Dynactin is required for bidirectional organelle transport

Abstract

Kinesin II is a heterotrimeric plus end-directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II-mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.

Figure 1.

Kinesin II and the dynactin complex interact in melanophore extracts. (A) Kinesin II was precipitated from melanophore extracts with five different polyclonal (left) and one monoclonal (right) antibodies against the 95- and 85-kD subunits of kinesin II, respectively. Blots were probed with monoclonal anti-p150. Quantification of these blots shows that different kinesin II antibodies pull down 2–6% of the total p150 in the extract. (B) Dynactin was precipitated with polyclonal (left) or monoclonal (right) antibodies against p150. Blots were probed with monoclonal anti–kinesin II. Quantification of these blots shows that p150 antibodies pull down ∼1% of the total kinesin II in the extract. C indicates control samples precipitated either by normal rabbit IgG (left) or an unrelated monoclonal antibody (right).

Figure 2.

Direct interaction of p150 and XKAP. (A) 2.5 μg each of GST–C-XKAP and GST–N-XKAP were separated on a polyacrylamide gel and overlaid with 2 μg/ml p150^{Glued 1–811}. Purified p150^{Glued 1–811} binds to GST–C-XKAP but not GST–N-XKAP. (B) Blot overlay assay using 2 μg/ml purified p150^{Glued 600–811}. In this assay, p150^{Glued 600–811} showed robust binding to 1 μg each of purified DIC and GST–C-XKAP but not GST–N-XKAP. In A and B, the left panel is a Coomassie stain of purified proteins, and the right panel is blot overlay. (C) C-XKAP binds endogenous dynactin from melanophore extracts. GST–C-XKAP bound to glutathione agarose beads pulls p150 and dynamitin from cell extracts, whereas GST–N-XKAP does not.

Figure 3.

Kinesin II and dynein compete for binding to dynactin. (A) Polyclonal antibodies against DIC and the 95-kD subunit of kinesin II are each able to pull down p150 from melanophore extracts, but they do not pull down each other. Antibodies used for immunoprep are shown at the top. Antibodies used to probe the blot are indicated on the sides. (B) The ability of p150 to bind to purified DIC via blot overlay diminishes in the presence of increasing amounts of C-XKAP but not N-XKAP. Purified DIC was overlayed with myc- p150^{Glued 600–811} alone or in the presence of a 1:1 or 1:2 molar ratio of C-XKAP or N-XKAP. Blots were probed with an anti-myc antibody.