22S AXONEMAL DYNEIN IS PREASSEMBLED AND FUNCTIONAL PRIOR TO BEING TRANSPORTED TO AND ATTACHED ON THE AXONEMES

In an earlier study we reported the isolation of a cytoplasmic dynein from the cytosol of Paramecium multimicronucleatum. In this study we r eport the isolation and characterization of two cytosolic axonemal dyn eins (22S and 12S) as well as a 19S cytoplasmic dynein from the cytoso l of whole or deciliated cells using preformed bovine brain microtubul es. These three dynein species were characterized according to mass, m orphology, vanadate photocleavage patterns, CTPase/ATPase ratios, K-m and V-max values, temperature optima and reactivity with a mAb. For co mparison, 22S and 12S axonemal dyneins (ADs) were also isolated and pu rified from the demembranated axonemes. The 22S and 12S soluble dynein s appear to be related to ciliary ADs in that the 22S soluble dynein i s three-headed while the 12S is a one-headed dynein, as determined by negative staining. Ciliary ADs and their corresponding 22S and 12S sol uble dyneins isolated from the cytosol also have similar K-m and V-max values as well as vanadate photocleavage patterns and temperature opt ima. A mAb raised against the soluble 22S dynein reacted with the 22S ciliary dyneins but not the 12S axonemal or the 19S cytoplasmic dynein . All isolated dyneins supported similar microtubule gliding rates but had different ionic requirements for the translocation buffer. These results suggest that: (i) the two soluble 22S and 12S dyneins are prec ursor molecules of the ciliary dyneins, (ii) the subunits of the outer arm dynein are already assembled in the cytosol as a three-headed bou quet, and (iii) the 22S and 12S soluble dyneins are functional prior t o being transported and attached to the axonemes of the cilia. (C) 199 4 Wiley-Liss, Inc.