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Dynactin is required for bidirectional organelle transport.

Deacon SW, Serpinskaya AS, Vaughan PS, Lopez Fanarraga M, Vernos I, Vaughan KT, Gelfand VI - J. Cell Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801.

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.

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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 p150Glued 1–811. Purified p150Glued 1–811 binds to GST–C-XKAP but not GST–N-XKAP. (B) Blot overlay assay using 2 μg/ml purified p150Glued 600–811. In this assay, p150Glued 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.
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fig2: 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 p150Glued 1–811. Purified p150Glued 1–811 binds to GST–C-XKAP but not GST–N-XKAP. (B) Blot overlay assay using 2 μg/ml purified p150Glued 600–811. In this assay, p150Glued 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.

Mentions: The fact that dynactin interacts with cytoplasmic dynein via p150Glued raised the possibility of a similar interaction with kinesin II. Based on previous work (Vaughan and Vallee, 1995), we performed blot overlays on purified melanosomes using recombinant p150, residues 1–811. These assays revealed binding to a ∼100-kD protein in purified melanosomes, consistent with the molecular weight of XKAP (unpublished data). To confirm this result, we prepared Escherichia coli expression constructs encoding GST fusions with the NH2-terminal 350 aa of XKAP (N-XKAP) or the COOH-terminal 263 aa (C-XKAP) and purified these proteins for use in blot overlays. In these assays, p150Glued1–811 bound to GST–C-XKAP but not to GST–N-XKAP (Fig. 2 A). To more accurately map the region of the p150Glued –XKAP interaction, a more limited p150Glued construct encoding residues 600–811 was used in blot overlays. p150Glued 600–811 bound to purified DIC's and GST–C-XKAP, whereas no binding was observed to GST–N-XKAP (Fig. 2 B).


Dynactin is required for bidirectional organelle transport.

Deacon SW, Serpinskaya AS, Vaughan PS, Lopez Fanarraga M, Vernos I, Vaughan KT, Gelfand VI - J. Cell Biol. (2003)

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 p150Glued 1–811. Purified p150Glued 1–811 binds to GST–C-XKAP but not GST–N-XKAP. (B) Blot overlay assay using 2 μg/ml purified p150Glued 600–811. In this assay, p150Glued 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.
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Related In: Results  -  Collection

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fig2: 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 p150Glued 1–811. Purified p150Glued 1–811 binds to GST–C-XKAP but not GST–N-XKAP. (B) Blot overlay assay using 2 μg/ml purified p150Glued 600–811. In this assay, p150Glued 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.
Mentions: The fact that dynactin interacts with cytoplasmic dynein via p150Glued raised the possibility of a similar interaction with kinesin II. Based on previous work (Vaughan and Vallee, 1995), we performed blot overlays on purified melanosomes using recombinant p150, residues 1–811. These assays revealed binding to a ∼100-kD protein in purified melanosomes, consistent with the molecular weight of XKAP (unpublished data). To confirm this result, we prepared Escherichia coli expression constructs encoding GST fusions with the NH2-terminal 350 aa of XKAP (N-XKAP) or the COOH-terminal 263 aa (C-XKAP) and purified these proteins for use in blot overlays. In these assays, p150Glued1–811 bound to GST–C-XKAP but not to GST–N-XKAP (Fig. 2 A). To more accurately map the region of the p150Glued –XKAP interaction, a more limited p150Glued construct encoding residues 600–811 was used in blot overlays. p150Glued 600–811 bound to purified DIC's and GST–C-XKAP, whereas no binding was observed to GST–N-XKAP (Fig. 2 B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801.

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.

Show MeSH