The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1).
p150Glued was first identified as a polypeptide that copurifies with cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, and has more recently been shown to be present as a member of the oligomeric dynactin complex, which includes the actin-related protein centractin (Arp-1). Dynactin is thought to mediate dynein-driven vesicle motility, as well as nuclear transport, in lower eukaryotes. The mechanism by which dynactin may function in these cellular processes is unknown. To examine the role of the dynactin complex in vivo, we overexpressed the rat cDNA encoding p150Glued in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150Glued polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150Glued were determined to be sufficient for microtubule association. We also tested for a direct interaction between p150Glued and centractin. In vitro translated centractin was specifically retained by a p150Glued affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved motif from the C terminus of p150Glued. These results demonstrate that p150Glued, a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin.
Waterman-Storer
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CM
Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia 19104-6046.
Karki
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S
Holzbaur
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EL
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008868:Microtubule Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D013329:Structure-Activity Relationship
D000199:Actins; D000072159:Dynactin Complex; D046911:Macromolecular Substances; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D010455:Peptides; C076834:centractin; D004398:Dyneins