In vitro motility from recombinant dynein heavy chain.
The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.
Mazumdar
M
M
Worcester Foundation for Biomedical Research, Shrewsbury, MA 01545, USA.
Mikami
A
A
Gee
M A
MA
Vallee
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002465:Cell Movement; D018076:DNA, Complementary; D004398:Dyneins / Q000378:metabolism*; D007313:Insecta; D008870:Microtubules / Q000502:physiology; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D004398:Dyneins