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-GFP 3 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:
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