Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein.
The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.
Sakato
Miho
M
Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030-3305, USA.
King
Stephen M
SM
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002696:Chlamydomonas / Q000378:metabolism* / Q000502:physiology*; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding
D000255:Adenosine Triphosphate; D004398:Dyneins; D002118:Calcium