Figure 1: Effect of Arl3(Q71L) and LC8 on dynein–dynactin interaction.
From: Arl3 and LC8 regulate dissociation of dynactin from dynein
(a) Microtubule pull-down assays using purified cytoplasmic dynein, dynactin and recombinant proteins: Arl3(Q71L) and LC8. After microtubule pull-down assay performed with or without microtubules, supernatants (S) and pellets (P) were analysed by WB for examination of the change in the ratio of dynein and dynactin. Upper WB: addition of Arl3(Q71L) or LC8 increased dynein amounts in the supernatant (lanes 5–10). Addition of both Arl3(Q71L) and LC8 simultaneously (lanes 11 and 12) resulted in the significant increase of dynein in the supernatant. However, addition of Arl3(Q71L) and/or LC8 did not affect dynactin binding to microtubules (lower panel). Lower graphs: the change of the ratios of dynein (left) or dynactin (right) in the supernatant were quantified (n=5). (b) GST pull-down assay; 0.4 μM Arl3(Q71L) or Arl3(T31N) prebound to glutathione sepharose were reacted with 0.5 μM dynein, dynactin and LC8, respectively (lanes 5–12). As shown in lanes 5–8, Arl3(Q71L) bound to dynactin, but not to dynein or LC8. Input lane indicates 10% of each protein used for GST pull-down assay. (c) Schematic diagram of P150Glued subunit of dynactin with previously mapped interaction sites. Three GST-tagged P150Glued fragments were used in d. (d) HIS pull-down experiments. As shown here, P150Glued fragment aa 549–925 was bound to Arl3(Q71L). Input lanes indicate 10% of each protein used. (e,f) Immunoprecipitation (IP) assays. Intrinsic Arl3- and LC8-depleted MEF cell lysates (Supplementary Fig. 1g,h) were immunoprecipitated with an anti-DIC (e) or -P150Glued antibody (f). Precipitates were treated with Arl3(Q71L) or LC8 alone or in combination. In e, co-precipitated dynactin was apparently reduced after addition of Arl3(Q71L) or LC8 alone, especially in together (lanes 2–5). Similarly, in f the amount of co-precipitated dynein was significantly reduced by addition of Arl3(Q71L) or LC8 alone or in combination (lanes 2–5). Input lane indicates 5% of cell extract used for immunoprecipitation assay. Lower quantitative graphs (n=4) indicate the reduction of co-precipitated proteins. P-values were calculated using analysis of variance, mean±s.e., **P<0.01, ***P<0.001.
