Figure 4. Multiple Myo4 motors are bound... | Rockefeller University Press

Figure 4.

$Figure 4. Multiple Myo4 motors are bound to a single localization element. (A) Myo4-HA coprecipitates with Myo4-myc when bound to U3 RNA. MYO4-HA was expressed in wild-type cells (lanes 1 and 2) or cells expressing MYO4-13xMYC (lanes 3–6), and U3 RNA expression was induced (lanes 1, 2, 5, and 6) or not induced (lanes 3 and 4). RNaseA was added to the indicated cell extracts (lanes 2, 4, and 6). Myo4-myc was immunoprecipitated from each cell extract, and the precipitants were analyzed by a Western blot with anti-HA or anti-She2 antibodies as indicated. Input shows equal amount of initial cell extracts probed with anti-HA antibody. (B) Myo4-myc and Myo4-HA coprecipitation requires She2. MYO4-HA was expressed with MYO4-13xMYC in the presence of SHE2 (lanes 1 and 2) or in she2Δ cells (lane 3), and U3 RNA expression was induced (lanes 2 and 3) or not induced (lane 1). Myo4-myc was immunoprecipitated and analyzed as in A. (C) The 15S U3 RNP complex consists of multiple Myo4 motors. MYO4-HA was expressed with MYO4-TAP, and U3 RNA expression was induced. TAP-purified complexes were loaded on 10–50% sucrose gradients and the collected fractions were analyzed by Western blotting. Note that the Myo4p blot detects both Myo4-TAP and Myo4-HA.$

Multiple Myo4 motors are bound to a single localization element. (A) Myo4-HA coprecipitates with Myo4-myc when bound to U3 RNA. MYO4-HA was expressed in wild-type cells (lanes 1 and 2) or cells expressing MYO4-13xMYC (lanes 3–6), and U3 RNA expression was induced (lanes 1, 2, 5, and 6) or not induced (lanes 3 and 4). RNaseA was added to the indicated cell extracts (lanes 2, 4, and 6). Myo4-myc was immunoprecipitated from each cell extract, and the precipitants were analyzed by a Western blot with anti-HA or anti-She2 antibodies as indicated. Input shows equal amount of initial cell extracts probed with anti-HA antibody. (B) Myo4-myc and Myo4-HA coprecipitation requires She2. MYO4-HA was expressed with MYO4-13xMYC in the presence of SHE2 (lanes 1 and 2) or in she2Δ cells (lane 3), and U3 RNA expression was induced (lanes 2 and 3) or not induced (lane 1). Myo4-myc was immunoprecipitated and analyzed as in A. (C) The 15S U3 RNP complex consists of multiple Myo4 motors. MYO4-HA was expressed with MYO4-TAP, and U3 RNA expression was induced. TAP-purified complexes were loaded on 10–50% sucrose gradients and the collected fractions were analyzed by Western blotting. Note that the Myo4p blot detects both Myo4-TAP and Myo4-HA.