THE INVOLVEMENT OF THE INTERMEDIATE CHAIN OF CYTOPLASMIC DYNEIN IN BINDING THE MOTOR COMPLEX TO MEMBRANOUS ORGANELLES OF XENOPUS OOCYTES

Cytoplasmic dynein is one of the major motor proteins involved in intr acellular transport. It is a protein complex consisting of four subuni t classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new m onoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chai n could block the function of cytoplasmic dynein. When cytoplasmic ext racts of Xenopus oocytes were incubated with either one of the monoclo nal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was b locked at an antibody concentration as low as 15 mu g/ml. In contrast to these observations, no effect was observed on organelle movement an d tubular network formation in the presence of a control antibody at c oncentrations as high as 0.5 mg/ml. After incubating cytoplasmic extra cts or isolated membranes with the monoclonal antibodies m74-1 and m74 -2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dyn ein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-c oil domain, in close proximity to the p150(Glued) binding domain. In a n IC affinity column binding assay, both antibodies inhibited the IC-p 150(Glued) interaction. Thus these findings demonstrate that direct IC -p150(Glued) interaction is required for the proper attachment of cyto plasmic dynein to membranes.