from extracts were affinity purified on glutathione-Sepharose. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to Myo2p heavy chain. pGST-cam1, negative control with GST-Cam1p. pGST-rlc1, positive control with GST-Rlc1p. (D) GST-Rng3p purified from S. pombe and recombinant GST-Rng3p purified from Escherichia coli. SDS-PAGE gel stained with Coomassie blue. Lower band in the S. pombe lane represents a breakdown product. (E–J) Actin filament gliding assays. Time-lapse fluorescence micrographs of filaments labeled with rhodamine-phalloidin (also, see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). Trajectories are indicated with white dots marking the trailing end of filaments at 2-s intervals. Bar, 5 μm. Conditions: indicated concentrations of Myo2 and GST-Rng3p 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) Crude one-step purified Myo2 (0.25 mg/ml impure protein). (F) Three-step–purified 75 nM Myo2 preincubated with 250 nM native S. pombe GST-Rng3p. (G) Three-step–purified 75 nM Myo2 preincubated with 250 nM recombinant GST-Rng3p. (H) Three-step–purified 75 nM Myo2 alone. (I) S. pombe GST-Rng3p (250 nM) alone. (J) recombinant GST-Rng3p (250 nM) alone. (K) Dependence of the actin-activated ATPase activity of three-step–purified Myo2 as a function of the concentrations of native (open circles) and recombinant (closed circles) GST-Rng3p. Conditions: 30 nM Myo2 and 10 μM actin filaments in 2 mM ATP, 3 mM MgCl2, 0.1 mM CaCl2, and 75 mM KCl. Error bars show SD. (L) Dependence of the number of actin filaments captured by two-step–purified Myo2 on the concentration of recombinant GST-Rng3p. All filaments in a 130 μm2 frame of a fluorescence micrograph were counted. (squares) 20 nM two-step–purified Myo2. No gliding was observed in the absence of GST-Rng3p. (circles) 200 nM two-step–purified Myo2. These samples supported gliding in the absence of GST-Rng3p. (horizontal line) 150 nM crude Myo2 purified after overexpression from MLP 374 (3nmt1 promoter-myo2 plus pGST-cdc4 and pGST-rlc1). These samples supported robust gliding.
Crude Myo2, purified on glutathione-Sepharose but not chromatographed further, bound to a glass coverslip, and supported the gliding motility of actin filaments at a velocity of ∼0.5 μm/s, somewhat slower than skeletal muscle myosin (Fig. 3 E, Table I, and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200404045/DC1). Myo2 purified through three steps failed to bind or move actin filaments in this assay (Fig. 3 H). Given the good ATPase activity of this purified Myo2, we hypothesized that purification separates Myo2 from an activator required for motility activity. We ruled out Cdc4p and Rlc1p as the missing activators because both copurified with Myo2 through three purification steps, and because addition of excess purified Cdc4p or Rlc1p failed to activate purified Myo2 (unpublished data).