Dynein and p25 of dynactin are codependent for the HookA–dynein–dynactin interaction. (A) A diagram showing the dynactin complex (Schroer, 2004) and the heavy chain (HC) and intermediate chain (IC) of dynein. (B) Western blots showing that HookA–dynein–dynactin interaction is defective in a dynein HC conditional null mutant, alcA-nudA^HC, and an Arp1 conditional null mutant, alcA-nudK^Arp1. Cells were grown in rich medium YG that contains glucose to prevent expression of the nudA dynein HC gene and the nudK Arp1 gene. (C) A quantitative analysis of the Western results shown in B. The ratio of pulled down dynein HC or dynactin to HookA-GFP was calculated. Values of all the mutants are relative to the wild-type values, which are set at 1. Mean and SD values were calculated from multiple independent pull-down experiments, and the number of experiments is indicated as n. For the ratio of dynein to HookA (dynein/HookA), the mean ± SD values for alcA-nudA^HC and alcA-nudK^Arp1 are 0.1 ± 0.17 (n = 3, P < 0.001) and 0.08 ± 0.13 (n = 3, P < 0.001), respectively. For the ratio of dynactin to HookA (dynactin/HookA), values for alcA-nudA^HC and alcA-nudK^Arp1 are 0.04 ± 0.07 (n = 3, P < 0.001) and 0.01 ± 0.01 (n = 3, P < 0.001), respectively. (D) Western blots showing that the HookA–dynein–dynactin interaction is defective in the alcA-nudA^HC and the Δp25 mutant. (E) A quantitative analysis of the Western results shown in D. Values of all the mutants are relative to the values of ΔC1-HookA in the wild-type background, which are set at 1. For the ratio of dynein to ΔC1-HookA (dynein/ΔC1-HookA), the mean ± SD values for alcA-nudA^HC and Δp25 are 0.04 ± 0.08 (n = 3, P < 0.001) and 0.01 ± 0.03 (n = 4, P < 0.001), respectively. For the ratio of dynactin to ΔC1-HookA (dynactin/ΔC1-HookA), values for alcA-nudA^HC and Δp25 are 0.1 ± 0.09 (n = 3, P < 0.001) and 0.06 ± 0.12 (n = 4, P < 0.001), respectively. (F) Western blots showing that the HookA–dynein–dynactin interaction is defective in the Δp25 mutant. For the pull-down experiments presented in F, a supernatant of the 100,000 g high-speed centrifugation was used. (G) A quantitative analysis of the Western results shown in F. Values of the mutants are relative to the values of ΔC1-HookA in the wild-type background, which are set at 1. For the ratio of dynein to ΔC1-HookA (dynein/ΔC1-HookA), the mean ± SD value for Δp25 is 0.12 ± 0.2 (n = 3, P < 0.001). For the ratio of dynactin to ΔC1-HookA (dynactin/ΔC1-HookA), the mean ± SD value for Δp25 is 0.02 ± 0.03 (n = 3, P < 0.001).