Three-headed outer arm dynein from Chlamydomonas that can functionally combine with outer-arm-missing axonemes.
A procedure was developed for isolating Chlamydomonas outer-arm dynein that can functionally combine with the axoneme of an outer-arm-missing mutant, oda1. Previous studies showed that the outer-arm dynein of this organism, containing three heavy chains (alpha, beta, gamma), dissociates upon extraction with a high-salt-concentration buffer solution into an 18-S particle containing the alpha and beta heavy chains and a 12-S particle containing the gamma heavy chain. It was found, however, that the three heavy chains did not dissociate if the high-salt extract was centrifuged in the presence of Mg2+; the three chains constituted a single species (23-S dynein) sedimenting at about 23 S and displayed a three-headed bouquet configuration in electron micrographs. Furthermore, the 23-S dynein had the activity to bind to the axonemes of oda1 and increase the reactivated motility of detergent-extracted cell models; its addition increased the beat frequency from 28 Hz to 53 Hz, a frequency comparable to that of wild-type axoneme. The 18-S and 12-S dyneins, on the other hand, were unable to increase the motility of oda1 axonemes even when added together. The new protocol thus enables purification of outer-arm dynein that retains its functional activity. It will provide a useful experimental system with which to study the mechanism of outer-arm function.
Takada
S
S
Department of Molecular Biology, School of Science, Nagoya University, Aichi.
Sakakibara
H
H
Kamiya
R
R
D000818:Animals; D002499:Centrifugation, Density Gradient; D016825:Chlamydomonas reinhardtii / Q000378:metabolism* / Q000648:ultrastructure; D004398:Dyneins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D005407:Flagella / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron
D004398:Dyneins