THE P150(GLUED) COMPONENT OF THE DYNACTIN COMPLEX BINDS TO BOTH MICROTUBULES AND THE ACTIN-RELATED PROTEIN CENTRACTIN (ARP-1)

p150(Glued) 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-l), 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 p150(Glued) in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150(Glued) polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150(Glued) were determined to be sufficient for microtubule association. We also tested for a direct interaction between p150(Glued) and centractin, In vitro translated centractin was specifically retained by a p150(Glued) affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved moth from the C terminus of p150(Glued). These results demonstrate that p150(Glued), a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin.