25 ?-resolution structure of a cytoplasmic dynein engine reveals a seven-member planar ring

25 ?-resolution structure of a cytoplasmic dynein engine reveals a seven-member planar ring. used in this study are detailed in Table 1. These strains (except the and double mutants constructed with this laboratory) are available from your Genetics Center (Duke University or college, Durham, NC). Cells were cultured in R medium (M medium plus 0.0075 M sodium acetate) having a light/dark cycle of 15 h/9 h and constant aeration (Witman, 1986). Table 1. Strains used in this study (1991)Porter (1992)(1991)LeDizet and Piperno (1995)(1994)(1997)(1993)(1991, 1993)(1991)(1991)(1982)Rupp (1996)(1981)(1981) Open BMS-794833 in a separate window Preparation of Flagellar Axonemes and Dynein Flagellar axonemes were prepared by standard methods (Witman, 1986). Intact outer arm dynein ( HCs) and an – HC subparticle that lacks the HC engine unit were extracted from and mutant strains, respectively. Dyneins were purified by sucrose denseness gradient centrifugation in the presence of Mg2+ and at low hydrostatic pressure as previously explained (Takada cells were cultivated to a denseness of 1 1.0 106 cells/ml in 500 ml liquid BMS-794833 medium, harvested, treated with autolysin, and resuspended in immunoprecipitation (IP) buffer (30 mM HEPES, pH 7.4, 5 mM MgSO4, 0.5 mM EDTA, 25 mM KCl, 1 mM dithiothreitol [DTT]) plus a 1/100 volume of protease inhibitor cocktail (P8849, Sigma, St. Louis, MO) to a total volume of 0.5 ml. The cell suspension was homogenized with an equal volume BMS-794833 of acid-washed glass beads (diameter 1 mm) by vortexing for 1 min. The homogenate was clarified inside a TLA100.2 rotor (Beckman, Fullerton, CA) at 33,000 rpm for 2 h at 4C. The clarified cytoplasmic extract was supplemented with 75 mM NaCl and 0.05% Triton X-100 and incubated with Rabbit polyclonal to Complement C4 beta chain CT240 antibody (generated with this study) or preimmune serum for 1 h at 4C and for 1 more hour after the addition of 10 l settled volume of ImmunoPure Immobilized protein G Plus beads (Pierce Biotechnology, Rockford, IL). The beads were washed three times with IP buffer comprising 75 mM NaCl and 0.05% Triton X-100 and once with IP buffer only. The immunoprecipitates were eluted by adding 2 gel loading buffer (0.1 M Tris-Cl, pH 6.8, 0.2 M DTT, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) and boiling. Twenty micrograms of cytoplasmic components and equivalent quantities of immunoprecipitates were analyzed by electrophoresis and immunoblotting. Ca2+ Effects on HC Subparticle Sedimentation The purified HC subparticle was fractionated inside a 5C20% sucrose gradient in HME buffer (30 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM EGTA) containing 1 mM DTT and 1 mM phenylmethylsulfonyl fluoride either in the absence of Ca2+, or at DNA polymerase (Stratagene, La Jolla, CA) and cloned into the pMAL-c2 vector (New England Biolabs, Ipswich, MA); residues 1-442, 1-754, BMS-794833 1-1089, 1-1486, 1432-1848, 338-754, 691-1089, 691-1486, 875-893, 875-1167, 875-1182, 890-1167, 890-1182, 1014-1486, and 1164-1182. This resulted in fusion of these regions to the C-terminus of maltose-binding protein (MBP) via a hydrophilic linker comprising a Factor Xa cleavage site. Fragments 338-754, 691-1089, 1014-1486, and 691-1486 were either expressed very poorly BMS-794833 or showed very limited solubility and could not be used further. The control MBP-LacZ protein derived from the original pMAL-c2 vector; the MBP-LC4 create was explained previously (King and Patel-King, 1995). To generate an N-terminal 10 His-tagged LC4 create, full-length LC4 was amplified with DNA polymerase using the original LC4 cDNA (King and Patel-King, 1995) as template and cloned into the pET16b vector (Novagen, Madison, WI). Recombinant proteins were overexpressed in strains XL1-Blue (Stratagene) or BL21(DE3)pLysS (Novagen). MBP fusion proteins were purified by amylose affinity chromatography (New England Biolabs). His-tagged LC4 was purified using His-Bind Resin (Novagen). Recombinant LC4 was acquired by digesting MBP-LC4 with Element Xa and separating the products by anion exchange chromatography using a HiTrap ANX FF column (Amersham Biosciences, Piscataway, NJ) on a Biologic chromatography workstation (Bio-Rad Laboratories, Hercules, CA). The MBP-LC4 and MBP- HC stem website N1 (residues 1-442) proteins were used as the immunogens to obtain rabbit polyclonal antibodies CT61 and CT240, respectively. Sera were blot-purified against the appropriate recombinant proteins lacking the MBP moiety before use; for some preparations of CT61 His-tagged LC4 was used. Other antibodies used include rabbit polyclonals against LC1 (R5932; Benashski (1991). After trypsin digestion, peptides were recognized by mass spectrometry in the University or college of Massachusetts Medical School Proteomic Mass Spectrometry Facility (Worcester, MA). Bad Stain Electron Microscopy.