CYTOPLASMIC DYNEIN BINDS DYNACTIN THROUGH A DIRECT INTERACTION BETWEEN THE INTERMEDIATE CHAINS AND PL50(GLUED)

Cytoplasmic dynein is a retrograde microtu bule motor thought to parti cipate in organelle transport and some aspects of minus end-directed c hromosome movement. The mechanism of binding to organelles and kinetoc hores is unknown. Based on homology with the Chlamydomonas flagellar o uter arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in Linking the motor protein to or ganelles and kinetochores, In this study two different IC isoforms were used in bl ot overlay and immunoprecipitation assays to identify IC-binding partn ers. In overlays of complex protein samples, the ICs bound specificall y to polypeptides of 150 and 135 kD, identified as the p150(Glued) dou blet of the dynactin complex. In reciprocal overlay assays, p150(Glued ) specifically recognized the ICs. Immunoprecipitations from total Rat 2 cell extracts, rat brain cytosol, and rat brain membranes further id entified the dynactin complex as a specific target for IC binding, Usi ng truncation mutants, the sites of interaction were mapped to amino a cids 1-123 of IC-1A and amino acids 200-811 of p(150Glued). While cyto plasmic dynein and dynactin have been implicated in a common pathway b y genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin a s a dynein ''receptor''. Our data also suggest, however, that this int eraction must be highly regulated.