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Vol. 8, Issue 10, 2077-2088, October 1997
Cytoplasmic dynein is one of the major motor proteins involved in
intracellular transport. It is a protein complex consisting of four
subunit classes: heavy chains, intermediate chains (ICs), light
intermediate chains, and light chains. In a previous study, we had
generated new monoclonal 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 chain could block the function of cytoplasmic dynein.
When cytoplasmic extracts of Xenopus oocytes were
incubated with either one of the monoclonal antibodies (m74-1,
m74-2), neither organelle movement nor network formation was observed.
Network formation and membrane transport was blocked at an antibody
concentration as low as 15 µg/ml. In contrast to these observations,
no effect was observed on organelle movement and tubular network
formation in the presence of a control antibody at concentrations as
high as 0.5 mg/ml. After incubating cytoplasmic extracts 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 dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued
binding domain. In an IC affinity column binding assay, both antibodies
inhibited the IC-p150Glued interaction. Thus these
findings demonstrate that direct IC-p150Glued interaction
is required for the proper attachment of cytoplasmic dynein to
membranes.
Institute of Biochemistry and Molecular Cell Biology,
Biocenter, University of Vienna, A-1030 Vienna, Austria;
Cell Biology Graduate Group, University of Pennsylvania,
Philadelphia, Pennsylvania 19104;
§Cell Biology Group,
Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts
01545;
Department of Animal Biology, University of
Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania
19104; and
¶Institute of Zoology, University of Rostock,
D-18051 Rostock, Germany
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